Annual Report 06-07English

ANNUAL REPORT 2006 - 2007

Department of Biotechnology Ministry of Science & Technology Government of India

Annual Report 2006-2007

Department of Biotechnology Ministry of Science & Technology Government of India

CONTENTS Chapter-1:

An Overview

1

Advisory Structure Human Resource Development Biotech Facilities and Programme Support Research and Development - Agricultural Biotechnology • Crops • Biofertilisers • Biopesticides and Crop Management - Bioresource Development and Utilization • National Bioresource Development Board • Medicinal and Aromatic Plants • Plant Biotechnology • Animal Biotechnology •

Aquaculture & Marine Biotechnology

• Seribiotechnology - Basic Research in Modern Biology - Medical Biotechnology - Stem Cell - Bioengineering - Human Genetics and Genome Analysis - Environmental Biotechnology - Mission Mode Programme on Bioenergy and Biofuels Biotechnology for Societal Development Bioprocess and Product Development - Food and Nutrition Biotechnology - Large Cardamom -Product Plan - Microbial and Industrial Biotechnology - Biotechnology Patent Facilitating Cell - Small Business Innovation Research Initiative (SBIRI) for Public Private Partnership - Biosafety issues Bioinformatics Biotechnology Parks and Incubators International Collaboration Autonomous Institutions - National Institute Of Immunology, New Delhi - National Centre for Cell Science, Pune - Centre for DNA Fingerprinting and Diagnostics (CDFD), Hyderabad - National Brain Research Centre (NBRC), Manesar, Haryana - National Centre for Plant Genome Research (NCPGR), New Delhi - Institute of Bioresources and Sustainable Development, Imphal - Institute of Life Sciences, Bhubaneshwar Public Sector Undertaking

I

2 2 2 2 2 2 3 3 4 4 5 5 5 6

6 7 7 8 9 9 10 10 10 10 11 11 11 11 11 11 11 12 12 12 13 13 13 13 14 14 15 15 15

Contents

International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi Administration and Finance Chapter-2:

Advisory Structure

17

Standing Advisory Committee-Overseas Biotechnology Research and Promotion Committee Inter-Disciplinary Research Committee National Bioethics Committee Chapter-3:

Human Resource Development

Biotech Facilities and Programme Support

Centres of Excellence in areas of Biotechnology Biotech Facilities Programme Support Chapter-5:

Research and Development

19 20 21 22 22 24 25 25 29 29 32 35 37

Agricultural Biotechnology • Crops • Biofertilisers • Biopesticides and Crop Management Bioresource Development and Utilization • National Bioresource Development Board • Medicinal and Aromatic Plants • Plant Biotechnology • Animal Biotechnology • Aquaculture & Marine Biotechnology • Seribiotechnology Basic Research in Modern Biology Medical Biotechnology Stem Cell Bioengineering Human Genetics and Genome Analysis Environmental Biotechnology Mission Mode Programme on Bioenergy and Biofuels

DBT Annual Report 2006-07

17 17 17 17 19

Postgraduate M.Sc./ M.Tech Teaching Programme Training Programmes Scholarships and Awards Biotechnology Overseas Associateship Visiting Scientists from Abroad Programme DBT-TWAS Fellowship for Ph.D Biotechnology Popularization Support to seminars/symposia/conferences Chapter-4:

15 16

II

37 37 49 51 57 57 69 74 81 87 94 97 102 115 117 118 122 129

Chapter-6:

Biotechnology for Societal Development

133

Programmes for SC and ST Population Programmes for Women Progra mmes for Rehabilitation of Tsunami Affected People Programmes for Rural Areas

133 135 140 140

Chapter-7:

Bioprocess and Product Development

Biotechnological Approaches for Food and Nutritional Security Large Cardamom -Product Plan Microbial and Industrial Biotechnology Biotechnology Patent Facilitating Cell Small Business Innovation Research Initiative for Public Private Partnership Biosafety Issues

143 143 147 147 151 153 153

Chapter-8:

Biotechnology Information System Network

159

Chapter-9:

Biotechnology Parks and Incubators

165

Chapter-10:

International Collaboration

167

-Indo-Denmark collaboration -Indo-Finland collaboration -Indo-Germany collaboration -Indo-UK collaboration -Indo-US collaboration -Collaboration with IAVI -Indo-Australia collaboration New Bilateral Programmes Multilateral Collaboration Chapter-11:

167 167 168 168 168 173 173 173 174

Autonomous Institutions

177

National Institute of Immunology, New Delhi National Centre for Cell Science, Pune Centre for DNA Fingerprinting and Diagnostics (CDFD), Hyderabad National Brain Research Centre (NBRC), Manesar, Haryana National Centre for Plant Genome Research (NCPGR), New Delhi Institute of Bioresources and Sustainable Development, Imphal Institute of Life Sciences, Bhubaneshwar Chapter-12:

Public Sector Undertaking

177 179 182 185 187 190 191 195

Bharat Immunologicals & Biologicals Corporation Limited, Bulandshahr Indian Vaccines Corporation Limited, Gurgaon

III

195 195

Contents

Chapter-13:

International Centre for Genetic Engineering and Biotechnology (ICGEB)

Human Health Plant Biotechnology Chapter-14:

197 197 199

Administration and Finance

201

Administration Establishment Progressive Use of Hindi in the Department Parlianmentary Matters Vigilance Unit Other Activities Finance

201 201 202 203 203 203 204

Annexures Annexures I -

205 Gender Budgeting Cell

205

Annexures II - Important Committees and Task Forces

206

Annexures III - DBT- Sponsored Universities/Institutions Offering Regular Teaching Courses in Biotechnology in India

229

Annexures IV - Biotechnology Overseas Associateship (2005-06)

237

Annexures V - Bioinformatics Infrastructure Facilities

242

Annexures VI - Databases available with BTISnet Centres

246

Annexures VII - Software available with BTISnet Centres

247

AnnexuresVIII - Bioinformatics Training/Workshop Organized During 2006-07

248

Annexures IX - Publications / Patents Of Department’s Autonomous Institutes

250

Annexures X - Statement of Budget Estimates

263

Abbreviations

264

DBT Annual Report 2006-07

IV

Chapter-1

An Overview Biotechnology is a set of rapidly emerging and far reaching new technologies with great promise in areas of sustainable food production, nutrition security, health care and environmental sustainability. Our vision is to use powerful tools of biotechnology to help convert the country's diverse biological resources to useful products and processes that are accessible to its masses for economic development and employment generation. Biotechnology is globally recognized as a rapidly emerging, complex and far reaching new technology. Biotechnology can, over the next two decades, deliver the next wave of technological change that can be as radical and pervasive as that brought about by IT. The recent and continuing advances in life sciences clearly unfold a scenario energized and driven by the new tools of biotechnology. The convergence of advances in biology genomics, proteomics, bioinformatics and information technologies is driving the emergence of a new bioeconomy.During the last five years, biotechnology industry has been growing at a rate of 40% and in 2005-06 exceeded US$ 1.5 billion in turnover. Though the growth was achieved mainly through leadership in biogenerics and contract manufacturing, research leading to innovative product development did not lag behind. The social impact of such growth is evident from India assuming a dominant place in vaccine exports, diagnostics, transgenics (Bt Cotton) and a number of biotherapeutics. There is a projection of an annual turnover of US $ 10 billion for India by 2010 and a speculated about 25% annual growth rate between 2010 and 2015.During the year 2006-07, the impetus has been on programmes of national relevance with special emphasis on strengthening of infrastructure, creation of centers of excellence, capacity building and developing mission mode programmes and public-private partnerships. Over 450 R&D projects have been supported during the year with approximately 200 universities and research laboratories being provided the necessary support in

terms of both capacity building and infrastructure strengthening.In the area of health care, new vaccines and diagnostics have been indigenously developed and are under clinical trials. A major initiative has been taken to develop stem cell research in the country and 6 centres have received programme support. A road map has been formulated and city clusters established to forge interdisciplinary collaboration, crucial to this sector. Cutting edge research in areas of tissue engineering, bio-medical devices, biomaterials, nanobiotechnology and RNAi is being supported. A special initiative for devices and formulation required for a national programme on maternal, neonatal and child health has been initiated. In the area of agriculture biotechnology the focus is on nutritional enhancement, increased productivity and development of crops resistant to biotic and abiotic stresses. Establishing Centres of Excellence has received special attention during the current year to achieve reengineering of certain institutes for greater innovation and focus. The societal development programme has received special attention and benefited more than 20,000 SC/ST population, women and rural population during the year. The efforts were focused to create enably circumstances for increasing access of common people to new technologies and products and promoting the mass use of these technologies for healthcare, nutritional security, employment generation and environmental well being.Life science and biotechnology sector is characterized by dynamic changes in flow of new idea and conception and development of new tools for research. Rapid responses are required to meet these challenges. A number of new initiatives has been taken up during the year to achieve this goal, which include increased number of Ph.D., post-docs including overseas fellowships, rapid grants for young investigators, innovation fellowships etc. A major success during the current year has been the launch of public/private partnership scheme Small Business Innovation Research Initiative (SBIRI),

1

An Overview

which promotes highly innovative early stage, pre proof-of-concept and late stage development research emphasizing on important national needs. During the year, the Autonomous Institutes have concentrated on technology and product development beside4s basic science. New International Collaborations have been forged with Denmark, Netherlands, US, Finland, UK, etc. Several of these are dedicated to tailored agricultural and vaccine and diagnostics technologies for regional/local needs.

strengthen their teaching. To encourage medical and engineered students to take up research work, 26 premier institutions have been identified and 10 fellowships each will be provided. In addition to the ongoing scheme for hands on training of mid career scientists, new schemes for training of college teachers and postgraduate teachers have been initiated. The Department is also providing a number of awards and scholarships to recognize the outstanding contributions of scientists in the field of biotechnology in the country.

Advisory Structure

Biotech Facilities and Programme Support

The standing Advisory Committee - Overseas, SAC (O) of the Department consisting of non-residential Indians settled abroad was reconstituted with the approval of the Hon'ble Minister for Science and th Technology. The 17 meeting of the Standing Advisory Committee-Overseas (SAC-O) will be held on March 15, 2007 which will be preceded by a twoday meeting on HIV/AIDS.

Centre of Excellence in Areas of Biotechnology A programme to augment and strengthen institutional research capacity in areas of biotechnology through support for establishment of Centres of Excellence (COEs) continued during the year. Three types of COEs are planned to be supported: (i) Basic biology emphasizing new opportunities and emerging fields (ii) Centres for Science, Engineering and Technology that would address the interphase between engineering, physical sciences, biology, medicine, agriculture or forestry, and (iii) Translational Centres directed towards innovation in the areas of medicine, agriculture, environment, animal and food biotechnology sectors. Award decisions will be based on scientific and technical merit as determined by peer-review, the Programme Advisory Committee recommendations, the prospect of enhancement of the basic research capability in medical school system and translational capacity in basic science institutions and the availability of funds. Both excellence in technology development and excellence in science will be considered in the proposals for “Centre of Excellence”. The proposals with merit which may not qualify for support as 'Centre of Excellence' may be considered for support as “Programme Support” or an individual 'R&D Project'.

Human Resource Development The Department is implementing M.Sc./M. Tech teaching programmes in general, marine, agricultural, animal, medical, pharma, industrial, biotechnology and molecular and human genetics. The students for these programmes are selected on all India basis and around 1000 students are admitted every year. The department is also facilitating industrial training of these students for a period of six months. A new programme for creation of database on placement of students in last 5 years as well as assistance in placement has been initiated. The Department has assigned a study to IIM, Bangalore for evaluation of ongoing teaching programmes. A study has been assigned to Ernst & Young to assess the availability and requirement of manpower at different levels in the country. DBT JRF programme and DBT PDF programme is being implemented through Poona University, Pune and Indian Institute of Science, Bangalore respectively. The number of JRFs has been increased from 100 to 230 from 2007. Top ranking 100 students in merit will be given option to join any institute of their choice in the country for pursuing Ph.D. 2 JRFs will be provided to each PG teaching institution to

DBT Annual Report 2006-07

Research and Development Agricultural Biotechnology Crops

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In a project to identify, map and transfer desirable alleles at QTLs for yield and yield components and stability and also to generate QTL near isogenic lines of rice, agronomic evaluation of BC2F4 Near Isogenic Introgressions lines (NIILs) as many as 200 BC2F5 progenies were evaluated for the second consecutive year during summer 2006 in multilocation trials. A new triticale line involving Himalayan rye and indigenous wheat genotypes has been synthesized to be further utilized as a diverse source for obtaining certain important wheat-rye translocations. In project on functional genomics of rice aiming at discovery and functional validation of genes, novel genes conferring bacterial blight (BLB) resistance have been discovered in accessions of wild species like O. longistaminata, O. nivara, O. glaberrima and O. barthii; land race accession Ac32753 and few mutant lines of IR64. Gall midge resistance genes Gm1 and Gm4 are being fine mapped to within 10cM of 2 MB region of the genome. In the network project on programme on development of Salinity and dehydration stress tolerance in rice, a gene encoding fructose 1,6 bisphosphate was cloned to full length from Portresia (PCFR) and this enzyme was found to be active in the presence of NaCl. In the project on multi-site Evaluation of Transgenic Mustard (DMH-11) based on barnase - barstar system, the National Research Centre of Rapeseed-Mustard, Bharatpur conducted the trials along with four checks, viz.CMS based hybrid (DMH-1), National Checks (Varuna and Kranti) and a zonal check, at 10 locations during the year 2006 Higher yield of DMH-11 over the best check variety was recorded in 6 out of 9 locations. In the project on development of Transgenics Cotton for Resistance to Insect Pests, around 300 independent transgenics lines in cotton (Coker 310FR) carrying the cry1Ac gene for attaining resistance to Helicoverpa armigera developed. In most of the transgenics, the cry1Ac gene is under the control of the double enhancer CaMV 35S promoter. Improvements have also been made in the transformation protocol of cotton which allows the use of Imidazolinone as a selection agent instead of kanamycin by using a double mutant acetolactate synthase gene as marker.

Biofertilisers With growing environmental concerns, the sole dependence on chemical inputs based agriculture is being replaced by integrated approach involving conjunctive use of both organic and inorganic sources. In this context, biofertilizers have been well accepted as economical, cost effective, renewable and safe organic source of plant nutrients to sustain crop productivity. Moreover, with recent focus on organic/bio-dynamic farming, the demand of biofertilizers is likely to grow at a much faster rate than before. At this juncture, it is realized that microbial inoculants are 'ecological inputs' whose effects are 'subtle and not dramatic' like chemical inputs. Hence, inoculation with good quality inoculants is a must and should be treated as an insurance against failure of nodulation. The shelf life both in the storage and transit needs to be improved with due consideration to various 'abiotic' stresses. The quality oriented production and marketing network will certainly make biofertilizers a viable enterprise for ultimate customer satisfaction. Keeping these in view, programmes on development of liquid biofertilizers and biofertilizers based Integrated Nutrient management packages for plantation crops and medicinal plants have been generated. In addition biofertilizers strains developed through transgenosis will be evaluated in contained conditions. Biopesticides and Crop Management Integrated pest management system has become much more sophisticated through biotechnological interventions. A number of potent and cost effective methods of biological pest control have been successfully developed. Genetic improvement of various species of entomopathogenic nematodes for enhanced efficacy and tolerance to environment e.g. temperature etc. has been achieved and found to be effective against insect pests of pigeon pea, rice stem borer, gram pod borer cardamom root grub, sucking pests of cotton etc. Conservation and augmentation of two predators viz. Dipha aphidivora and Microuns igorotus have been achieved, which suppressed sugarcane wolly aphid populations. Pheromones were found to be quite effective against various

3

An Overview

species of bollworms, Pomegranate fruit borer and sucking moths of sweet orange. Pheromone dispensers were also developed and found to be suitable for Indian condition in comparison to imported ones. Molecular characterization of antibiotic producing novel plant growth promoting rhizobacteria, has been done. Insecticidal toxin genes of various plant species and various novel bacterial strains is being done to develop a potent biopesticides formulation. The multicentric programme on the management of Parthenium launched to control the weed and for its possible economic potential is progressing well.

activities continuing specially for rearing technologies, DNA Barcoding etc. Projects have been supported for prospecting of novel genes, molecules, enzymes etc. from plants, microbes, fungi, lichens for production of potential products of industrial importance. Novel genes/ promoters, transcription factors are also being identified so as to develop transgenics for biotic / abiotic stress and understand different metabolic engineering pathway(s) operative in a system. A biofertilizer formulation based on novel salt tolerant nitrogen fixing and phosphate solubilising strains of bacteria was developed and transferred to the industry.

Bioresource Development and Utilization National Bioresource Development Board

A major new initiative during the year has been the launch of a Network programme on "Zingibers" and “Honey bee resources”. Under the Zingiber network, programmes have been supported on biochemical and molecular characterization in relation to commercially useful traits, prospecting for selected secondary metabolites and domestication of some underutilized species of ornamental value. Under the Tea Research Network, a major new initiative has been taken on generation and analysis of Expressed Sequence Tag (EST) and also integrated genetic linkage map and marker assisted selection. A network programme has been launched for the Indian Coffee Genome Research under which cDNA libraries and ESTs are being developed. Under the Sugarcane Genomics, a major achievement has been the development of PCR based diagnostic kits for red rot and smut diseases which is presently undergoing validation. Under the Bamboo Demonstration programme, nearly 380-ha has been planted with tissue culture material. In addition, R & D programmes have also been supported for developing and standardizing tissue culture protocols for other priority bamboo species.

Programmes under the Board continued in the area of Biodiversity Characterization and Inventorization, Bioprospecting, improvement and utilization of resources and Capacity Building. The Fourth Meeting of the Board Chaired by the Hon'ble Minister th S&T and ES, Shri Kapil Sibal was held on 25 July 2006. The digitized inventory of the primary and secondary data of bioresources (plant, animal, microbial and marine) “Jeeva Sampada” and the maps and atlas prepared under the project on Biodiversity characterization of the landscape using remote sensing tools for Central India, Eastern Ghats and Mangrove regions were released by the Hon'ble Minister. A web portal Indian Bioresource Information Network (IBIN) has also been launched as a single window access to spatial and non-spatial data. This unique effort is the first of its kind which overlays all spatial information with ground level species information, the address to providing details of the genetic level studies being conducted. IBIN site was also launched by the Hon'ble Minister. Study on Mapping and Quantitative Assessment of Plant Resources continued for Eastern Himalayan Region, Western Ghats and Eastern Ghats. The Country's first Butterfly Park at Bannerghatta Biological Park, th Bangalore was inaugurated on 25 November 2006 by Hon'ble Minister Science & Technology and Earth Sciences, Shri Kapil Sibal. The Park houses more than 2000 butterflies at any given time representing 42 species. The uniqueness lies in the research

DBT Annual Report 2006-07

Under the Capacity Building Programmes, during this year 21 Vacation training programmes were organized in different parts of the country benefiting about 600 children on sustainable utilization of bioresources. A Bioresources Nature's trail has been established at Kerala Forest Research Institute (KFRI) sub-centre at Nilambur in an area of 5ha.

4

germplasm and biodiversity characterization. Field demonstration of Bamboo was further extended to cover around 200 hectares in different agroclimatic zones of the country. The activities envisaged under the programme support on Micropropagation Technology Plants were implemented.

Medicinal and Aromatic Plants Four gene banks have been further strengthened with respect to collection, conservation and characterization of more number of germplasm accessions. A rapid and highly reproducible protocol for in vitro propagation of Picrorhiza scrophulariflora has been developed. High yielding lines of Nothapodytes nimmoniana with more than 1% camptothecin were identified from Western Ghats. Evaluation of the performance of elite tissue culture plantlets vis-à-vis stem cuttings of vanilla (Vanilla planifolia) in farmers' field over an area of 20 ha in Tripura state has been initiated. Cell-cultures of Commiphora wightii were grown in 2-litre stirred tank and 6-litre airlift bioreactor for guggulsterone production. A network project on development of standardized herbal product for bovine mastitis has been initiated. Purified pectic polysaccharide from Aegle marmelos have shown significant in vivo antileishmanial activity. Root extract of Clitorea ternatea and taraxerol showed significant inhibition of acetyl cholinesterase activity and cognitive enhancing property. RAPD and minisatellite profiles of the sandalwood (Santalum album) populations of the southern regions of India have been generated. Work on cloning and characterization of regulatory elements of genes involved in picrosides biosynthesis in Picrorhiza kurrooa has been initiated. The full-length 4,11-diene synthase gene involved in sesquiterpene biosynthesis regulation in Artemisia annua has been cloned. Four genes of isoquinoline alkaloid biosynthetic pathway in Papaver somniferum have also been cloned.

Basic research was identified as an important area where impetus has been on understanding the basic mechanisms as to how plants develop and adopt to various environmental conditions. Projects were supported to address issues like how a signal when perceived by a plant is received till a final response is obtained, how internal machinery of a plant operates etc. Salinity and drought tolerant genotypes of Brassica have been identified and physiologically characteised under laboratory conditions. Transgenic plants with both over-expression as well as RNAi constructs developed and are being analysed with respect to their behaviour under osmotic stress. The interaction between plant pathogens and their host plants is extremely complex and this has been identified as a priority area. Studies were supported to study the Molecular basis of determinants of host-pathogen specificity; Early events in pathogenesis initiation with respect to adhesion, penetration mechanisms, hydrolytic enzymes etc.; Signal transduction cascade involved in early events both in host as well as pathogen; Gene expression profiling of plant(s) and pathogen(s) during pathogenesis; Innate immunity / non-host resistance; Systemic Acquired Resistance; Induced Systemic Resistance etc. and Mechanism of Pathogenicity Under the international Solanaceae Genome Project launched alongwith 10 partner countries to sequence tomato genome, India has been assigned the responsibility of sequencing 12 Mb region of chromosome 5. So far 1.5 Mb region has been sequenced. In addition to genome sequencing an effort was made to support research in the understanding functional genomics aspects also, the thrust has been on disease resistance, nutritional improvement and fruit ripening.

Plant Biotechnology During the year, studies continued in the areas of plant tissue culture, molecular characterization and transformation for desirable traits especially post harvest. Tissue culture protocols were standardized for important forest trees - pine, bamboo, red sanders etc; horticulture crops grapes rootstock, papaya, apple root stock and citrus and plantation crops cashew, vanilla, clove. Studies on delayed fruit ripening and increased shelf life continued on tomato, grapes, banana and bell pepper. Studies also continued on molecular characterization of important forest trees, horticulture and plantation crops for

Molecular Taxonomy studies continued to receive support for those taxa which could not be segregated morphologically at family, genera, species and sub5

An Overview

species were studied for over expression of superoxide dismutase enzyme useful for bioremediation and salt tolerance. Bioactive molecules were explored for antibacterial, antiviral and anticancer agents. Role of bacterial plasmid gene was studied in pathogenesis of Epizootic Ulcerative Syndrome and its virulence. Applications with the use of phytase were explored from yeast as an alternate fish feed supplement. Neuro-peptide synthesis was explored from Indian cone snails and conus peptide sequence was worked out. A bioreactor was under development for microbial based treatment system for seafood industrial discharge. Vaccine development for fish for Aeromonas showed promising leads. Bacteriophage therapy in improvement of shrimp larvae was pursued as an alternative to antibiotics in aquaculture. Oligonucleotide probe for monitoring vibrio counts in hatcheries were designed. Organ development and differentiation were studied using perivitelline fluid of Indian Horse Shoe Crab. Development of cell lines from Seabass showed promising leads. Shrimp genomics was pursued with focus on expression of immune function associated proteins from shrimp.

species, level. The taxonomic confustion between Amaranthaceae & Chenopodiaceae has been resolved using DNA fingerprinting. Animal Biotechnology New programme were initiated in the area of animal nutrition and development of newer animal vaccines. Standards were developed for estimation of mycotoxins in animal feed and distributed to various laboratories for routine analysis. A novel and potent anthrax vaccine which includes mutants of lethal factor and edema factor was developed which provides better efficacy in vivo. An attenuated buffalopox virus vaccine was developed and its field trial is underway. Phage display technique was used as an alternative to hybridoma to produce mono specific antibodies against recombinant gag antigen of Bovine Immunodeficiency Virus. Diagnostics for peste des petits virus and buffalopox virus were developed and validated successfully. A RT-PCR assay was standardized for specific detection of Border disease virus and a nested PCR was also developed for differentiation of Border disease virus, Bovine viral diarrhea virus 1 and 2. Multicentric programme on Buffalo Genomics was implemented with focus on identification of genes of economic importance. Structural and functional aspects of 3D scaffold of bovine origin for cardio mycocyte culture are being studied. Efforts are also on to develop biomaterial of bovine origin for reconstruction surgery in animals.

Seribiotechnology Screening of silkworm germplasm for baculovirus resistance in silkworm (Bombyx mori) has resulted in identification of three each of bivoltine and multivoltine strains under a network project. Microsatellite analysis carried out in muga silkworm (Antheraea assama) populations indicate genetic variability in hill populations as compared to plain area populations. A Bombyx mori gene that code for antiviral protein has been partially characterized. A collaborative project has been initiated on epidemiology, spatial and temporal dynamics of diseases of muga silkworm. Under the Indian initiative on International Consortium on Lepidopteran Genome Project, functional annotation of unique putative genes of muga silkworm has been carried out. A total of 67 mulberry accessions have been conserved in vitro and 238 accessions have been successfully cryopreserved. Field evaluation of mulberry trangenics (with HVA-1 gene) for abiotic stress tolerance has been initiated. A few epicuticular

Aquaculture & Marine Biotechnology During the year, various projects on biosurfactants, of marine enzymes, bioactive molecules, immune response, neuro-peptide synthesis, bioreactors were pursued. Progress on vaccine development for fish, bacteriophage therapy genetic characterization of marine organisms, cell lines development, shrimp genomics, fish feed supplement, biosafety and water quality and monitored were implemented. Biosurfactant were screened using marine Acinetobacter genospecies. A prototype for raceway based shrimp production technology was utilized for nursery rearing of shrimp. Studies on occurrence of human pathogenic viruses in coastal marine waters were carried out. Marine cyanobacteria and chlorella

DBT Annual Report 2006-07

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wax related gene fragments having homology with Arabidopsis have been cloned from mulberry. Under a network project, screening of mulberry germplasm for disease response to powdery mildew, tukra and nematode has been completed.

IIT Kharagpur revealed a positive correlation between ROS and sperm morphology and its DNA damage; Scientists at ICGEB, New Delhi characterized CIPK protein and showed that the protein contain autophosphorylation activity; Scientists at IIT, Kharagpur attempted to make folatenanoparticle conjugate by grafting folic acid through some biocompatible nonpolymeric coupling agent and Scientists at University of Madras, Chennai are using biophysical techniques, chiefly X-ray crystallography, but also computer modelling, UV spectroscopy and gel mobility, to study the structures of DNA junctions, such as three-way and four-way junctions, as well as unusual DNA packing modes that lead to novel microstructures.

Basic Research in Modern Biology Support to R&D projects having fundamental questions was continued to provide new vistas to the knowledge required for understanding the intricacies involved in applied research. Fifty-two projects across various disciplines were sanctioned. Significant achievement during the year are : NIPER, Mohali showed promising results for improvement of the oral bio-availability of cyclosporine and reduction of nephrotoxicity associated with the commercial formulation; Studies were carried out at Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum, using an in vitro cell culture model to evaluate the response of adult rat cardiac fibroblasts to hypoxia; IISc Bangalore revealed that UdgB plays a significant role in mycobacteria; Scientists at IMTech., Chandigarh showed that phoP promoter activity is negatively autoregulated by PhoP through sequence-specific interaction(s) involving 3 direct repeat subunits with a 9-bp consensus binding sequence; Studies carried out at IISc, Bangalore indicated that both MBP and preMBP are more prone to aggregation under crowded conditions with preMBP showing a greater extent of aggregation; Scientists at School of Life Sciences, JNU studied delineation methods to explore the physiological role of SMARCAL1; Scientist at IISc., Bangalore revealed that the stabilizing contacts in the folded conformations of glycodelins are different; Scientists at CDRI, Lucknow used NMR spectroscopy to solve structure of Mycobacterium tuberculosis, Escherichia coli, and Homo sapiens peptidyl-tRNA hydrolase. A structural model based on E. coli Pth crystal structure, was generated; Crystallographic analysis of PfFabZ of Plasmodium falciparum carried out at IISc., Bangalore revealed new strategies in the design of novel antimalarials; Studies done at JNU, New Delhi suggested that D. discoideum under oxidative stress exhibits PARP mediated caspase independent paraptotic cell death; Oxidative stress induced DNA damage in ICSI being investigated at

Medical Biotechnology Concerted efforts have been made towards development of vaccines and diagnostics for the major infectious and non-infectious diseases specially in the area of tuberculosis, avian influenza, chicken guinea, rotavirus, typhoid, malaria, HPV . New DBT-ICMR collaborative projects were initiated on HIV/AIDS and microbicide research. About fourteen projects have been implemented through this joint effort. Brain storming sessions were organized on future R&D efforts on Avian Influenza (H5N1) and Chikungunya and other viral infections. Emphasis was also laid on setting up of Virus Research Centres/ Networks projects. A Double-Blind Randomized Placebo Controlled Dose Escalating Phase Ib/IIa study to evaluate the safety and immunogenicity of live attenuated rotavirus vaccine (vero cell based 116E) in healthy non malnourished infants 8-20 weeks of age was initiated. As an initial step, infants are screened for eligibility (healthy, non-malnourished) for receiving the vaccine. A total of 187 infants have been enrolled into the study and vaccinated. The study is progressive well. The cell bank and technology for production of recombinant malaria vaccine candidates i.e. PfMSP-119 and PfF2 were transferred to BBIL, Hyderabad, for developing master cell bank and master working cell bank for characterization. The BBIL has successfully produced cGMP grade material of three consistent batches at 10L scale for

7

An Overview

human clinical trial and initiated toxicological studies. Typhoid vaccine development technology was transferred to M/s. USV Limited, Mumbai for further cGMP grade production, preclinical and clinical studies.

the life sciences. The potential of stem cell technology to develop therapy for many untreatable diseases through cellular replacement or tissue engineering is widely recognized. Keeping in view its potential therapeutic applications, both basic and translational research are being promoted by the Department in various institutions, hospitals and the industry. Till date, more than 55 programmes have been identified and supported on various aspects of stem cell research. These include generation of human embryonic stem cell lines, differentiation of pancreatic progenitor cells to insulin secreting cells, isolation of multipotent adult progenitor cells from bone marrow and their clonal expansion, use of banana lectins for stem cell preservation, haematopoitic stem cells (HSC) for haplo-identical HSC transplantation, use of limbal stem cells for ocular surface disorders, isolation and characterization of mesenchymal and liver stem cells, in vitro differentiation of human embryonic stem cells to neural and non- neural lineages, cardiac stem cells, embryonic stem cells etc,.

In HIV/AIDS research, a novel interaction of SMAR1 with the host TAP (HIV-1 Tat activating protein) has been observed which has potential to block the transcription complex formation. Also, a human mannose receptor has been identified as CD4 independent HIV receptor on spermatozoa which may determine the possible risk of sexual transmission of HIV. An indigenous lentivirus based gene transfer vector has been developed with novel features of versatile multiple cloning site with expanded cloning capabilities. Sandwitch ELISA and RT-PCR based assay systems have been developed for SARS virus. The network programme on the development of vaccine candidates for Human Papilloma Virus has been implemented and progressed well during the year. The department has implemented projects to study the effect of Curcumin for various cancers such as oral pre-cancerous lesions/OSMF; Head & Neck cancer; and cervical cancer. A herbal formulation, TM BASANT for clearance of early cervical lesions associated with HPV is also being investigated.

Disease specific brainstorming sessions included in the area of cardiac, stroke, limb ischemia and orthopaedic to explore the potential applications of stem cells in these areas. “CMC-DBT Centre for Stem Cell Research” has been established at CMC, Vellore to carry out basic and translational research. Multicentric clinical study has been implemented on acute myocardial infarction and pilot study has been initiated on acute ischaemic stroke to determine safety and efficacy of bone marrow mononuclear cells. Stem cell research facilities including clean rooms to handle stem cells have been created at PGIMER, Chandigarh, SGPGIMS, Lucknow, KEM Mumbai and LVPEI, Hyderabad. A training centre to provide training for embryonic and adult stem cells has been supported jointly at NCBS & JNCSAR, Bangalore. Current Good Manufacturing Practices (cGMP) workshop/ training course was also organized. A number of scientists and clinicians were invited to participate in this training course.

Necessary steps have been initiated to start masters course/fellowship in translational health sciences and clinical sciences to create trained manpower in the area at selected medical schools. Steps have also been initiated to set up 5-6 clinical trial research training centres to train graduates and post graduates students of medical schools and life sciences. In order to reduce the age at which scientists get their first Principal Investigator grant and expand opportunities for young scientists (upto 37 years of age), DBT invited proposals under Rapid Grant for Young Investigators scheme, in which about 45 proposals were recommended out of 144 proposals for support and the same were implemented.

Currently the challenges in this area include: availability of human resource of desired expertise; adequate infrastructure; interdisciplinary network of researchers and clinicians for theme-based

Stem Cell Stem cell biology is a promising and emerging field of

DBT Annual Report 2006-07

8

research; appropriate regulatory mechanisms; well defined basic research leading toclinical/ translational research, focused centres and institutions.

development, technology transfer and commercialization. Product development, however, continues to remain a major challenge. Human Genetics and Genome Analysis

The Department and Indian Council of Medical Research have jointly formulated draft guidelines for stem cell research. The guidelines are currently being placed for public debate.

The Human Genetics & Genome Analysis programme which is under implementation since 1990-91 has established major infrastructure to pursue post genomic research activities in the country and also to keep pace with international efforts to exploit the available human, animal and microbial genomics available in public domain. A total of 21 genetic diagnosis-cum-counseling units have been established since 1991-92 providing continuous patients services to affected families to reduce common genetic disorder/disease burden. So far,more than one lakh families got benefited from these units and saved foreign exchange by providing diagnostic services in the country. In order to develop trained manpower in the area, established four training centres (CMC, Vellore; AIIMS, New Delhi; IIH, Mumbai; and SGPGIMS, Lucknow) to train clinician scientists and technicians working at various medical colleges/institutions.

Bioengineering Bioengineering has been identified as a potential area of research by the Department. In order to identify priority areas, create road map, strengthen R&D activities and infrastructure for bioengineering research in the country, the Department organized a number of brainstorming meetings. The key areas identified are: tissue engineering, biomaterials for therapeutics, medical devices, bioinstrumentation and biosensors. A number of workshops were organized in the identified areas. Brainstorming meetings were also organized on “Devices and equipments for maternal, new born and child health care” and “Indigenous production of surfactants for the treatment of pre-mature babies”. It was felt that there is a need to initiate mission mode programmes at institutions having adequate facilities in collaboration with the industry; to strengthen R&D activities for the development of biomaterials especially for drug delivery, cellular/molecular imaging technology; disposable biosensors at low cost for rapid diagnosis of diseases, MEMS biosensor using multi-parameter approach; fabrication of medical devices and bio-instruments, development of implants, etc. As an outcome, several network groups of clinicians and basic researchers have been formed. Multi-centric projects have been generated and implemented in the key areas of bioengineering. A separate task force has been constituted to consider projects in this area.

Several projects in the area of human genetics, human genome diversity, functional, structural, microbial, biocomputing, pharmacogenomics, clinical proteomics were implemented involving large number of clinicians, molecular geneticists and anthropologists. A strategy plan/roadmap document th for the 11 Plan was prepared to initiate major programmes in human genetics and genomic network projects including genetic education in the country. Several projects/programmes have been generated through brain storming session in the area of clinical proteomics, pharmacogenomics and RNAi and many projects were supported during the period. Environmental Biotechnology

There is an enhanced awareness among the scientists, clinicians and industry in the country about bioengineering and product development. Efforts have been made to form network of academia, clinicians and industry in this area so that all stakeholders are associated in all stages of development, namely research, product gy transfer

Concerted efforts have been made to identify priority areas for focussed R& D in the area of Environmental Biotechnology and Biodiversity Conservation. Major 10 areas have been identified under which many research projects have been sponsored. Efforts were also made to generate collaborative, multi9

An Overview

institutional network projects. Since, environmental pollutions are of different nature because of diversified industrial activities in the country, biotechnological interventions can provide the solutions for the environmental pollution problems through development of technologies, techniques, tools and processes. During the year, four brain storming sessions have been organized for generation of focussed, multi-institutional R&D projects in the area of Environmental Genomics, Environmental Biotechnology and Biodiversity Conservation. In addition two Task Force meetings were convened.

developed for chemical hydrolysis of the lignocellulosic wastes from plants like Lantana camara and Prosopis juliflora into fermentable sugars and subsequent conversion into ethanol. The lignin degrading fungal isolate has been characterized, recombinant microbial strains have been identified which show enhanced ethanol recovery. They are being scaled up and industry interaction for commercial utilisation is further being explored. Biotechnology for Societal Development Demonstration and training programmes on proven and field-tested technologies were continued. The projects implemented could help in increasing the skills and income of SC/ST people, rural folk and women through product and process development and employment generation and improvement of their health status. More than 1,16,000 people have been benefited through around 135 ongoing projects on cultivation of aromatic and medicinal plants, mushroom, biological control of plant pests and diseases, solid waste management, vermiculture and vermicomposting, biofertilizers, aquaculture, quail farming and human healthcare etc. This year, 20 new proposals were funded.

During the year, progress of ongoing R&D projects and 18 completed projects were reviewed, 15 new R&D projects have been supported by Task Force. Sub-Committee of the Working Group for formulation of XI th Five Year Plan was constituted and its meeting was held in June, 2006 for finalization of priority areas for consideration in XI th Five Year Plan. Mission Mode Programme on Bioenergy and Biofuels The programme on biofuels continued with support for Bioenergy, Biodiesel and Bioethanol. Bioenergy plantations have been established at the nursery and demonstration levels in different agroclimatic zones. The plants have been developed through various propagation methods. The self-help groups were trained in maintaining nursery and vegetative propagation of the selected species.

Bioprocess and Product Development Food and Nutrition Biotechnology During the year, the main emphasis during the year was on development and use of nutraceuticals and probiotics for holistic health. The Department after indepth consultation with national and Canadian experts has worked out the logistics to establish a National Agri-Food Biotechnology Institute (NABI), and the Bioprocessing Unit (BPU) as its autonomous institution. Both NABI and BPU are planned to come up along with an Agri-food Park designed to house start-up companies. All these three NABI, BPU, and the Park will form the Agri-food Cluster at Mohali, Punjab. Further, taking into account the demand of trained manpower in the area of food and nutritional science ,the Department took the initiative for seeking letters of intent for creation and/ or remodeling of Departments for an integrated Master's and Doctorate programme in Nutritional

Under the Jatropha Micromission,1000 accessions have been collected from different parts of the country and tested for oil content. More than 50% show oil yield of more than 35%. 5 lakhs plants of superior material have been produced in the nursery. Nearly 300 ha have been brought under cultivation. Operational guidelines for collection,characterization and production of quality planting material have been brought out. Programmes are being initiated on improvement of Jatropha through marker assisted selection and metabolic pathway engineering. Under the programme on production of Bioethanol from lignocellulosic waste, methodology has been DBT Annual Report 2006-07

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Science or Food Science & Technology.

few assets that can increase in value over time. They motivate researchers who enjoy challenges; and they also help record and develop new ideas. The Department recognises this and facilitates patenting of research results from publicly funded programmes. The public also benefits as they receive new, useful ideas when the invention develops into a marketable product.

Large Cardamom Product Plan Field evaluation of the performance of tissue cultureraised large cardamom vis-à-vis open pollinated seedlings continued on farmers' field in Uttarakhand state. About 34.45 ha area has been field planted during 2006 season using open-pollinated seedlings and tissue culture plantlets. Four training programmes for project personnel and farmers on cultivation and management of large cardamom have been organized.

Small Business Innovation Research Initiative (SBIRI) for Public Private Partnership The department has launched a scheme 'Small Business Innovation Research Initiative (SBIRI) in September, 2005 to bring biotech industry at the forefront of technological revolution. Under the scheme, innovative, risky R&D projects for establishing proof of concept as well as development and commercialization of research leads having market demands are focused. The department has received tremendous response from the private sector. So far 34 projects are recommended in principle for support under the scheme.

Microbial and Industrial Biotechnology The technologies for production and application of various enzymes having industrial importance such as keratinase, pullulanases, cellulase, laccase, protease etc. have been developed. Emphasis has also been given on production of enzymes like hydrolase, L-asparaginase, phytase, chitinase etc. and medicinally important fungal products such as fumagillin, lovosatin and ezetimibe. The new projects relevant to health sector are focused on development of a novel vesicular drug delivery system for psoriasis and biochip based diagnostics for detection of genetic diseases. Projects on hyper production of dyes/pigments from selected lower fungi for application in textile dyeing industry, development of membrane bioreactor for the synthesis of structured lipids, preparation of an amperometric biosensor for determination of triglycerides, development of immunodiagnostic kit for the detection of Karnal bunt in wheat lots, production of wine from mango, and design and optimization of a circulating fluidized bed biomass gasifier have been implemented. Besides the identified thrust areas, proposals with novel/innovative ideas for product related discovery science and product development have been invited from prospective investigators through a Call for Proposals.

Biosafety issues Under Biosafety programme main emphasis is given to facilitate and implementation of biosafety procedures and guidelines for ensuring safety from the use of Genetically Modified Organisms (GMOs) and products thereof in research and application to the users as well as to the environment in the institutions and industries where recombinant DNA work is being undertaken through Institutional Biosafety Committees (IBSCs), Monitoring-cumEvaluation Committee (MEC) and Review Committee on Genetic Manipulation (RCGM) and other institutional structures. Apart from considering the applications submitted by various organizations involved in the recombinant DNA technology, RCGM has taken several policy decisions such as standardization of protocol for conduct of multilocation field trials, data collection parameters, nomenclature of transgenic crop/ gene/ event and new monitoring mechanism for Bt. cotton. Applications made by several applicants in pharma/ agriculture sector for import of transgenic materials including transgenic seeds, conduct of pre-clinical

Biotechnology Patent Facilitating Cell Patents are powerful tools, which can be used to assert supremacy in the present worldwide knowledge driven scenario. A patent is also one of the

11

An Overview

toxicity studies, contained single/ multi-location field trials on transgenic crops were examined by the RCGM and appropriate decisions were taken. Thirteen Central Monitoring Teams were constituted to interact with the Director of Research of the respective State Agricultural Universities (SAUs) regarding monitoring of the field trials conducted on Bt. cotton, Bt. brinjal, Bt. cabbage, Bt. rice and Bt. okra under the jurisdiction of the respective SAUs. Monitoring-cum-Evaluation Committee (MEC) constituted under RCGM in the Department of Biotechnology met five times during the year to review the field trial data generated in multi-location field trials. MEC & RCGM played an important role in the release of 38 new Bt. cotton hybrids by GEAC and marketing approval of indigenously developed several r-DNA pharmaceuticals. Keeping in view the Hon'ble Supreme Court Order, RCGM deferred the applications for contained open field trials till the Supreme Court takes a final decision on the conduct of transgenic crop field trials.

collaborative projects are being considered so as to improve the contribution of bioinformatics in wet lab biotechnology research. The Centres of Excellence in Bioinformatics such as JNU and University of Pune have upgraded their Diploma courses in bioinformatics to M.Tech in Computational and Systems Biology and M.Sc. in Bioinformatics, respectively. A national level Bioinformatics Certification (BINC) Examination was initiated this year to recognize the quality human resource available in the country in Bioinformatics. An unprecedented success made in organsing the 5th International Conference on Bioinformatics (InCoB2006) in India. Over 1,000 registrants for this conference with 400 posters, 20 papers were published in BMC Bioinformatics with Impact Factor 4.96 and others in J BioScience. The 2nd ASEANINDIA workshop on Bioinformatics was also conducted in this year for the benefit of 30 ASEAN country scientists including scientists from India.

Bioinformatics

Biotechnology Parks and Incubators

The BTISnet program of this department has today developed into an extensive nationwide Network covering over 120 institutions, spread geographically all over the country. The Network is engaged in providing support to Biotechnology research, creating human resource in Bioinformatics and carrying out research in different areas of Bioinformatics. Scientists of this network have published more than 1000 bioinformatics research papers in peer reviewed journals in last five years and helped in publishing more than 3000 research papers in biology and biotechnology. Fifty two Bioinformatics Facilities (BIF) were established towards introducing innovation in Biology Teaching through Bioinformatics (BTBI). These facilities will be a centralized resource of individual institutions to support bioinformatics tools and resources for the enhancement of learning capabilities in Biology and Biotechnology.

The Biotech Parks and Biotech Incubation Centers provide an excellent template for promotion of Biotech startup companies. The Park at Lucknow has progressed well and has attracted several new partners in setting up ventures at the Park. The projects in Andhra Pradesh, Kerala, Himachal Pradesh and Karnataka are being actively performed to emerge as models of Public Private Partnership. International Collaboration International collaborations in biotechnology are an important vehicle for expanding the knowledge base and developing expertise which would leverage the growth of research and development in the country. There is a renewed interest in collaboration with India amongst the developed countries.Good progress has been made following the MOU which were signed with Denmark and Finland and joint projects have been funded. Joint projects have also been funded with The Biotechnology and Biological Sciences Research Council BBSRC, UK. In new collaborations, the Department signed two memoranda with Agriculture and Agri- Food, Canada

Initiated focused multi-institutional consortium projects in bioinformatics to address specific problems through bioinformatics approach. B i o i n f o r m a t i c s a n d e x p e r i m e n ta l b i o l o g y

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and the National Research Centre Canada respectively. The ongoing bilateral agreements and collaborations have also been significant, with joint projects being funded with Germany, Norway and USA. Bilateral interactions have been initiated with Sweden, Ukraine and EU. The multilateral collaboration including cooperation amongst SAARC countries were pursued.

to reduce apopotosis in CD34+ cells. The differentiation of mouse embryonic stem cells into dopaminergic neurons has been achieved. In diabetes research, chick pancreatic β islets has been found to be an excellent screening model for physiological and pharmacological studies. In cancer biology area, a distinctive nuclear-mitochondrial mutational profile and varying stem cell dynamics have been identified which seem to be associated with tumorigenesis. As a potential therapeutic anticholesterol agent, methgl- β-cycolodexterin in combination with other cytotoxic drugs towards reduction of drug dosage is being evaluated. Studies on signal transduction revealed that cox-2 is a potential agent for protate tumor suppression. In infection and immunity studies, selenophosphate synthetase gene has been cloned and characterized. Successfully isolated and characterized dendritic cell types 1 & 2. Genome sequencing of poxviruses and herpesvirus showed that members of these families encode structural homologs of human regulators of the complement activation to mask themselves against the hosts complement attack. Studies on HIV biology indicate that Hsp40 as a crucial player in Nef mediated enhancement of HIV gene expression and replication. Leads from chromatin architecture and gene regulation studies on HIV have advanced the knowledge on mechanism of global gene regulation. During the reporting period, 46 scientific papers were published in high impact factor journals and 7 patent applications have been filled.

Autonomous Institutions National Institute of Immunology, New Delhi The Institute continues to make inroads to basic research related to the immune system with a commitment that the knowledge gained would contribute to newer and more effective ways of addressing the health needs of the country. During the year, more than 50 peer reviewed manuscripts and 5 reviews have been published. There is about 25% increase in the number of original peer-reviewed publications over the last year especially in high impact journals that include: Nature Immunology, Immunity, EMBO Journal, Journal of Clinical Cancer, Cell Death and Differentiation, Journal of Biological Chemistry and European Journal of Immunology etc. The Institute continued with the concept of 'end-toend' research in the biosciences and have signed MoU with AstraZeneca India, Bangalore, and Cadila Pharmaceuticals, Ahmedabad on a technology related to novel molecules that inhibit Mycobacterial FadD proteins and can have the potential as antimycobacterial drugs. National Centre for Cell Science, Pune

Centre for DNA Fingerprinting and Diagnostics (CDFD), Hyderabad

The Centre has emphasis on R&D activities in the areas of cell biology including stem cell biology, signal transduction, cancer biology, diabetes, infection and immunity and chromatin architecture and gene regulation. The national cell repository supplied 1154 cell lines 128 scientific institutions in India. Training and teaching programmes were also conducted. In the cell biology research, for the first time a nuclear pore protein has been found to be associated with interphase microtubules. A protein molecule from perivitelline fluid of Indian horse shoe crab has shown cardiac promoting activity. In stem cell research, arachidonic acid (omega 6) and its metabolities found

The Centre has been providing services for DNA fingerprinting, diagnostics, new born screening and bioinformatics based on modern high-technology DNA-based methods of direct benefit to the public, as well as in performing fundamental research of international standards in frontier areas of biological science. The Centre is providing DNA fingerprinting services to various Government & Law Enforcement Agencies and signed MoUs with State/Central Forensic Science laboratories to popularize this technology for the benefit of the society. There are presently fifteen groups working on diverse research areas related to genetics, molecular and cell biology,

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An Overview

cancer biology, pathogen biology, HIV biology, Immunology, etc. CDFD also has a Sun Microsystem's Centre of Excellence in Medical Bioinformatics. Based on novel technology developed by the Centre, a new joint activity has been initiated this year at the CDFD as “APEDACDFD Centre for Basmati DNA Analysis” with funding through APEDA. The Centre will test and certify export consignments of basmati rice for their purity, and is expected to contribute in increasing the value and quality of such exports from the country. The major thrust areas of research in the Centre continue to be studies on infectious disease pathogens including M. tuberculosis, H. pylori, HIV, and HPV; silkmoth genetics and genomics; computational biology and bioinformatics; and fundamental studies on transcription and signal transduction. Transgenic silkworms have been created that are resistant to baculovirus, causative agent for destroying the worms, by using RNAi technology. Important results in K-Ras signaling pathways in cancer epithelial cells have been obtained and a novel and convenient tool for Human Papilloma Virus detection has also been developed.

research, developmental neurobiology and basic research towards understanding of neurological and psychiatric disorders. National Centre for Plant Genome Research (NCPGR), New Delhi The NCPGR is engaged in developing transgenic plants with improved agronomic characters and nutritive qualities. Nutritional Genomics is one of its priority areas under which a protein rich AmA1 GM potato has been developed. The GM potato lines showed enhanced photosynthetic activity in addition to its increased amino acid contents. The Centre has also made progress on transformation of other food crops such as rice , sweet potato and cassava with AmA1 gene for improving their protein contents. A low oxalate tomato has been developed transferring OXDC gene, which has been successfully tested in the restricted open field trial. The Centre is actively working on chickpea genomics with an idea to identify genes for disease resistance, agronomic traits and seed quality and place them on the genetic linkage map of the legumes. A foc-1 locus that renders chickpea resistance to Fusarium oxysporum f. sp. Ciceri (foc) has been mapped in the vicinity of several molecular markers; which may help in chickpea breeding programme. The Centre has undertaken studies on comparative genomics with focus on wheat, foxtail millet, tomato, chilies, Medicago and Brassica and could map QTLs for grain weight for wheat. The NCPGR has joined an international Solanaceae Genome Network programme for sequencing the chromosome 5 of tomato, and made a good progress on sequencing and annotation for the allocated part.

National Brain Research Centre (NBRC), Manesar, Haryana NBRC was established as a Centre of Excellence in Brain Research with state-of-art facility in the country to consolidate, network and undertake basic research of high caliber in neurosciences and also to generate highly trained human resource. The mandate of the centre is also to have establish linkages with national and international organizations involved in neuroscience research. So far, the centre has established interlinks 47 neuroscience groups/institutions in the country to promote multidisciplinary research and providing the facilities of a digital library. The Functional Magnetic Resonance Imaging (fMRI) facility of the centre was made th operational on 29 September, 2006. As a deemed university, NBRC is continuing its M.Sc. Ph.D programmes for research fellows.

Another area of genomics project concerns manipulation of genes for α-mannosidase and βhexosaminidase to increase the shelf-life of fruits and vegetables.. The Centre has cloned these genes from tomato as well as capsicum plants and their RNAi analogues have been synthesized for genetic manipulations to understand the roles of counterpart enzymes.

The major areas that have been identified for research include computational neuroscience, system and cognitive neuroscience, stem cell

DBT Annual Report 2006-07

On moving to its new building , the Centre has no constraint of space as a result of which it has added

14

many sophisticated instrumentation facilities and expanded its research programmes. The NCPGR has come up a national research centre only of its kind in the country entirely dedicated to core research in advanced areas of Plant Biotechnology including molecular biology, genetic engineering and genomics with an applied emphasis. In addition, it is making an important contribution to the development of trained scientific manpower for the development of these advanced fields in the country.

sporozoite carrying capacity of these vectors. Studies on bio-prospecting were continued with a view to tap the vast potential of thermopiles. A diverse group of bacteria belonging to the genera Thiomonas, Comamonas and Chromobacterium were isolated from previously unexplored hot springs. A chemolithoheterotrophic, thiosulfate oxidizing, gram negative bacterium (designated strain S10) was isolated and identified. 16S DNA sequence data and the total fatty acid analysis suggested it to be a new species of genus Thiomonas for which the name Thiomonas bhubaneswarensis has been proposed.

Institute of Bioresources and Sustainable Development, Imphal

During the year, three workshops on training were organized on DNA technologies, functional genomics and proteomics research and studies of abiotic stress responses and stress inducible genes. Ten publications have been brought out in November 2006 with an average impact factor of 3.02. Six additional non-technical posts were also sanctioned during the year. The construction activities for the new research building, animal house and research scholar's hostel have been initiated and the contract awarded to M/s RITES Ltd.

Work on the database on microorganisms with special reference to cyanobacteria available in Manipur has been initiated. A Distributed Information Sub-Centre (Sub-DIC) under the Bioinformatics Network has been set up at the institute. In vitro multiplication and hardening of tissue culture plantlets of Kaemferia galanga is in progress. Hybridization of two rare vandaceous orchids Aerides vandarium and Vanda coerulea achieved. Genetic differentiation of tree bean (Parkia timoriana) cultivars grown in Manipur were analyzed. About 10 lakhs of spawn and 20,000 fingerlings of Osteobrama belangeri (Pengba) an endemic high value fish were supplied to the farmers. Three training programmes on the use of tools and techniques for bioresource development and utilization were organized.

Public Sector Undertaking There are two public sector undertakings i.e.Bharat Immunologicals & Biologicals Corporation Limited, (BIBCOL) and Indian Vaccines Corporation Limited, (IVCOL). The BIBCOL located at Bulandshahar manufactures Oral Polio Vaccine being used in the National Immunization Programme. The IVCOL was established as a joint venture company. Efforts are being made to revive it with new products mix and financial pattern.

Institute of Life Sciences, Bhubaneshwar Cutting edge technology in molecular biology continued to serve as a useful tool for acquiring insights into biology of the aging process, pathogenesis of chronic myeloid leukemia, infectious diseases such as cholera, malaria and filariasis and in plant and environmental biotechnology. A septaplex PCR assay was developed for rapid identification of species-specific virulent and sxtpositive strains of V. cholerae and one hundred strains of V. cholerae O1 were tested to document the validity of assay. A multiplex PCR assay to detect Anopheles fluviatilis sibling species developed during the course of the year will be used to understand feeding habits (Anthropophilic index) and

International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi ICGEB continued its research efforts in identified areas of human health, agriculture and product development. A high through-put microtiter assay based on the heme detoxification pathway of Plasmodium has been developed for screening chemical combinatorial libraries and crude extracts of marine organisms. Several bioactive proteins from the secretome of insect pathogenic bacterium,

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An Overview

Xanthomonas nematophila have been identified. A HCV test based on designer diagnostic HCV multiepitope protein developed by the centre has been marketed in India. Three workshops were organized in the field of malaria, virology and plant transformation. An International symposium on tuberculosis research was also organized. Two patents have been field.

management to enhance its performance. It effectively utilises the inherent employee skills to achieve both long term and short-term goals of the Department. It provides training and development opportunities to improve the employee skills, improves working environment and thus increases the job satisfaction of the personnel. The vigilance section ensures that the officers and the staff in this department take quick and correct decisions, which ultimately translates into benefit to the Nation as it enhances the perception of India on the international platform.

Administration and Finance The Administration Division in the Department of Biotechnology provides assistance through infrastructure, logistical support and human resource

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Chapter-2

Advisory Structure Standing Advisory Committee-Overseas

biotechnology

_

Inter-disciplinary Research Committee

The Standing Advisory Committee Overseas, SAC(O) of the Department consisting of nonresidential Indians settled abroad was reconstituted with the approval of the Hon'ble Minister for Science th and Technology. The 16 meeting of the Standing Advisory Committee-Overseas (SAC-O) was held on 13-14th March, 2006. The major areas discussed were: Translational Research, UNESCO Centre, Initiatives in tuberculosis, Plants as biofactories and Human Resource Development.

During the year, an Inter-disciplinary Research Committee was constituted to consider project proposals which were inter-disciplinary in nature. Two meetings of the committee were held and a total of 41 projects were considered for support in the area of agriculture, biotechnology. environment, basic research, biotech product & process development and animal and aquaculture National Bioethics Committee

The SAC(O) recommended that scientific leadership should be rewarded, a pool of centrally selected scientists should be created in centers of excellence and universities and create positions/ conditions (e.g. joint grants) where outstanding scientists working abroad can have laboratories in India, with students, staff and post doctoral fellows. SAC(O) also suggested that since the portfolio of DBT had considerably widened and the responsibilities multiplied, it may be appropriate to consider reorganization of DBT.

The Department has setup a “National Bioethics Committee” with the approval of Hon'ble Minister of Science and Technology. The terms and reference of this committee are: (a) To consider any amendment(s) in the “Universal Declaration on Human Genome and Human Rights”; (b) To liaison with the “International Bio-ethics Committee” of UNESCO; (c) To consider and recommend all the proposals for Gene Therapy Research; (d) To develop guidelines for bio-ethics and e) To monitor and review progress on “Gene Therapy Research” programme.

The 17th meeting of the SAC(O) will be held on March 15, 2007, this will be preceded by a two-day meeting on HIV/AIDS.The meeting of HIV/AIDS is being organized with the help of SAC(O) members and would have participation of the best scientists from USA.

This committee is providing inputs to the Permanent Representative of India to UNESCO from time to time based on the inputs received from orther Ministries. This committee has also discussed documents received from UNESCO. During the year, the committee discussed documents on: (a) “Assisting Bioethics Committee” (ABC), and (b) Project on “Ethics Around the World-Rotating Conferences”. ABC project is currently being developed by UNESCO for activities to be undertaken by the Social

Biotechnology Research and Promotion Committee (BRPC) During the year, two meetings of the BRPC were held. A total of 12 proposals were considered for support in the area of medical and agriculture

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Advisory Structure

and Human Sciences Sector of UNESCO in support of establishment of ethics and bioethics committees at all levels i.e. national, regional, local as well as assistance to existing committees as part of capacity building action of UNESCO in the field of Bioethics.

DBT Annual Report 2006-07

The committee felt that there is a need to encourage this kind of activities to interact with the international groups. The committee was also in favour of pursuing the area “Ethics Around the World-Rotating Conferences”.

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Chapter-3

Human Resource Development The Department is implementing an integrated programme of human resource development in biotechnology to generate adequate and appropriately trained manpower required as well as upgrade skills of existing manpower for overall development of Biotechnology in the country. Under this programme, the Department is implementing a number of schemes for teaching and training.

quality of students and to maintain All India nature , students are selected through common entrance test conducted by JNU, New Delhi. The programme has an inbuilt component of visiting faculty to fill the gap areas in in-house expertise and provision of studentship and summer training for students. The progress of these programmes is vigorously monitored by annual course coordinators meet, task force and respective advisory committees.

The progress under different schemes is as follows :

The annual meeting of course coordinators was held on 31st July Ist August, 2006 at Bhubaneswar to review the progress of postgraduate teaching programme, DBT JRF and DBT-PDF programme. The DBT UGC task force on HRD has been reconstituted (annexure I) and task force met on 28th 29 August, 2006 to consider 22 new proposals for support by UGC and 14 new proposals for support by DBT. During the year, advisory committees of NBRC, Gurgaon, HAU, Hissar, Goa University, Goa, Bhagalpur university, Bhagalppur, Burdwan College, Gopalbag, Jamia Milia Islamia, Delhi have been held. One new course on agricultural biotechnology by ND university , Faizabad has been supported and 2 new proposals to start M.Sc. biotechnology in NEHU, Shillong, and agricultural biotechnology in UAS, Bangalore have been approved. One course for advanced PG diploma in intellectual property rights , biosafety and regulatory by M.S. University, Baroda is under consideration for financial support by the Department.

Postgraduate M.Sc/M.Tech Teaching Programme The Department of Biotechnology has initiated a number of M.Sc./M.Tech. teaching courses in different universities and institutes in biotechnology. The universities / institutions have been selected on the basis of existing expertise and infrastructure, strong ongoing R&D programmes and nearby institutions engaged in R&D in biotechnology. Selection has been made in close collaboration with agencies like University Grants Commission, Department of Ocean Development, Ministry of Human Resource Development and respective state governments to ensure continuation of the programme. The department provides critical inputs in terms of equipment, upgradation of infrastructural facilities, recurring grant for books and journals, consumables , travel, contingency, visiting faculty, studentship etc. The courses were started in 198586 in four universities and have been gradually increased to 63 courses at the end of 10th five year Plan based on the need to increase the number of general biotechnology courses as well as start new courses in marine, agriculture, animal, medical , pharma, biochemical engineering, etc. Details of institutions running the courses, no. of seats, mode of selection of students, course coordinator, studentship are given in Annexure-I. To ensure

The total intake of students in the postgraduate courses is around 1000 per annum. Of ongoing 63 courses, 24 have been started in 10th Plan. Till date 17 universities/R&D institutions have been provided one time financial support under non-recurring grant for strengthening their ongoing PG teaching courses, of which 7 have been supported during 10th Plan.

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Human Resource Development

A study for evaluation of ongoing PG teaching programme in terms of availability of infrastructure , faculty, selection of students, revision of course curriculum, quality of teaching programme, placement of students, need to start new courses in gap areas has been assigned to IIM , Bangalore.

availability and requirements of manpower in schools, colleges, universities, R&D institutions, industries, government, and NGOs as well as quality and gap analysis of teaching programmes visà-vis requirement of employers. The study would involve compilation of information about human resources produced in 2006 at 10+2 level in biology and biotechnology stream in CBSE and state boards undergraduate, post graduate, Ph.D. and post doctoral level in biotechnology and allied fields such as applied life science, botany, zoology, microbiology , biochemistry, biophysics, molecular biology, professional degrees such as B.Pharma / M. Pharma, B.Tech/ M.Tech., MBBS/ MD, BVSC/MVSC , B.Sc. / M. Sc. In agricultural science, biochemical engineering, down stream processing, veterinary, pharmacy, marine, bioinformatics, human genetics, IPR / biosafety and molecular biology etc. information on present manpower employed, future need , turn over, products produced in sector based industry in manufacturing and service sector will also be collected.

The Department is considering to initiate new courses in food & nutrition, clinical pharmacology and product development, bio-instruments and biomedical standards, bioenterprise management and financing, regulatory affairs etc. Placement Analysis The Department has compiled data on first placement of 2000 students who have passed out. Students coming out of these programmes qualify in UGC-CSIR National Entrance Test (NET) for Junior Research Fellowship (JRF), DBT Biotechnology eligibility test (BET) for JRF and are pursuing research in leading laboratories in the country e.g. TIFR, BARC, IISc, NII, CCMB, JNU etc. A number of students find placement in leading industries such as Biocon, Dr. Reddy's Laboratories, Shantha Biotech, Panacea, Advanced Enzyme Technology, Bharat Serum, Intas Pharmaceuticals Ltd. Serum Institute, US Vitamins, Wockhardt, Zydus Cadila Pharmaceuticals Ltd. etc. Analysis of first placement of approximately 1000 students produced during 1985 -1995 shows 54% students opt for Ph.D. within the country, almost ¼th students opt for jobs in research, teaching and industry. Analysis of 2000 students produced during last 5 years depicts almost similar trend. Percentage of general biotechnology students working in industry has increased from 12 to 17 while as expected, almost half M.Tech students join industries. The Department has assigned a study for compilation of database on first placement of students passed out in last 5 years and assist students in finding placement in industries by organizing regional interviews.

Training programmes Short Term Training Courses for Mid Career Scientists The department has an ongoing scheme for conducting short term training courses for 12-16 participants for the duration of 2- 4 weeks. Realizing the need to upgrade the skills of mid career scientists engaged in research in biotechnology. These courses are intended to provide hands on training in biotechnology in leading R&D institutions in the country. Every year, about 10-12 short term courses are conducted. During the year, 7 courses have been sanctioned. Short Term Training Courses for College Teachers involved in Undergraduate Teaching in Life Sciences and Biotechnology There is need to upgrade the skills of college teachers involved in undergraduate teaching in life sciences and biotechnology to improve the quality of teaching at undergraduate level as biotechnology is

Need Assessment Study The Department has approved a study to be conducted by Ernst & Young to find out the mation

DBT Annual Report 2006-07

20

a rapidly advancing area and teachers employed several years ago do not get opportunity to improve their skills. Realising this need, Department has initiated a new programme to organize short term training courses for college teachers. The programme was advertised during the year, 16 proposals were received and 5 proposals have been supported.

industries as is evident from increase in number of applicants , number of selected candidates. Number of industries offering this training has also steadily grown. During the last 4 years, 402 students have availed this training. Several leading biotechnology companies like Monsanto, Workhardt, Aurigene Discovery Technologies, Gangagen Biotech, Lifecare Innovations, Dabur, Dr. Reddy's Lab, Panacea Biotech, JK Agrigenetics, Auroprobe Labs, Nicholas Piramal, Jubilant Biosys, Pepsi Foods, Pepsico Holdings, ABL Biotech Ltd., Mahyco etc. have offered training to the students. This industrial training has facilitated in permanent placement of students in industry with a success rate of approximately 25%.

Refresher Courses for Faculty Involved in Post Graduate Teaching in Life Sciences & Biotechnology Considering the need to upgrade the skills of faculty members involved in postgraduate teaching in biotechnology, the department has initiated a programme to run refresher courses for upgradation of skills of faculty members involved in PG teaching. On the basis of an average of six faculty members per teaching programme, about 500 faculty members need to be offered faculty improvement programme. These courses will primarily cater to faculty of DBT sponsored programmes. However, provision has been made for non-DBT sponsored institute's faculty also. The programme was advertised and 3 proposals were received. 3 proposals have been supported.

Scholarships/Felloships and Awards DBT Junior Research Fellowship Programme

(DBT- JRF)

To fill the gap between PG teaching courses and post doctoral fellowship (PDF) programme of the Department, JRF programme has been started from the year 2004. Under this programme, 100 JRFs are selected through Biotechnology Eligibility Test (BET) conducted by University of Pune, Pune and fellowships are provided for an initial period of 3 years which may be extended upto five years depending upon the progress. Contingency grant of Rs.30,000/- per fellow per annum is also provided. It has been decided to provide JRF in two categories from 2007 . Top ranking 100 students in merit will be given option to join any institute of their choice in the country for pursuing Ph.D. 2 JRFs will be provided to each PG teaching institution to strengthen their teaching. To encourage medical and engineered students to take up research work, 26 premier institutions have been identified and 10 fellowships each will be provided.

Biotech Industrial Training Programme On an average, 1000 postgraduate students are passing out every year out of DBT supported teaching programmes. The Department of Biotechnology is facilitating practical exposure to biotechnology postgraduates for a period of six month in industry to bridge the gap between the skill sets of students produced by universities and requirements of industry. This programme is mutually beneficial to the students and industry. Industrial exposure ( in R&D, quality control, production & marketing) orients students to needs of industry increasing their acceptability for placement. It also provides an opportunity to the companies to assess the performance of trainees. This programme has completed 13 years and has become increasingly popular among students and

DBT Postdoctoral Fellowship (DBT-PDF) Programme The department initiated a programme for providing 100 PDF per year in 2001. Under this programme, fellowship of Rs. 11,000 per month in the first year

21

Human Resource Development

and Rs. 11,500 per month in the 2nd year and contingency grant of Rs. 50,000 per PDF per annum is provided for a period of two years. Selection of PDFs is done by Indian Institute of Science, Bangalore. This fellowship can be extended upto 5 years on merit / progress on NIH pattern.

Associateship for specialised training of young scientists In niche areas of biotechnology The screening-cum-selection committee met twice in response to the advertisement for the associateship which was advertised twice in this year. 31 applications were received in response to the first advertisement of which 12 applicants were awarded the associateship for specialized training in niche areas. In response to the second advertisement, 59 applications were received and 27 applicants have been awarded the associateship in the areas of Medical Genetics; Stem Cell Research; Transgenic Animal Models; Agriculture Biotechnology; Bioengineering and Seribiotechnology.

Biotechnology Overseas Associateship To match the increasing demand for highly skilled manpower, it is necessary to commensurately expand the capacity building in high tech areas of biotechnology. There is a gap in the expertise available in cutting edge areas of research and techniques. State-of-the art infrastructure is available within the country and it is imperative that skilled manpower is also available to optimally and constructively utilize these facilities.

Visiting Scientists from Abroad Programme Under this scheme, eminent scientists working in the frontier areas of biotechnology in research laboratories abroad are invited for a period upto three months to research institutions / universities in India to initiate collaborative research programmes or conduct training courses or to participate in teaching activities. During this year, seven scientists from abroad have visited India under this programme at ICGEB, NBRC Manesar; National Instt. of Technology, Karnataka;, Hyderabad; Mangalore and Coimbatore and information and expertise were exchanged in the areas of comparative immunology, stem cell & tissue engineering, neuron-generative diseases and cancer immunology. The visits also provided opportunities for dissemination of information on various state of the art techniques.

The biotechnology overseas associateship programme has provided an opportunity to scientists for undertaking advanced research in frontier areas of biotechnology in leading research institutions outside of India. The overseas associateship is awarded under two categories namely long term (for a period of one year) and short term (for a period of 36 months). The stipend amount per month for longterm is US $ 2000 and for short-term US $ 2400. Biotechnology Overseas Associateship programme was extended towards a new scheme for the associateship for advanced training of young scientists in niche areas in biotechnology. The areas identified are (i) medical genetics (ii) stem cell research (iii) nanobiotechnology (iv) transgenic animal models (v) agriculture biotechnology

Scholarships and Awards National Bioscience Award for Career Development

Biotechnology Overseas Associateship Award 235 applications were received for the Biotechnology Overseas Associateship award 2005-06, of which 121 were shortlisted by a screening committee and finally 82 applications were recommended for the award by the selection committee which includes 61 short-term and 21 long-term.

To recognize outstanding contributions of young scientists below 45 years of age pursuing basic and applied research in biosciences and biotechnology, National Bioscience Award for Career Development was instituted in 1999-2000. As per the scheme, each award carries a cash prize of rupees one lakh, citation and a research grant of rupees three lakhs

The advertisement for the award for the year 2006-07 has been issued.

DBT Annual Report 2006-07

22

per year for a period of three years. The scheme has a provision to give upto ten awards every year subject to availability of suitable candidates. For the career development awards for 2006, 63 nominations have been received and 7 scientists have been selected.

award namely Innovative Young Biotechnologist Award (IYBA) during the year 2005. The purpose of this scheme is to recognize potential young biotechnologist and promote them to carry out research in hardcore biotechnology subject with Innovative ideas. Through this award the scientists those are not working on regular basis will be getting a fellowship of Rs. 25000/- per month and grants-inaid of Rs. 30.00 lakhs for a period of 3 years to be investigated a specific innovative project. The candidate those are in the regular employment will receive an amount of Rs. 1.00 lakh as cash award per annum and grants-in-aid for a period of 3 year.

National Women Bioscientist Award Department has instituted three National Women Bioscientist awards every year to encourage and recognise significant research contributions of women bioscientists. Senior Women Bioscientist Award is given to a senior women scientist for life time contributions, excellent research and application for the benefit of the society. The award carries Rs. 1 lakh along with citation and a gold medal. Two young women scientists below the age of 45 years are awarded Young Women Bioscientist Award for pursuing a brilliant research career in biology. Contributions made in the last 5 years are considered mainly to select candidates for award. The awards carry cash amount of Rs.50,000/- along with citation and gold medal. During 2006-07, 14 nominations under senior category and 29 nominations under young category have been received. For the national women bioscientist award for 2006, 1 woman scientist under senior category and 2 women scientists under young category have been selected.

IYBA 2005 For the IYBA Award 2005, a total of 69 applications were received from various parts of the country based on wide advertisement in various means in which 42 applications were found eligible, meeting the minimum criteria. The review of these proposals was carried out through online with IYBA expert committee members. Out of 42 eligible applications, 15 were short-listed and they were invited for presentation before the IYBA expert committee. Finally 4 candidates were awarded during the year 2005. The following are those awardees.

Biology Scholarships Twenty-five biology scholarships are awarded to top students in Biology/Biotechnology at the CBSE 10 +2 level examination. This scholarship is awarded to encourage them to take up Biology as a career. Each scholarship carries a cash amount of Rs.15,000/-, a gold plated medal and a certificate of merit. The awards are presented at the National Science Day th function held on 28 February every year. From 2006-07, 2 candidates from each state board will also be given DBT biology scholarship.

1)

Dr. Amita Gupta, Delhi University, South Campus, New Delhi

2)

Dr. Samir Vishwanath Sawant, NBRI, Lucknow

3)

Dr. D. Sundar, Pondicherry University, Pondicherry.

4)

Dr. Gitanjali Yadav, NCPGR, New Delhi

IYBA 2006 For the IYBA 2006, the Department has received a total of 82 applications which are under online pear review. Once after this, the IYBA selection committee will select suitable candidate for this award and likely the number of awards can be increased upto 25 this year.

Innovative Young Biotechnologist Award (IYBA) : Background The Department of Biotechnology has launched a

23

Human Resource Development

awardee will, in addition, receive a contingency of Rs.5.00 lakh per annum.

Tata Innovation Fellowship To recognize the contribution of scientists in the area of Life Sciences, Agriculture, Biomedical Sciences and various fields of Bio-engineering, who are engaged in discovery, innovation and invention, the Department of Biotechnology (DBT) has instituted a scheme “Tata Innovation Fellowship” this year. This competitive fellowship is to recognize scientists with an outstanding track record in biological sciences and a deep commitment to find Innovative solutions to major problems in health care, agriculture and other related areas. The aim is to reward interdisciplinary work of high quality with emphasis on translation and innovation. The fellowship is open to Indian Nationals residing in India who are below the age of 60 years. 20 (maximum at any point of time). The amount of the fellowship will be Rs. 20000/- per month in addition to regular salary from the host institute. In addition, each Fellow will receive a contingency grant of Rs. 5.00 lakh per annum.

Emeritus Research Scientist Award In order to utilize the expertise of scientifically active superannuated scientists who have made significant contribution in basic and applied biotechnology and related fields, the Department of Biotechnology (DBT) instituted a scheme “DBT Emeritus Scientist Award”. The main aim of this scheme is to facilitate continued research by scientists particularly those who have been actively engaged in innovative science projects during the preceding five years of superannuation. As an emeritus scientist a person may also contribute towards creation of strategies and road maps in specific areas in biotechnology involving young scientists or promoting counseling or advice to biotechnology industry for innovative progress as well as help industry as a resource person and drive them through with his / her innovative ideas which could boost their research planning and strategy. Emeritus Scientist awardee could also mentor young scientists, guide them through their research projects, participate in training programmes and brain storming meetings. Each Emeritus Scientist (ES) would be entitled to honorarium of Rs. 20,000/-; Rs. 50,000/- as contingent grant per annum and Technical / Secretarial Assistance.

Ramalingaswami Fellowship This year, the department has instituted “Ramalingaswamy Fellowship” for scientists of Indian origin working outside the country in various fields of biotechnology, life sciences, bio-engineering and translational health science and all other related disciplines who are interested in taking up scientific research positions in India. Ramalingaswami Fellows could work in any of the scientific institutions / universities in the country and would also be eligible for regular research grant through extramural and other research schemes of various S&T agencies of the Govt. of India. The main objective of the scheme is to support scientists of Indian origin who wish to return to the home country and pursue research of high caliber in Life Sciences/Agricultural Sciences, Biotechnology, Bio-engineering, Translational Health Science, etc. All areas of Life Sciences & Biotechnology, Agriculture Science, Engineering and Health Care will be covered by this fellowship. The amount of the fellowship will be of Rs.50,000/- per month for the first 3 years and will be increased to Rs. 60,000/- per month during the last two years. Each

DBT Annual Report 2006-07

DBT-TWAS Biotechnology Fellowships for Ph.D and Post- doctoral Research DBT along with the Third World Academy of Sciences (TWAS) has launched a DBT-TWAS Biotechnology Fellowship Scheme for students of the Third World Countries, to help them to pursue their Ph.D/Post-doctoral research programmes in Universities/National Laboratories in India. While TWAS bears the travel cost of the students, DBT provides the monthly stipend and living expenses for the students. The selection of students is done by an Expert Committee constituted by TWAS which has Senior Scientists and also a DBT representative. So far, 8 students have availed

24

this fellowship and 9 students have been selected for Ph.D and post-doctoral research for the current year.

India and two internaitonal trade fares in Chicago and Malaysia. Support to Seminar /Symposia / Conferences

Biotechnology Popularization

Financial support to the Universities and R&D institutions is provided for organizing the national / international seminar / symposia / conferences in various areas of Biotechnology.

The objective of this programme is to popularize the potential of Biotechnology and its application in various fields, amongst students, scientific community and the general public. The department provides grant for organizing popular lectures by experts in Biotechnology; bringing out popular books in biotechnology in English and Indian languages; publication of science research journal in the area of biology / biotechnology; celebration of National Science Day by the institutes and universities which are conducting Post Graduate teaching programmes in Biotechnology with the support of the department. The department also participates in S&T exhibitions and trade fares held in India and abroad.

In the year, 100 national and international seminar / symposia / conferences have been supported. Financial support of Rs. 15.00 Lakhs to the 94th Indian Science Congress held during Jan 3-7, 2007 at Annamalai University, Annamalai Nagar has been provided. The Department also provides travel support to the scientist for attending overseas international conferences to opportunity for give exposure through interaction with international scientific community. This helps in generating new ideas / research projects in the area of biotechnology. This year, travel support has been provided to 120 scientists

During the year, five Popular lectures were organized and department participated in four exhibitions in

PLACEMENT OF STUDENTS (1985-95)

Working (India Non-BT) 2%

Industry 12%

Ph. D (India)

Working (India BT) 14%

Ph. D (Abroad)

Ph. D (India) 54%

Working (Abroad) Working (India BT) Working (India Non-BT) Industry

Working (Abroad) 5% Ph. D (Abroad) 13%

DATA AVAILABLE

:

DATA NOT AVAILABLE : TOTAL No. :

19 25

958 Students (Depicted Above) 261 1219

Human Resource Development

PLACEMENT OF STUDENTS (2000-2005) Ph.D (India) Ph.D.(Abroad) Working

Others 4%

Industries 17%

Industries Others

Ph.D (India) 50%

Working 15% Ph.D.(Abroad) 14%

Total No. of Students = 1979

M.Tech. Biochemical Engineering & Biotechnology and Pharmaceutical Biotechnology (2000-2005)

Others 3%

Ph.D (India) 26%

Ph.D.(Abroad) 15%

Industries 46% Working 10%

DBT Annual Report 2006-07

Ph.D (India) Ph.D.(Abroad) Working Industries Others

26

Biotech Industrial Training Programme 1600

1400

1367 1296

1200

1000 866 800

600 501 400 300

176

200 95

45

60

80

2001-2002

2002-2003

2003-2004

0

No. of applicants

2004-2005

2005-2006

No. of Trainees placed

Biotech Industrial Training Programme 80 70 67 60

60

50 45 40 30 20

29 20

10 0 2001-2002

2002-2003

2003-2004

2004-2005

2005-2006

No. of companies offering training

27

Human Resource Development

Chapter- 4

Biotech Facilities and Programme Support combination of antigens that can potentially be used as vaccine candidates against tuberculosis alongwith identification and validation of genes involved in the pathogenesis of Mycobacterium tuberculosis which are essential for the survival of pathogen in the host. Identification of new drug targets and understanding of the mechanism(s) of pathogenesis will also be carried out. The system biology of the Mtb-macrophage interaction will be studied by combining mathematical modeling with experimental approaches.

Centres of Excellence in the area of Biotechnology During the year 2006-07, the concept papers were invited through two rounds of call for establishment of Centres of Excellence through an open advertisements and DBT website. A total of 163 letters of intent were received in response to the advertisement. Out of these, 38 were short-listed to be invited as full proposals by a screening committee. Thirty three full proposals were received from the invited Team Leaders. The same were peer reviewed and a total of 22 full proposals have so far been considered by a Programme Advisory committee (PAC) constituted by the Department. So far, the following four Centres of Excellence have been supported during the year:

(c) Centre of Excellence for Novel paradigms of inhibitor design against key metabolic pathways to decimate infectious agents at National Institute of Immunology, New Delhi; The major aims are development of anti-malarial and anti-tubercular leads for therapeutics, exploration of peptidomimetic antibiotics, to utilize glycosylation in the design of peptidomimetics and to dissect biosynthetic machinery involved in the biosynthesis of mycobacterial lipids and develop potential antituberculosis agents.

(a) Centre of Excellence for Genetics and Genomics of Silkmoths at Centre for DNA Fingerprinting and Diagnostics, Hyderabad The major focus is to develop transgenic silkworms for baculovirus resistance, identification, characterization and functional evaluation of micro RNAs from silkworms, and analysis of immune related genes.

(d) Centre of Excellence for High-Throughput Allele Determination for Molecular Breeding at International Crops Research Institution for SemiArid Tropics, Patancheru

(b) A Virtual Centre of Excellence for Coordinated Research on Tuberculosis: Development of Alternate Strategies at International Centre for Genetic Engineering & Biotechnology (ICGEB), New Delhi, University of Delhi South Campus, New Delhi; Acharya Narendra Dev College, University of Delhi, New Delhi; Sri Venkateswara College, University of Delhi, New Delhi; Institute of Mathematical Sciences, Chennai and Central Jalma Institute for Leprosy and other Mycobacterial Diseases, Agra

The goal of the centre is to develop effective and efficient Diversity Array Technology (DArT) platforms for enhancing the efficiency of basic research and breeding programmes for a range of Indian crops such as sorghum, millets, chickpea, pigeonpea, groundnut etc. High-throughput allele mining for drought responsive and agronomic trait (drought and salinity) related genes using Targeting Induced Local Lesions in Genomes (TILLING) in various crop species will also be established. It will provide high-

The major focus of this COE is to identify antigens or

29

Biotech Facilities and Programme Support

throughput molecular marker service (SSR, DArT) to Indian researches towards crop improvement and will also organize training programmes to students / scientists within India.

assembling the framework for human antibody light and heavy chain genes is underway. Optimization of various assays crucial for testing antibody binding and neutralization of TNG-α and EGFr is in progress.

Programme Support

Putative homologous of genes encoding proteins of the soluble triglycerol biosynthetic complex (TBC) of Rhodotrula glutinis have been identified in Pichia pastoris at Indian Institute of Science, Bangalore. Gene cloning, recombinant protein expression and biochemical characterization of these recombinant proteins is in progress. A single stranded DNA binding protein that binds to specific upstream sequences of alcohol oxidase (AOX 1) promoter of the methylotrophic yeast, Pichia pastrosis has been isolated and identified as zeta crystallin (ZTA1). About 92 fungal isolates from garden soil and sugarcane baggase were screened for their Diacylglycerol (DAG) content. Four isolates showed a significant accumulation of DAG. The amount of DAG in one of the isolates was found to be very high (27% on dry weight basis). Several clones of M13 phage from the phage display library have been identified which bind specifically with either the hyphal or yeast form of Candida albicans. The clones have been sequenced and peptide sequences have been determined. Under the programme on cancer biology and therapeutics at the same institute, efforts have been initiated to identify gene expression signatures in various forms of breast cancers and breast cancer stem cells. About 96 breast cancer tissues and 82 normal breast tissue have been collected for microarray analysis. In order to elucidate the role of AP2 α in the progression of breast cancer, a total of 314 breast cancer cases (retrospective) alongwith the respective survival data of various clinical forms were collected. Immunohistochemical staining for all these samples have been completed. Towards developing cancer cytotoxic drugs based on metal complexed compounds, two series of ruthenium and platinum complexes were prepared. The cytotoxicity of the complexes and the corresponding ligands has been established by MTT assay on cancer and normal cells.

The following five projects have been supported in 'Programme Support' mode during the year: (i) Studies on Human Genetic Disorders at Banaras Hindu University, Varanasi; (ii) Research on Micronutrient deficiencies (including essential amino acids and fatty acids in reproductive age women) at St. John's Research Institute and Medical College, Bangalore; (iii) Immunotherapy of Cancer and Leishmaniasis at National Centre for Cell Science, Pune; (iv) Translational Research on Transgenic rice at University of Calcutta, Kolkata; (v) Biotic Stress in plants: A concerted approach based on functional genomic, proteomic and transgenic techniques at Bose Institute, Kolkata. In addition, three individual R&D projects have also been supported during the year: (i) Identification of genes from Arabidopsis for engineering of apomixes at Centre for Cellular and Molecular Biology, Hyderabad (ii) Identification of new molecular targets for the development of anticancer agents at Indian Institute of Chemical Biology, Kolkata (iii) Therapy of infectious and chronic diseases: targeted gene delivery and long-term specific modulation of gene expression at All India Institute of Medical Sciences, New Delhi and University of Delhi, South Campus, New Delhi. The salient achievements of the projects supported under the 'Programme Support' mode during the year 2005-06 are summarized below: Recombinant clones expressing erythropoietin (EPO), and interferon gamma (IFN-G) have been constructed and an expression clone for interleukin-2 (IL-2) is being designed toward developing technologies for therapeutic proteins at Institute of Microbial Technology, Chandigarh. Current work is also focusing on optimization of protein yields and folding for wild-type chains of all these three proteins from E. coli. Synthesis of the constructs coding for therapeutic monoclonal antibodies to be used for

DBT Annual Report 2006-07

The 'Programme Support' at M.S. Swaminathan

30

Research Foundation, Chennai aims at characterization and validation of the mangrove genes in the transgenic rice system for abiotic stress 0

tolerance. Fifteen putative transcription factor cDNA clones were identified from the sequenced Avicennia m a r i n a E S Ts . A g r o b a t e r i u m m e d i c a t e d transformation of potential transcription factors isolated from A. marina with constitutive and stress inducible promoters in rice system has been undertaken. One of the EST clones (Am 244) have been shown to be upregulated in roots of A. marina seedlings upon treatment with 500 m M of sodium chloride. Studies showed that Am 244 has a possible role in the response to salt stress in A. marina. Am 244 is found to be a putative plasma membrane protein and its transcript is expressed in both leaves and roots.

6 12 24 48 12w 24w 0 6 12 24 48 12w 24w

Expression studies with salt stress for the two transcription factors Plasmolysis with 0.8M Mannitol confirms plasmamembrane localisation of GFP-PR244 in the transgenics

Plasmolysis with 0.8M Mannitol confirms -- Mannitol Mannitol

++ Mannitol Mannitol

A E x p er s s i o n o f P 5 C S IP

CP WT 1

2

3

4

WT 1

2

3

4

W t - Wil d t y p e I P - I n d u c i b l eo pmro t e r C P - C o n s t i t u t i v eo m p ro t e r

N o n s ters s St re s s

B . P or l i n e l e v e l s i n w i l d t y p e a n d t r a n s g e n i c p l a n t s

WT

WT

Tr a n s g e n i c l i n e s ( C P )

Tr a n s g e n i c l i n e s ( I P )

N o n - s ters s

St re s s 2 w e e k s a f tree rc o v e r y

Tr a n s g e n i c p l a n t s e xs spirn g P 5 C S (opl irn e b i o s y n t h e t i c g e n e ) o n c o n s t i t u t i v e ( C a M V 3 5 S ) a n d i n d u c iobm l eo t( eArB wR e reE )d epvre l o p e d . E x p re s s i o n o f P 5 C S ( A )o,l ipnre a c c u m u l a t i o n ( B ) w a s s e e n o n l ys t ru e snsd ewrh e n e xepsrs e d o n i n d u c i b l oe mport e ra n d t r a n s g e n i c s s h o w e d i m p ro v e d t o l e r a n c e t o d e s i c c a tei sosn (sCt r) . C o n s t i t u t i v e l ye sesxepdr p l a n t s s h o w e d edaescerd gorw t h u n d ecro n tor l c o n d i t i o n s ( C ) .

31

Biotech Facilities and Programme Support

plasmamembrane localisation the transgenics

of GFP-PR244 in

ii) Creation of P3 facility for studying dangerous pathogens with special reference to anthrax causing pathogen Bacillus anthracis at Jawaharlal Nehru University, New Delhi.

At University of Agricultural Sciences, Bangalore, work on transcriptome profiling of drought stress responsive genes from groundnut and their functional characterization has been initiated. Among the genes isolated from Arachis hypogaea, nearly 70% had homologues in Arabidopsis thaliana as revealed by genome analysis. The functional relevance of some of the stress responsive genes was validated. A genomic library of groundnut was developed in lambda as a genomic resource for cloning genes of interest. A few full-length stress responsive transcription factors and functional genes have been cloned from both finger millet and groundnut for functional validation. Trangenics expressing DREB2A, DREB1A and NAC genes in groundnut and finger millet have been developed and are being evaluated for drought tolerance and preliminary studies showed stress adaptive response. Work on introgression of specific drought tolerance traits such as water use efficiency and root characteristics through molecular breeding to improve drought tolerance in rice has been initiated.

iii) NMR facility for structure biology at National Institute of Immunology, New Delhi The significant achievements at the ongoing facilities are as under: International Depository Authority (IDA), Institute of Microbial Technology, Chandigarh: The Microbial Type Culture Collection (MTCC) and gene bank supported during 1987 jointly by DBT and CSIR became 1st IDA in India and 7th in Asia and 34th in the world in October, 2002. Besides IDA deposits, cultures for regular purposes and safe deposits are maintained in the IDA. Repository for Filarial Parasites and Reagents at Mahatma Gandhi Institute of Medical Sciences (MGIMS), Sevagram, Maharashtra The activities and achievements of the facility are as follows:

Biotech Facilities

Maintenance of Brugia malayi infection in rodents (Mastomys & jirds): there are about 322 Mastomys and 285 jirds maintained in a CPCSEA registered animal house with about 24 Mastomys and 42 jirds carrying filarial infection. For breeding and rearing of Aedes aegypti mosquito colony (as vector for transmission of filarial infection to animal models), an Insectarium is also being maintained. Apart from being sources of parasite material & reagents, the animals have been used for filarial vaccine development studies involving Anna University, Chennai and BHU, Varanasi.

The sophisticated biotech facilities have been set up in research Institutes / Universities spread across the country for wide spread use by scientists, institutions, industries and students engaged in biotechnology activities. The facilities undertake production and supply of Biologicals, reagents, microbial cultures and experimental animals. The facilities also conduct regular training programmes for capacity building in areas of instrumentations, handling of small animal houses, bioprocessing, microbial taxonomy and molecular Biology. The department has supported establishment of following facilities during the year 2006-07:-

Maintenance of Filarial parasite Bank: presently there are about 40 million B. malayi mf, 500 male and female adult worms (from infected animals) and 1000 infective larvae (collected from infected mosquitoes) and about 2000 W.bancrofti mf collected from different endemic zones. The parasites from the bank from time to time have also been used for research work in the facility and supplied to VCRC (ICMR),

i) Automated DNA Sequencing and Controlled Environment Plant Growth Chamber Facility at National Centre for Plant Genome Research, New Delhi.

DBT Annual Report 2006-07

32

Programme support for priority areas in Plant Biotechnology at Bangalore University

Pondicherry, Jiwajee Univ., Gwalior, Anna Uni. Chennai, Dept. of Biotechnology, RTM Nagpur University, Nagpur, IIT Kharagpur & BHU, Varanasi.

The activities wise achievements are:

Filarial serum bank: the bank has collection and storage of about 504 bancroftian filarial sera of different patient groups from different endemic zones (viz., Vidharbha, Raipur, Calicut, Mangalore, Bhubhaneshwar & Rourkela) and as well from nonendemic normal individuals. Sera from the bank have been supplied to BHU, Varanasi and Anna University, Chennai for diagnostic work.

Mass propogation, establishment of germ plasm bank of rare, endangered and endemic orchids of Western Ghats: thirty different rare, endangered and endemic orchids species has been collected from Western Ghats. Out of them, 10 rare orchids were multiplied through in vitro seed germination. Conservation, mass propogation, nursery technology and establishment of germplasm bank of rare medicinal plants of Sothern Western Ghats; twenty five different rare critically endangered and high value target species of medicinal plants has been collected.

Databases on diagnostics and management of filarial cases has been developed that provide information pertaining to region-wise clinical manifestations, filarial antigen and antibody positivity in different patient groups, duration of drug treatment requires for different clinical groups etc.

Ex situ conservation of forest genetic resources of Southern Western Ghats; the establishment of gene bank and collecting of species is in progress.

National Laboratory Animal Centre (NLAC) at Central Drug Research Institute, Lucknow

National Facility for Marine Cyanobacteria ( N F M C ) a t B h a r a t h i d a s a n U n i v e r s i t y, Tiruchirappalli

The National Laboratory Animal Centre being a prime source of research animals in the country to fulfill the requirements of animals to the out-side institutions, universities and industrial establishments for research purposes. During the year upto October, 2006, 140 cell lines has been supplied, 581 samples of fecal, blood, urine etc. of d i ff e r e n t a n i m a l s p e c i e s e x a m i n e d a n d microbiological monitoring was undertaken in respect of 835 samples. About 28000 animal lines have been supplied both within institution and to about 35 institutions both in public and private sector. In addition, chest radiography and tuberculin testing services were rendered in 138 case and procuring & utilizing 96 non human primates.

In NFMC, 400 marine cyanobacteria belonging to 25 genera and 65 species are maintained and available for research purposes. From the survey of Nicobar and Lakswadweep Islands, 110 and 112 strains of marine cyanobacteria have been recorded. From Palk Bay region, 50 marine anoxygenic photosynthetic bacteria are isolated and the identification is in progress. Identification tools have been developed with isoenzymes patterns, RAPD, 16sRNA , ITS, cpc and introns as markers. The facility has supplied 30 germplasm cultures to 30 Research Institutions and instrumentation facilities offered to 96 scholars outside the institution.

National Infrastructure Facility on Animal House at National Institute of Nutrition, Hyderabad

Centre for Genetic Engineering and Strain Manipulation at Madurai Kamaraj University, Madurai

The center has been involved in breeding and supplying of different species and strains of animals all over the country. During the year, a total of 19139 animals were bred and 16341 animals were supplied. The center has also provided 510 ml of different blood products so far.

The significant advances have been made with regard to study on M. leprae infection / reaction, regulation of chitinase expression and regulation of 33

Biotech Facilities and Programme Support

antibiotic biosynthesis in Streptomyces. Major achievements include identification of biomarkers in serum of leprosy patients; identification of daunorubicin (anti-cancer antibiotic from Streptomyces) development of strain over producing chitinase; patenting of two skin genes; 6 international publication with impact factor 3 and above and training of 10 students /scientists from industry/institution. The facility has developed technologies for production of chitinase, amylase and antibiotics.

The equipment has been installed and the facility has been extensively used for teaching, training and research. Three training programmes have been conducted on software, maintenance and operations of flowcytometry and 20 scientists /students have been trained..

DNA Sequencing Facility at University of Delhi, South Campus, New Delhi

Sun Fire E20K High End Server and related software for hosting various tools and database received from EMBnet and APBionet has been installed for developing new tools and services for Bioinformatics and Biotechnology Research. The upgradation has a very positive impact on quality and quantity of bioinformatics related publications from CDFD.

Upgradation of the existing computing infrastructure at the Bioinformatics facility at Centre for DNA Fingerprinting and Diagnostics (CDFD), Hyderabad

The existing facilities have been optimally utilized and more than 1200 users are benefited. A 48 capillary automated DNA sequencer has been installed as phase III of the facility. Toxicology facilities for GM Products at Shriram Institute for Industrial Research (SRI), New Delhi

Transgenic Green House Facilities at University of Agriculture Sciences, Bangalore and Tamil Nadu Agriculture University, Coimbatore

The Toxicology facility was inaugurated on 23.11.2006 and evaluations of GM products has already been started. Remodeling of existing animal house is completed and has been made functional. Toxicological studies for DBT sponsored projects namely Natural Dye & Dunelliella Biomass has already been completed and Safety evaluation of the GM Tomatoes are in progress. These studies have been conducted on 50 % of the SRI's scheduled charges.

The green house with incinerator has been constructed with the objectives of testing of the transgenic crops already developed by these universities for evaluating their performance outside the laboratory, and to obtain Bio-safety data for large Scale cultivation and testing of the transgenic materials developed outside the Universities to test their performance and to obtain the Bio-safety data for large scale cultivation to derive their benefits.

High precision microcalorimetry facility for monitoring biomolecular stability and interactions at Jawaharlal Nehru University, New Delhi

Prof. S.P. Roychaudhari Drosophila Stock Centre at University of Culcutta, Kolkata Drosophila stock centre maintains over 400 different genetic strains D. melanogaster. These stocks are used for teaching in various Universities and Institutes. Some of the strains has already been enlisted Drosophila Information Service, USA. 200 M.Sc. and Post M.Sc. students from different Departments of Universities are trained by the Scientists of the Centre. A workshop entitled “Drosophila genetics: A new way to look teaching and Research resources” with 40 participants conducted.

The main objective of the facility is to determine the thermal stability and conformational transitions of proteins, enzymes, nucleic acids and lipids in terms of transition temperature, enthalpy, free energy and heat capacity of denaturation. The equipments have been commissioned and are being utilized by various users. Flowcytometry Facility at All India Institute of Medical Sciences, New Delhi

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34

production of D. asper is in progress 10000, plants have been dispatched and it is aimed produce over 40,000 plants by end of August, 2007. In addition other species of Bamboo are also being produced B. balcooa, B. nutans and Dendrocalamus hamiltonii, Mass multiplication of Anogeissus latifolia is being carried out for the industry. Presently, over 6,000 plants of this species are undergoing hardening in the nursery.

700 MHz NMR facilities for Biological Research (Jointly with DST) at IISc., Bangalore The facilities has been utilized for structure studies on cytochorme b5 based fusion protein system, Conotoxins, conformationally equilibrium forms of Holo-Acyl Carrier protein (PfACP) from Plasmodium falciparum. enzymes of the fatty acid biosynthesis pathway in the malarial parasite and Pantothenate Synthetase. During the year, two training programmes /seminars have been conducted with 68 participants and 42 resource persons. The facility has resulted in 37 publications in International journals.

Approximately 4.90 lakhs of imported citrus rootstocks varieties, C-35, US-812, Benton, Carrizo, Rich 16.6, Troyer Citrange and Rangpur lime were dispatched to Punjab Agri Export Corporation during Feb. 2006 Sep. 2006. Fresh initiations of the varieties, which are in demand, were made and the cultures were sent for virus-indexing. The results have confirmed the virus-free nature of the mother

Programme Support Programme Support for Micropropagation Research and Technology Development For large scale production and demonstration of tissue culture plants support continued to the two micropropagation technology parks and hardening units. A National Certification programme for insuring production of quality planting through tissue culture material was volved in consultation with Ministry of Agriculture. The salient achievements under the programme are given below: Micropropagation Technology park at TERI, New Delhi During the year, about 6.9 lakh tissue-cultured plants of various species were produced/ dispatched from DBT sponsored Micropropagation Technology Park at TERI. This includes forestry species (Paulownia, Dendrocalamus asper, D. strictus, Bambusa bambos, B. balcooa, etc), fruit crops (apple, citrus, strawberry), medicinal & aromatic plants (Aloe vera, Swertia chirata, Patchouli, Stevia, etc) and cash crops (Jatropha, hops). The tissue-cultured plants were distributed among various end users for field demonstrations and routine plantations. Production of a less-thorny type of Bambusa bambos, collected by Karnataka Forest officials was undertaken during the reporting period. More than 10,000 tissue cultured plants were dispatched from MTP. MTP shall be producing and dispatching approximately 15,000 plants of Bambusa bambos by July 2007. Mass

Tissue culture raised Acacia mangium plants in field : 3 years old

cultures. Currently, mass-production with freshly initiated rootstocks is underway as MTP is required to supply 2,00,000 plants in Feb-March 2007 and additional 2,00,000 plants in September-October 2007. Cultures have been initiated and plants being produced to meet target. In 2005, a new variety, 'Kamarosa' of strawberry was introduced into production. The plants of this variety were massproduced and transplanted at TERI's Supi Centre in March 2006, for field trials and runner production. In addition to 'Kamarosa', 23,000 plants of two other varieties, Chandler and Ophra were produced during

35

Biotech Facilities and Programme Support

the reporting period. Out of these, approximately 20,500 plants have been dispatched to various private growers. Approximately 55,800 plants of Aloe vera were dispatched to private growers during 2006. Over 12,000 plants of a natural sweetener, Stevia rebaudiana were dispatched to various private growers in and around Delhi-Gurgaon region. Largescale production of medicinal plant, Swertia chirata, was carried out and approximately 30,000 plants were dispatched. Approximately 4000 plants of 'Lahul Bitter' variety of Hops were supplied to a private group. This production was taken up as a contract production order.

40kg/plant). This has lead to generation of awareness among the farmers, that tissue culture propagules remain disease free for 3-4 generations, and exhibit early bearing, uniformity and higher yield. In turmeric, the average yield was approximately double as compared to the conventional plants. Two thousand ginger rhizomes were given for field trial to a farmer who conducted the trial near Narayangaon Dist. Pune. Rhizomes with a weight of 7-10 g have shown the highest survival rate. For forest trees species seven demonstration plots were established using tissue culture raised propagules of Teak and Bamboo in which two agencies, two research institutes, one farmer and one seed company are involved. A field trial on teak was carried out through Choudhary Plantation Ltd at Raipur, Chattisgarh. It was observed that the height of tissue culture raised trees was more than that of local variety. Tissue culture raised teak trees grew straight with fewer branches; hence the commercial bole realization will be more. Tissue culture teak trees had fewer branches on the lower portion and thus expenses of labour for pruning can be saved. The problem of nodes was also less. A clonal trial on teak using three clones of tissue culture raised propagules of teak has been conducted at Miraj, Sangli, Maharshtra through Shree Swami Samarth agency since June 2002. The plants are over 4 years old now. Field data recorded during the year indicate that Clone A and Clone B are exhibiting better growth than Clone C. This trial has again proved that tissue culture raised plants exhibit higher uniformity and biomass.

Micropropagation Technology Park, NCL, Pune Extensive R&D was carried out on the micropropagation of identified crops species. Following tree species were selected based on economic importance and requirement of user groups-Acacia mangium ,Casuarina equisetifolia, and Jatropha curcas. Extensive work on development and refinement of micropropagation protocols for economically important plants at tissue culture pilot plant, NCL, Pune have led to development of micropropagation technologies which have been tested for technocommercial feasibility by the way of large number of field verificatory trials, benefit to cost ratio analysis and technology transfer to industries. With a view to generate awareness about the superiority of tissue culture raised propagules among the farmers and user agencies, small scale demonstration plots using tissue culture propagules for horticulture crops and forest tree species have been established under this project. For horticulture species five demonstration plots were established using tissue culture raised propagules of banana, turmeric, ginger, chlorophytum and patchouli. It was observed that tissue culture raised plants showed higher growth as compared to conventional plants. Growth of Banana var. mahan was best amongst the banana tissue culture raised plants. Two ratoon crops of these tissue culture raised banana were taken. The average yield was 30-40 kg/plant. The explants were taken from these plants for tissue culturing by a local tissue culture laboratory. These plants also exhibited early bearing capacity and higher yield (30DBT Annual Report 2006-07

A field trial on two species of Bamboo viz Dendrocalamus strictus and Bambusa bambos is being carried out through Kerala Forest Research Institute, Thrissur. A total of 4000 no. of tissue culture raised plants of bamboo were supplied to KFRI. These plants survived the transporting shock and have been field planted in the month of November, 2006. A total of 3000 tissue culture raised Bambusa bambos plants have been supplied to West Bengal State Council of Science and Technology, Kolkata for field trial. These plants have been field planted in 5 hectare area and are reported to show high survival rates and good growth. 36

Chapter- 5

Research and Development Agricultural Biotechnology

augmented design. Also 141 progenies had numerically higher yield (10-20% higher) than the corresponding recurrent parent yield components leading to this increased yield in each line have been identified. In general grains per panicle was the most important trait contributing to increased yield.

Crops An R&D project is ongoing at PAU, Ludhiana to identify alleles at QTLs for yield and yield components and stability and also to generate QTL near isogenic lines (QTL-NILs) for pyramiding desirable QTL alleles into elite breeding lines. To achieve the set objectives, for generation of introgression lines more than 35 000 cross seed were generated during 2005 crop season that were planted during 2006. BC2F4 plants of the crosses involving Pusa44 have introgressions from 18 accessions and PR114 have introgression from 18 accessions. Introgression of desirable variability is in progress from additional 30-40 accessions. Desirable plants selected from BC2F4 have been selected for agronomic evaluation. For agronomic evaluation of BC2F5, a set of 25 NILs were evaluated in replicated trials. These lines are being analyzed for introgression using SSR markers. Out of the 200 progenies about 79 progenies showed numerically higher yield (10-20% in small plots) than the recurrent parent PR114. About 800 BC2F2 progenies were evaluated at CRRI Cuttack. Selected plants of these progenies were evaluated in multi-location trial in an

Panicle size of recurrent parent PR114 (3) and NIILs derived from O. nivara (1&2) and O. glaberrima (4&5)

In a project on microsatellites in wheat genomics continuing at CCS university, Meerut, two hundred seventy five (275) ESTSSRs were physically mapped to 672 loci with an average of 2.44 loci per ESTSSR. The distribution of loci in the three subgenomes, seven homologous groups, and 21 chromosomes were nonrandom. Minimum number of 20 ESTSSR loci was mapped on chromosome 4B and a maximum number of 46 ESTSSR loci were mapped on chromosomes. The distribution of loci in distal and proximal bins deviated significantly from the distribution expected on the basis of the relative lengths of these bins. Also gene enrichment strategies of methylation-filtration and reassociation kinetics were used to generate and analyse 2000 hypomethylated, 1026 high C0t (HC) and 1253 reassociated DNA (RD) sequences of bread wheat, which together constituted 1.61 Mb of genomic sequences. SSRs were detected in the above sequences. At CSK HP Agricultural University, Palampur interesting results were obtained in the project on bread wheat improvement through doubled haploidy breeding following wheat x maize system. Studies were conducted during the period under investigation to enhance the doubled haploid production frequency in wheat through in vivo application of colchicine. A new triticale line involving Himalayan rye and indigenous wheat genotypes has been synthesized to be further utilized as a diverse source for obtaining certain important wheat-rye translocations. Development of a doubl haploid population for mapping the genes responsible for vernalization and resistance to leaf and stripe rusts is

Comparison of Pusa44 (left) and Pussa44 introgression line (right) derived from a cross with O. glaberrima acc 100108

37

Research and Development

in progress. Besides multiplication of the elite winter and spring types of doubled haploids, multi locational evaluation was also conducted at different research stations of the University encompassing varied agroclimatic situations of HP including dry temperate. Seven doubl haploids of different maturity groups have been included in the Integrated Disease Screening Nursery (IDSN) of the All India Co-ordinated Wheat Improvement Project and subjected for further evaluations. Another project recently sanctioned at the same university is on application of molecular cytogenetic approaches and chromosome elimination techniques for genetic upgradation of bread wheat and other hill crops for various biotic and abiotic stresses. In the last couple of months, various elite triticale, wheat lines (hexaploid & tetraploid), wheat doubled haploids and triticale x wheat derived recombinants along with different maize lines (in the polyhouse) have been raised in the fields for developing fresh triticale x wheat hybrids and fix the already developed triticale x wheat derived recombinants through maize and Imperata cylindrica mediated haploid induction techniques. Besides all efforts are being made to establish and refine protocols for exercising in situ hybridization in the Lab so as to utilize it further for physical mapping of the alien gene introgressions in bread wheat.

Hyderabad, for development of transgenic groundnut expressing synthetic cry IE-C and a rice chitinase cDNA for insect resistance.The transgenic plants reported earlier were characterized by RTPCR, southern and western analyses. Western analysis on the transgenic plants using cry IC antibodies showed that the synthetic cry I E-C protein is expressed. Southern analysis using the PCR amplified npt II fragment as a probe indicated two integration sites on 3.5 kb and 6.0 kb fragments, which are longer than the expected sizes indicating integration of the transgenes into the groundnut genome. Insect bioassay on the transgenic plant progenies carrying the cry I E-C individually and in combination with rice chitinase cDNA indicated that the combination of genes had a better effect on insect mortality, combination plants exhibiting faster Spodoptera larval kill. Under project in progress at CPRI recombinant proteins of two putative subunits of potato RNase P, Pop5 and Rpp25, were over expressed in Escherichia coli and purified to homogeneity. RNase P assay was standardized using reconstituted E. coli RNase P protein (C5) and RNA (M1) subunits. However, both the antisera immunoprecipitated partially purified potato RNase P from floral buds. Standardization of purification procedure of potato RNase P to homogeneity and characterization of subunit composition of the enzyme from two different tissue sources are in progress.

In a project on molecular analysis of dehydration response in chickpea undertaken at NCPGR, New Delhi interesting results reported. Plants suffer from dehydration or water-stress and they respond by inducing expressions a set of genes. Some of these genes have been shown to provide tolerance to the plants when expressed in high amount. In this project towards screening for dehydration responsive elements in chickpea, induction of expression of 101 genes under dehydration stress in chickpea seedlings was carried out. One of the dehydration inducible chickpea genes (CaCIPK) is a CBLinteracting protein kinase (CIPK) homologue. CIPK is a recently identified serine/threonine kinase family. Computational analysis shows presence of a number of putative stress-responsive elements in the promoter. Different promoter deletion constructs with GUS gene as reporter have been transferred in tobacco.

The full-length and truncated versions of the BC1 and BV1 genes have been amplified from the full-length DNA B of the Madhya Pradesh isolate of the virus at MKU Madurai. These have been cloned in the binary vector pGA643 mobilized into Agrobacterium strain KYRT1 and the high-yielding NRC37 variety of soybean was transformed. PCR and Southern blots indicated the presence of the inserted DeltaBC1 gene in the transformed soybean plantlets. Although the wild type soybean could be regenerated from embryonic tip explants, the transformed plants did not form roots in the selection medium. Currently work is in progress towards establishing all the transgenic plants by micrografting. Molecular analysis of the transgenic plants will then reveal the presence and copy number of the transgenes.

Efforts were being made at University of Hyderabad,

Insecticidal crystal protein genes (cry) of Bacillus

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thuringiensis that are specific to Lepidoptera were selected out of 150 different cry sequences available at IARI, New Delhi. Sequences of all the three individual domains were extracted separately. As second and third domains are hyper variable among all delta-endotoxins and are implicated in insect receptor binding, they were aligned to study evolutionary relationships. Second domain of Cry proteins is responsible for insect specificity and receptor binding. The phylogenetic relationships of the second domains of Lepidoptera-specific genes show that Cry1J and Cry1Ac are closest to each other. On the basis of this study Cry1Ac the most effective toxin against Helicoverpa armigera and Cry1J that is closest to Cry1Ac second domain were used to design a chimeric Bt toxin. Twenty five tobacco transgenic lines were tested against neonates of Helicoverpa armigera by 'leaf feeding bioassay technique'. There was cent per cent larval mortality up to 3 days after feeding transgenic lines (T-9, 16, 22 and 25) and more than 90% in T-8, 12 and 18 transgenic lines. Seventeen tobacco transgenic lines (A-Q) were tested against neonates of Spodoptera litura but none of them showed any potentiality against the test insect. The crystal protein (Cry 1Jb) was bioassayed against H. armigera and S. litura on the basis of 'artificial diet surface incorporation' method.

used for the foreground selection and the polymorphic markers from a set of 500 SSR markers spanning all the bin locations were used for the background selection. The F1s were developed by using the recurrent parents as female parent and the first back cross was made with the F1 hybrid as the female parent to develop the BC1F1 seeds. The selected plants after MAS were used to develop the BC2F1 generation. At this stage the homozygosity of the selected plants were more than 90% and were similar to the recipient parents. Those selected plants were selfed to recover homozygous opaque 2 allele. The QPM version of CM 212 (VQL 1) and CM 145 (VQL 2) were used to develop the QPM version of the maize hybrid Vivek 9. The new hybrid (FQH 4567) possesses all the good yield attributes with enhancement in tryptophan content and elevated level of lysine. Three mapping population's viz., TAG 24 x GPBD 4, TG 26 x GPBD 4, VL 1 x mutant differing for late leaf spot (Phaeoisariopsis personata Berk & Vrx) and rust (Puccinia arachidis Speg) resistance have been generated in groundnut at UAS, Dharwad. The phenotyping of the RILs under artificial ephiphytotics has been completed for late leaf spot and rust. Screening of the parents VL 1 and Mutant (AFLP- 768 and SSR-47); TAG 24 and GPBD 4 (SSR-874); TG 26 and GPBD 4 (SSR- 874) for DNA polymorphism have been completed. Ninety-nine AFLP primers and 26 SSR markers (VL 1 and Mutant), 67 SSR markers (TAG 24 and GPBD 4) and 101 SSR markers (TG 26 and GPBD 4) were found to be polymorphic. Selective genotyping with 19 SSR polymorphic markers revealed association of 13E6 SSR marker with resistance to late leaf spot and rust and 5D05 with rust. The genotyping of individual RILs with the polymorphic markers is in progress.

The project work was initiated by using 82 maize inbreds procured from Almora, Ranichauri and Bjauara centres at CSKHPKV, Palampur. A field trial was conducted at Bajaura to evaluate these inbreds for different characters including diseases and purity. These inbreds have been subjected to RAPD analysis using 22 random primers at Palampur. The data are being processed. The SSR analysis of these lines has been also been started. On the basis of RAPD and SSR data, the lines will be assigned to different heterotic groups accordingly. A set of nine normal maize and five QPM inbred lines were analyzed for polymorphism with opaque 2 specific markers VPKAS, Almora. Based on the result, three normal inbred lines viz. V25, CM 212 and CM 145 and two QPM donors viz. CM 173 and CML 176 were chosen for the conversion programme. The QPM donors had tryptophan content ranging from 0.80 to 1.05% of the total protein. Three SSR markers, viz., phi 057, phi 112 and umc 1066 located as internal repetitive elements within the opaque 2 gene, were

Under programme for survey of new Bt protein, a total of 415 soil and 87 leaf samples were collected from seven districts of Kerala. The locations have been mapped based on GPS reading. 693 Bacillus sp. were isolated, out of which 219 turned out to be Bacillus thuringiensis. These were characterized for starch hydrolysis, urea hydrolysis, VP test, utilization of lecithin, esculin and sucrose. Three different types of crystal proteins were identified by staining with comassie brilliant blue and these included spherical, cuboidal and bipyramidal. Thirty random decamer

39

Research and Development

primers (Operon) under the series OPAA (OPAA120) and OPL (1-10) were screened for amplification by polymerase chain reaction. Five strains isolated from various parts of Kerala have been characterized by RAPD with 20 primers. Bioassay has been carried out with 58 isolates and 13 reference strains on larvae of the insect Diaphania indica, which is emerging as a problem for the cultivation of vegetables in Kerala. TNAU, Coimbatore collected soil samples from 759 different spots of fourteen different divisions of the Western Ghats forests in Tamil Nadu and out of 392 soil samples tested from various sites of Western Ghats, 285 samples were found positive for presence of B. thuringiensis isolates. From 285 soil samples, 343 new isolates of B. thuringiensis were identified based on the presence of crystalline inclusions. Forty nine of the new isolates of Bt were screened for toxicity against neonate larvae of Helicoverpa armigera and four of the new isolates were found to be on par with the reference strain, HD1 for toxicity against Helicoverpa armigera.

OSIAA4 lacks a critical domain II which is known to be responsible for proteasome-mediated degradation, a prerequisite for auxin-induceed responses. Cryptochromes(CRY) are blue/UV-A light sensing photoreceptors involved in regulating various growth and developmental responses in plants. The group has functionally validated BnCRY1 gene from Brassica napus, an oilseed crop, by regulating its expression in B. juncea transgenics. In contrast, the transgenics over-expressing CRY1 were distinctly short and accumulated anthocyanins at a higher level. These data provide functional evidence for a role of blue light up-regulated cry1 in controlling photomorphogenesis in Brassica species. The protocol for induction of somatic embryogenesis has been used to create a cDNA library of wheat (Triticum aestivum CPAN1676) for the isolation of early auxin inducible genes. For screening the library differentially, reverse northern for nearly 1500 clones were carried out using cDNA population derived from mRNA (isolated form the total RNA) of auxin treated and control samples. Database searches revealed the recovery of many previously known plant somatic embryogenesis (SE)-related genes, while the work on some of these novel genes is in progress, we have identified at least 15 ESTs from wheat (Triticum aestivum), which exhibit high sequence identity with Aux/IAA homologs in other species. One of these Aux/IAA genes, TaIAA1, has been characterized and encodes a nuclear localized protein, harboring all the four conserved domains characteristic of the Aux/IAA proteins. The expression of TaIAA1 is light-sensitive, tissuespecific. Isolation and characterization of abiotic stress-induced promoters is the need of the hour. Nearly 2 kb long rice hsp100 promoter was cloned upstream to gus reporter gene in pCAMBIA vector and the construct was transformed into rice. Putative transgenics have been analyzed for integrity and expression of the transgene by PCR, northern expression and for histochemical and fluorometric GUS expression. This analysis has suggested that hsp100 promoter is regulated by high temperature stress. Apart from high temperature, specific metals (such as arsenite and cadmium) also induced rice hsp100 promoter. This promoter has one distinct heat shock element. Experiments are underway to determine the functional role(s) of this element as

Plant Molecular Biology Programmes : Research projects related to plant molecular biology are being supported at the following four universities/institutions: University of Delhi, South Campus New Delhi: The CPMB at South Campus has four inter-related sub-areas of activity in the general area of plant molecular biology. The progress of work on these aspects is given as follows; analysis of inflorescence specific promoters from rice has revealed temporal pattern of gene expression under control of four promoters investigated. In case of OSIPP2, gus expression was evident after meiosis when microspores were released from callose wall and could be seen even after germination of pollen grains. Deletion constructs of promoters from OSIPK and OSIPA showed variable activity in Arabidopsis. Functional validation of OSISAP2 and OSIRPK revealed their activity in stress response and transgenics over-expressing these genes show stress tolerance. Performance of OSIRPK was better than OSISAP2. Stress tolerant rice with codA genes were investigated for mechanistic aspects in terms of transcription activity. Out of 31 AUX/IAA gene in rice,

DBT Annual Report 2006-07

40

well as other specific domains of the rice hsp100 gene promoter.

D

WL

parents viz., CO43 and W1263 as susceptible and moderately resistant to yellow stem borer respectively in rice. Microsatellite markers associated with YSB resistance at vegetative and reproductive stages were identified by deploying single marker analysis (SMA). In the project on mapping gene(s) associated with brown plant hopper resistance in rice, IR 50 and Ratu Heenati were identified as susceptible and resistant parents respectively to develop a mapping population to map the QTLs associated with BPH resistance. To identify the QTLs and validate the markers already identified the F5 progenitors were advanced to F6 generation by following SDS method. On genetic transformation of rice for insect resistance, genotypes viz., ASD16 and IR72 were transformed with cry1Ab gene through Agrobacterium mediated method. Three independent lines in ASD16 and one line in IR72 were generated. These lines were forwarded to T1 generation. With a view to developing marker-free transgenic cyr1Ab lines, IR 64 was transformed with Agrobacterium strain. Five out of six T0 lines transformed with Agrobacterium strain C58C1RifR (pGV2260::pSSJ1; pCAMBIA0390-cry1Ab) were found to be positive for hph, gusA and with cry1Ab demonstrated the integration of cry1Ab. Progeny analysis in T1 lines of KP-IR64-65 (expressing cry1Ab) revealed a stable inheritance of transgenes.

BL

Comparison of hypocotyl length between 8-d-old AsCRY1 (antisensecryptochrome 1) and WT seedlings grown in dark (D) or irradiated with white (WL) or blue light (BL). Note that the antisense-CRY1 seedlings are taller than the wild-type when grown in white or blue light; it has ramifications in engineering dwarfism in plants by over-expressing the blue light photoreceptor CRY1.

Tamil Nadu Agriculture University, Coimbatore Projects on molecular markers for leaf folder, brown plant hopper (BPH) and yellow stem borer (YSB) resistance and pyramiding insect resistance genes in rice are being pursued. For leaf folder, a recombinant inbred population of 250 lines was developed from the cross of IR36 (susceptible to leaf folder) and TNAU831311 (resistant to leaf folder) by single seed decent (SSD) method to map the genes associated with leaf folder resistance in rice. Marker phenotype association resulted in the detection of QTLs for leaf folder resistance on the linkage groups viz., 7 (RM5499, RM432 and RM11), 9 (RM257, RM242 and RM3909) and 10 (RM271and RM244). To validate the contribution of the QTLs identified over environments, a trial with 250 RILs has been laid out and the phenotyping of the RILs for the leaf folder infection is in progress. Towards mapping QTLs for yellow stem borer resistance in rice, a recombinant inbred population (250 RILs) and a doubled haploid population (250 DH lines) were developed using the

Madurai Kamaraj University, Madurai On development of transgenic blackgram, two different promoters of MYMV-Vig DNA A have been cloned and studied. A 842-bp medium AV1 (coat protein) promoter was found to have a strong and constitutive expression in leaf, stem and root portions of the transgenic tobacco plants. The expression level was comparable to that of CaMV 35S promoter. A 341-bp AC2 (Transcriptional activator protein) promoter was found to have prominent expression in the root portions and thus can be used for rootspecific expression of transgenes. Two blackgram plants transformed with MYMV-Rep antisense construct gave PCR amplification with AC1, nptII and gus primers. However, these plants did not show any signal in the southern blot analysis. These two plants were advanced to the T1 generation and a total of 42 T1 plants were analysed by PCR with nptII primers. All these T1 plants did not carry the transgene. From

41

Research and Development

a total of 326 cotyledonary node explants transformed with pLK3, three kanamycin-rooted plants have been obtained. To perform RNAimediated silencing of MYMV-Vig, we constructed a dsRNA-AC1 construct. This construct has a 369-bp fragment from the 3' end of the AC1 gene. The hairpin RNA construct was made by placing the AC1 fragment in sense and antisense orientations with an intron in between.

cytosolic MIPS. Upstream genome walking experiment showed the Oryza sativa MIPS sequence to possess a putative transit peptide for chloroplast localization. Thus the gene for the chloroplastic MIPS is identified as the same for that of the cytosolic MIPS (OsINO1-1 ) having an extension for the transit peptide which targets the translated product to the chloroplast. While this gene is mapped in the chromosome 3 in Oryza, a second gene for inositol synthase (termed OsINOI-2 ) has been identified and mapped in chromosome 10 after determining the exact exon and intron boundary in the process. In addition, upstream region of OsINO1 and PcINO1gene was cloned from genomic DNA library of Oryza sativa var. IR 64 and Poteresia coarctata respectively. The PcINO1 gene remarkably possesses some cis-acting elements found in stress regulated genes.

In another project on development of cardamom for virus resistance, plants were regenerated after bombardment with the NIb gene of cardamom mosaic virus. Out of the 86 plants which were regenerated, only three showed the amplification of the inserted gene after transferring to the greenhouse. The PCR products from these three plants were cloned and one of them was sequenced. The sequence was found to be identical to NIb gene. The transgenic plants have to be evaluated for virus resistance under field conditions.

Multi-institutional projects Functional genomics programme on rice:

Bose Institute, Kolkata (i) Gene expression profiling during flower and seed development and functional validation of identified genes

In project on engineering inositol metabolic pathway for raising salt-tolerant rice plants, several lines of trangenic rice plants (PB-1) have been obtained for cytosolic(3 lines for PcINO1 and 1 line for OsINO1) and chloroplastic (6 lines for Tp-PcINO1and 2 lines for Tp-OsINO1) introgression of the salt-tolerant inositol synthase from Porteresia (PcINO1). T1 plants were raised and analyzed for the stable integration of the gene. Photosynthetic efficiency of the plants was measured. After standardization of an in vitro regeneration protocol of an indica rice variety IR64, Agrobacterium mediated transformation of the same was carried out for cytosolic expression of PcINO1 and the OsINO1 gene. Though the transformation and regeneration efficiency was quite low in IR64 in comparison to the PB-1 variety, transgenics of the same as analyzed through PCR analysis. For PcINO1 transformed plants, gene specific primers were used that amplified a region of approximately 430 bp (described in the earlier report) and for OsINO1 transformed plants, hptII gene specific primers were used that amplified a region of approximately 1 kb.

After the completion of transcriptome profiling for eight stages of panicle and five stages of seed development along with four vegetative stages by using the Affymetrix rice GeneChip microarrays, a comprehensive bioinformatics exercise to delineate individual expression profiles of the genes encoding transcription factors (TF) and signal transduction components (STC) has been initiated. Resulting from these analyses, 50 genes have been short listed for validation of gene function and/or promoter activity. Vector constructs for 6 genes (both for gene silencing and overexpression) and 15 promoters have been made and work on plant transformation is underway. Eight new constructs have been transferred to the groups working on rice transformation and the work on the functional validation of these genes initiated. Transgenic plants for 6 promoter and 4 gene constructs have been obtained and are being evaluated for phenotypic aberrancies reporter gene activities. Meanwhile, some important TF and STC gene families viz., Aux/IAA, GH3, CDPK, MADS, Fbox, C2H2 Zn-finger etc., involved in flower and seed development have being analyzed and the results

MALDI TOF MS analysis of the salt-regulated ~ 60 kD chloroplastic MIPS, identified the protein as rice

DBT Annual Report 2006-07

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have either been published or are at manuscript preparation stage. A potential downstream target of the transcriptional regulator OsMADS1, OsMGH3 has been identified and the work on its functional validation initiated. Progress has also been made in developing a virus based gene silencing system for rice by using Rice tungro bacilliform virus (RTBV). Potential candidates for viral silencing suppressors have been identified and are being investigated by agroinfiltration using the standard Nicotiana benthamiana GFP silencing suppressor system. For the marker free transformations system, the genes encoding dianthin and Mungbean yellow mosaic virus (MYMV)-AC2 were evaluated as negative selection markers for deployment in gene targeting experiments in rice. Dianthin, which served as an effective negative selection gene in tobacco was found not to be toxic in rice. The work on (MYMV)AC2 evaluation is underway.

(ii) Identification and functional analysis of genes related to yield and biotic stresses in rice This project aims at discovery and functional validation of genes contributing to grain yield and tolerance/resistance to bacterial blight, blast, gall midge and brown plant hopper in rice. Genes contributing to grain yield are being identified from genotypes derived from indica and tropical japonica crosses which has given rise to New Plant Types (NPT) and from accessions of wild rice O. rufipogan. Mapping populations have been developed and both genotyping and phenotyping has been completed in the NPT derived material. The yield related quantitative trait loci (QTLs) have been introgressed into elite genotypes like Jaya and Pusa Basmati. Of the ten yield related QTLs from the wild species, several have been introgressed into the popular CMS line IR 58025A. Development of flanking markers for these QTLs has helped in delineating the genomic region and carry out gene content analysis and identify candidate genes Novel genes conferring bacterial blight (BLB) resistance have been discovered in accessions of wild species like O. longistaminata, O. nivara, O. glaberrima and O. barthii; land race accession Ac32753 and few mutant lines of IR64. These novel genes confer wide spectrum of resistance against diverse isolates of the pathogen. Besides these genes being introgressed into elite genotypes of rice, these are also being characterized through development of closely linked markers. Nature of induced resistance in rice against the pathogen has been studied using selective mutant forms of the pathogen. These studies suggested that proteins secreted through the Xoo T2S (strain deficient in Type 2 secretion system) are major contributors towards inducing rice defense responses during infection and that these responses are suppressed by proteins secreted through the T3S. Transcription h analysis of the candidate Pi- k gene conferring resistance against the rice blast was done using RTPCT and QRT-PCR. RT-PCR with axon specific primer amplified a single band of size ~ 990 bp with RNA derived from rice genotypes Tetep (resistant) and HP2216 (susceptible) after pathogen infection. There was no amplification without infection. An experiment on QRT-PCR to quantify the amplified products obtained from both resistant and susceptible rice lines challenged with the pathogen,

Number of genes encoding transcription factors (TFs) and signal transduction components (STCs) in rice. The genes up-regulated specifically during stages of panicle and seed development are represented by green and blue.

A

B

A. Functional characterization of OsMADS1 as a regulator of floral organ development. Panel on the left shows panicles with knockdown of OSMADS1 have floret defects in floral organ formation and in determinacy. Panel center shows the experimental scheme and panel on the right shows a cluster analysis of the microarray data on expression levels of mRNA in the developing panicles of wild type or knockdown panicles. B. Functional categorization of transcripts affected on loss of OsMADS1 identifies a large proportion of genes regulated by OsMADS1 are transcription factors or signaling molecules.

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Research and Development

Magnaportha grisea, showed significant difference in the amount of product amplified at 35th cycle of the reaction. The quantity of amplified product was more in Tetep than in HP2216. Gall midge resistance genes Gm1 and Gm4 are being fine mapped to within 10cM of 2 MB region of the genome. Candidate genes within the region are being identified through resistance gene analogue based primer development and through RTPCR approach using primers designed for the known resistance pathway genes. Simple sequence repeat markers are being developed for the rice gall midge as biotype diagnostic tools. Rice resistance against brown plant hopper has been characterized in several of the genotypes and using these genotypes, mapping populations have been developed to track down the resistance genes. A large number of polymorphic markers have been identified to develop linkage map as a pre-requisite for this.

marker linked to Yr17 were analyzed on a complete population (109 lines) derived from the cross Tc X Tc.Lr37. The rust response of the mapping population (Tc X Tc.Lr37) using PBW343 isolate showed monogenic inheritance at Lr37 locus (23:1 = 0.0120, P= 0.950 - 0.900). Linkage analysis using this data revealed that VentriUp/LN2, Xgwm359 and Xgdm5 formed a linkage group with Lr37 (Figure 2). Rest of the primers failed to show an association with a gene and formed a separate linkage group. The primer pair VentriUp/LN2 was used for validation on F2 populations derived from the cross PBW343 XTc.Lr37. Linkage analysis of the data using Mapmaker revealed co-segregation of the marker (VentriUp/LN2) with Lr37. Whereas, F2 population derived from the cross Tc X Tc.Lr37 showed a linkage distance of 33.5 cM and eliminated the possibilities of using this marker for MAS in this population. Molecular analysis of these genotypes was carried out using 104 SSRs, 28 EST-SSRs mapped on wheat chromosome 2D and 200 SSRs spanning rest of the wheat genome. Combine analysis of all 61 polymorphic primers formed a linkage group around the locus Yr16. The marker cfd50 and Xgwm 47 flanked Yr16 at 35.7 and 38.8 cM respectively (LOD 4.0).

Development of varieties with durable resistance to leaf and stripe rusts using molecular marker technology in bread wheat During 2005-06, progeny of 1117 selected F3, F4, F5 and F6 plants derived from the crosses of PBW343/Yr16//PBW343/Lr24 or Lr28 or Lr37 were sown at three locations and evaluated for disease resistance. A set of these lines were also grown under net house conditions for screening with linked molecular markers. In addition, 32 F5/F6 bulks from the crosses PBW343/Yr16//PBW343/Lr24 or Lr28 or Lr37 were evaluated for agronomic and economic traits in a randomized block design with three replications during normal season 2005-06. Nine F5/F6 bulks outperformed the parent and the checks for one or more of the yield contributing traits or yield itself. All the 196 families selected during 2005-06 were sown at off-season nursery in Wellington during May-Sept. 2006. Further selections were made on the basis of marker data, rust reaction and plant type. On molecular tagging of Lr37 and Yr16 genes, molecular analysis of Thatcher and Tc.Lr37 differing in presence and absence of Lr37 was carried out using 105 SSR (gwm, gdm, barc, wmc, cfa and cfd) and 17 EST-SSR (cfe, cwem, ksum) primer pairs specific to 2A chromosome. Of these, nine polymorphic primers namely gwm512, gwm382, gwm296, gwm359, gdm5, barc212, wmc382, wmc407 and cfd36 alongwith VentriUp/LN2, a

DBT Annual Report 2006-07

Development of molecular markers for wheat quality improvement. Under wheat quality improvement programme, genotyping of RILs has been completed with ~200 SSRs for grain weight (GW) population and with 180 SSRs for the pre-harvest sprouting tolerance (PHST) population; framework maps will become available soon. An earlier SSR map of the grain protein content (GPC) population prepared by Meerut University had a few large gaps. Within these gaps, 41 new SSR markers were placed and a revised map with 214 SSR loci was prepared. Three hundred sixty (360) EST-derived SSR/STS markers specific for a bin of 2DL containing an important QTL for GPC were developed. Out of these, 96 markers were so far tested for polymorphism; seven markers that were polymorphic are being used for high density mapping. Similarly, for a major QTL for PHST on 3AL, 144 arm specific SSR/STS markers were developed. In both cases, homozygous recombinants (30 containing QTL for GPC and 98 containing the major

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transcription factor DREB2A (a DRE binding factor) is being functionally validated using the transgenic approach by over- and under-expression in indica rice . Regarding genetic transformation of rice, the PgNHX1 gene overexpressing transgenic rice tolerates high level of NaCl and their seeds could also germinate in the presence of salt. The glyoxalase II gene also confers improved tolerance towards salinity stress in transgenic rice.

QTL for PHST) were also identified for eventual use in fine mapping of the QTLs. Significant progress has already been achieved in transfer of major QTLs for high GPC and PHST into 10 elite Indian bread wheat genotypes using marker-assisted selection (MAS) during three backcrosses already completed; BC3F1 generation will be raised in the coming rabi-season and success will be evaluated again. The RILs for bread quality were developed at DWR Karnal taking HI 977 and CPAN 3004 as good bread quality parents and HD 2329 as poor bread quality parent (in the first phase of the project during 1995-2000). These along with parents were grown at different agro-climatic conditions of North Western Plains Zone, Central Zone and Peninsular Zone. The RILs of both the crosses were analysed for various quality parameters like protein content, moisture content, hectolitre weight, sedimentation value, thousand grain weight including bread loaf volume and bread quality score.

Localization of OsGR-RBP5 homologous protein in tobacco BY2 cells. A. a- Tobacco BY2 protoplasts (under uninduced control conditions) viewed in bright field b- DAPI-stained protoplasts. c- protoplasts probed with anti OsGR-RBP5 antibodies. B. a- Tobacco BY2 DaPI-stained protoplasts (following 42o C, 1 h heat stress) b- protoplasts probed with anti OsGRRBP5 antibodies (arrowheads mark the nuclear regions) c-merged image of panels a and b.

Salinity and dehydration stress tolerance in rice: cloning of responsive genes, their promoters and development of transgenics Towards cloning of stress responsive genes, several genes have been cloned. Amongst them one of the gene encoding fructose 1,6 bisphosphate was cloned to full length from Portresia (PCFR) and this enzyme was found to be active in the presence of NaCl. This gene is being functionally validated. Another novel gene encoding glycine rich RNA binding protein (GR-RBP5) has been cloned. It has been shown that GR-RBP5 is nuclear-localized but under stress conditions, bulk of this protein appears in cytoplasm. On cloning of stress responsive promoters and regulatory factors through 'genome walking PCR' technique, promoters for various genes that gets induced 'early' or 'late' with the persistence of stress, and also which are down regulated have been cloned. Using the 'promoter deletion approach', the stress responsiveness of these promoters is being validated by analyzing the reporter gene activity in the transgenic system. The specific transacting factors that bind to the specific ciselements of the promoter have been cloned. The OsBZ8 (an ABRE binding factor) has been shown to be highly expressed in salt tolerant cultivars than in salt sensitive cultivars of indica rice. Another

Relative salt tolerance of wild type (WT) and glyoxalase overexpressing transgenic T1 generation rice plants grown in the continued presence of 200 mM NaCl. Note that WT plants could not sustain growth under this condition while the transgenic was able to grow and set seeds.

Improvement of sorghum, finger millet and pearl millet through application of biotech tools Sorghum, pearl millet and finger millet are important dual purpose crops in the arid and drier semi arid tropics of north and north western and southern India and are usually grown as rainfed crop. Drought and some diseases are the two most important

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Research and Development

some diseases are the two most important constraints in their production throughout these regions. Therefore, the development of drought and disease resistant cultivars are important objectives in most breeding programmes of these crops. Under Sorghum programme, two genotypes M35-1 and B35 were used as parents for developing the recombinant inbred line mapping population. The genotype B35 is a well characterize source of staygreen while M 35-1 is the most popular post rainy season leaf senescent cultivar grown on a vast area in the states of Maharashtra and Karnataka. Quantitative trait loci and corresponding flanking molecular marker for staygreen have been identify and verified by NRCS, Hyderabad. UAS, Dharwad is evaluating recombinant inbred line population for charcoal rot resistance and related traits across location and seasons to develop genetic linkage map using SSR and EST based markers. MAU, Parbhani is developing population for marker development for shoot fly resistance in sorghum. TNAU, Coimbatore is characterizing sorghum germplasm for drought tolerance using molecular markers. QTL mapping and MAS to improve drought tolerance, downy mildew resistance stover quality and under standing the oxidative stress adaptation in pearl millet are in progress. SSR markers are being developed to saturate linkage map of finger millet at UAS, Bangalore. Transgenic approach is also being followed for salinity tolerance and low-HCN in sorghum to control downy mildew in pearl millet.

45% yield heterosis respectively over national check variety Varuna. Hence the Department supported to conduct the multi-location field trials of this hybrid. The National Research Centre of RapeseedMustard, Bharatpur conducted these trials along with four checks, viz.CMS based hybrid (DMH-1), National Checks (Varuna and Kranti) and a zonal check, at 10 locations during the year 2006. It was observed that higher yield of DMH-11 over the best check variety was recorded in 6 out of 9 locations. Maximum heterosis was recorded at Delhi location (28%). Since the trial was not shown in time in all the locations, the data recorded for the yield and its components did not reflect the true potential of DMH11. Keeping in view the recommendation of the MEC, the trials are being repeated during the current rabi season at 10 sites in six states Crop Biofortification (i) Biofortification of wheat for micronutrients through conventional and molecular breeding approaches The Network Project through conventional and molecular breeding approaches was sanctioned in the month of December 2005. There are 5 subprojects ongoing at 4 institutions namely IARI, New Delhi and its Exptl Station Indore, IIT, Roorkee, PAU, Ludhiana and IARI, Pune. Cereal crops vary considerably for micronutrient content in grains and other plant parts. In wheat only 20% of Fe and 70% of Zn, that is taken up by the plant are translocated to the grain, and these are primarily deposited in aleurone layer. Large proportion of micronutrients deposited in the aleurone layer is lost during milling and less than 50% of whatever is consumed, is absorbed by the body due to presence of phytic acid. Some of the wild species of wheat with higher levels of micronutrients might be very efficient in micronutrient uptake and their partitioning to grains as compared to the cultivated germplasm that shows little variability for the traits.

Multi-site evaluation of transgenic mustard (DMH-11) based on barnase barstar system Heterosis breeding could be deployed for enhancing the crop productivity in mustard. Production of hybrids requires availability of suitable male sterility restorer system to facilitate hybrid seed production. University of Delhi, South Campus has developed a male sterility restorer system making use of barnase and barstar genes. The male sterility and restorer capacity were subsequently transferred to suitable combines through recurrent backcrossing for development of DMH-11 hybrid. The hybrid seed production of DMH-11 is being undertaken by the inventors for last 2-3 years after taking necessary regulatory clearances. Two contained open field trials of the DMH-11 at Delhi University research station during 2003-04 and 2004-05 sought 55% and

DBT Annual Report 2006-07

Activity wise progress is as follows : For screening of wheat and related germplasm for micronutrients iron and zinc (including carotenoids for beta carotene in case of durum) and for low phytate level, diverse germplasm consisting of old and new

46

cultivars, landraces, synthetics, wild relatives, their derivatives and cytogenetic stocks are being analysed for the micronutrient content. For chemical analysis of iron and zinc whole grain samples from cultivated and wild accession were taken. It was decided to share a common set of genotypes at all the centers for the purpose of inter centre collaboration of the AAS at different centres. A high correlation between flag leaf iron and zinc content with those of seed content of potential donors was observed suggesting that the flag leaf content could be used as an indicator for high grain micronutrient content during early generation selection. For total carotenoid estimations, germplasm consisting of 225 wheat accessions, including varieties and locals from T. aestivum, T. durum, T. dicoccum, mutant lines as well as less cultivated species of tetraploid wheat were grown at Hol farm during 2005-06 season using RBD design in two replications and harvested manually.

The Network Project on Maize Biofortification, involving three institutions (VPKAS, Almora; IARI, New Delhi; HPKV, Palampur) was sanctioned in December 2005. The salient accomplishments are as follows: For analysis of genetic variability for kernel micronutrient content of the Indian maize germplasm a total of 82 maize genotypes including 29 popular CM (Coordinated Maize) lines and 53 maize landraces collected from diverse agroecologies were analyzed for kernel micronutrients (Fe, Zn and P) using Atomic Absorption Spectrophotometer. The kernel Fe and Zn content in the material analyzed ranged from 15-76 mg/kg and 16-56 mg/kg, respectively. This study led to the identification of potential maize genotypes with higher Fe and Zn content. Towards Multi-location evaluation of maize germplasm for kernel micronutrient analysis an experiment was undertaken during Kharif-2006 at three locations (i) IARI, New Delhi; (ii) VPKAS, Almora; and (iii) HPKV Research Station, Bajaura in order to identify promising germplasm for different agro-ecologies. The materials comprised of a common set of 100 maize inbred lines/landraces collected from different sources and the analysis for micronutrient content is in progress at all the these centres.

A set of 68 lines, including 15 cultivated wheats, 10 accessions of T. dicoccum and 43 T. dicoccoides, were analyzed for phytic acid content in the grains in three replicates. In cultivated wheats, the phytic acid content ranged from 81.8mg/100g in CS to 99.5 mg/100g in C306. In T. dicoccoides accessions the phytic acid content ranged from 72.9mg in 601012 to 101.3 in 4637. Maximum phytic acid content (121.91mg/100g) was observed in T. dicoccum acc. DDK 1018. For Interspecific hybridization to transfer high micronutrient content traits, crosses were made between wild species (putative) donors and hexaploid recipient parents. A number of interspecific hybrids were treated with colchicine for chromosome doubling and as many as nine synthetic amphiploids were generated which will be used for transferring useful variability as translocation, substitution and addition lines. Towards mechanism of micronutrient uptake and partitioning and accumulation investigation a set of related species and cultivated varieties has been grown in the field with recommended dose of fertilizer and spacing. Plants will be uprooted at different growing stages and micronutrient studies will be carried out in the plant parts and in the soils to find differences among the genotypes in the partitioning.

For molecular characterization of selected maize lines based on kernel micronutrient content a set of 20 promising maize inbred lines were selected for molecular analysis. Analysis for molecular polymorphisms in the selected set of maize inbred lines has been partially completed using 30 SSR markers covering various bin locations in the maize genome. Also transfer of Low Phytate gene in the genetic background of Quality Protein Maize (QPM) lines attempted. Out of six low phytate lines, two viz. A619 1-1 and A619 2-2 were crossed with VQL 1 and VQL 5, QPM lines developed at VPKAS, Almora. The seeds, thus obtained will be utilized during the ensuing season. The same set of low phytate donors were used to study polymorphism between the donors and recipients. The amplified products did not give any polymorphism. The amplified products of both the donors and the recipients were digested with more than 10 restriction enzymes. Only two enzymes viz. MspI and DPN II were found to have restriction sites within the amplified products.

(ii) Development of micronutrient-enriched maize through molecular breeding

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Research and Development

For utilization of Multiple Aleuron Layer (MAL) lines for improving micronutrients, a set of seven MAL (Multiple Aleurone Layer) lines obtained from CIMMYT Gene Bank is being explored for kernel micronutrient enrichment and development of suitable mapping population for the identification of SSR markers linked to the MAL genes. (iii) Rice bio-fortification with enhanced iron and zinc in high yielding non basmati cultivars through marker assisted breeding and transgenic approaches

in rice seeds At IGAU, Raipur, the rice lines with high iron and zinc levels are grown in the field for seed multiplications and further confirmations of mineral value. The plants were grown in the field during wet season 2006 with different levels of fertilizer applications. The mineral analysis of these materials is in progress. Six rice lines showing higher level of grain iron and zinc concentrations were crossed with the five modern rice cultivars F1 seeds (5-35 seeds) were harvested from each cross and are being analysed for mineral content evaluation.

Six independent integration events of ferritin transgenic lines were developed by Agrobacteriummediated transformation in the indica rice Oryza sativa at the MSSRF, Chennai. Matured nontransgenic and T2 homozygous transgenic seeds were used for histochemical and immunoblot analysis. At the TNAU, the plasmid "1301FerGlu1" has been mobilized into Agrobacterium strain LBA4404 through triparental mating. The transconjugants were confirmed for the presence of binary plasmid by restriction analysis and backtransformation. The Agrobacterium LBA4404 (1301FerGlu1) was used to mobilize the gene of interest into ASD16 and ADT43 using mature seed derived calli as target tissue. The cultures are in various stages of selection and regeneration.

At UAS, Bangalore, a field experiment has been conducted at Shettigere, Doddajala, Bangalore with 279 short duration, 325 medium duration and 320 long duration rice genotypes representing the breeders' active gene pool and germplasm to know iron, zinc and phytic acid contents in different parts of rice plant. All genotypes have been replicated thrice, under aerobic cultivation system at Shettigere, Bangalore. All necessary management practices have been implemented. Phenotypic characters of different rice genotypes have been documented. Samples for collection of DNA, iron and zinc have been collected. For the development of DNA markers based on the known metal transporter genes, attempt has been made to design the primers for expression analysis of these genes in elite and modern rice lines. The c-DNA sequences of these 43 metal genes were downloaded from the rice genome and primers were designed using Primer 3 programme.Atomic Absorption Spectrophotometers, grain milling and polishing facilities have been created at MSSRF and IGAU for the precise estimation of minerals.

Southern blot analysis was carried out with genomic DNA from nontransgenic and T2 homozygous PCRpositive plants. Genomic DNA digested with EcoRI and probed with ferritin cDNA showed the expected hybridization signal of 800 bp in all the transformed plants. This confirmed integration of the ferritin cDNA in the rice genome. Perl's Prussian blue staining of ferritin transgenic rice grain sections showed the distribution of iron accumulation throughout the alureone and sub aleurone layers and also in the central region of starchy endosperm. However, in non- transgenic grains, blue colour formation of iron accumulation was restricted only to aleurone layer and the intensity of the colour development was also very low. This histochemical analysis of iron in rice specifically showed temporal and spatial deposition of storage iron. This finding showed correlation with the Western blot analysis of expression of exogenous ferritin gene. In all the transgenic southern positive plants (seeds), a 28-kDa-ferritin protein was detected, confirming that ferritin protein accumulates

DBT Annual Report 2006-07

Generation of virus-resistant rice for India: Diversifying transgenic resistance to popular varieties This network project has been implemented at three institutions viz. University of Delhi South Campus, New Delhi, Bidhan Chandra Krishi Viswavidyalaya, Kalyani and Tamil Nadu Agricultural University, Coimbatore. The salient achievements reported during last one year are; Transgenic rice seeds (RTBV-O-Ds2) have been multiplied in the transgenic glasshouse at UDSC. Seed setting is

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complete and will be distributed to the collaborating partners for back-crossing. Majority of equipment at all three centres have been acquired. At BCKV, the varieties to be used in the back-cross have been identified. Procedures for the award of contract for the construction of poly-house for back-crossing and resistance testing have been initiated. Tungro incidences in several districts of West Bengal have been studied. At TNAU, the construction of the controlled field testing facility has been initiated. The varieties for back-crossing have been selected. To help in the resistance evaluation, E. coli cells, expressing the RTBV and RTSV coat proteins as fusion proteins were constructed at UDSC. These have been transferred to TNAU centre, where immunization is in progress.

simplified the earlier protocol developed and used in our laboratory as the transgenic embryos could be directly germinated on MSO medium and transferred to pots without the need of grafting. Our preliminary observations also show that a higher percentage of transgenics have normal morphology when selected on imidazolinone as compared to selections on kanamycin. Thus, different promoters as well as innovative modifications in the gene per se need to be tested. Testing large number of such modifications by developing transgenics in cotton is not easy in the current scenario. In order to overcome this an expression system for cotton has been developed which allows to test expression levels of any transgene cassette quickly.

Development and analysis of cotton transgenics for resistance to insect pests (Bollworm complex)

Biofertilizers With growing environmental concerns, the sole dependence on chemical inputs based agriculture is being replaced by integrated approach involving conjunctive use of both organic and inorganic sources. In this context, biofertilizers have been well accepted as an economical, cost effective, renewable and safe organic source of plant nutrients to sustain crop productivity. Moreover, with recent focus on organic/bio-dynamic farming, the demand of biofertilizers is likely to grow at a much faster rate than before. At this juncture, we must realize that microbial inoculants are an 'ecological inputs' whose efforts are 'subtle and not dramatic' like chemical inputs. Hence, inoculation with good quality inoculants is a must and should be treated as an insurance against failure of nodulation. The shelf life both in the storage and transit needs to be improved with due consideration to various 'abiotic' stresses. The quality oriented production and marketing network will certainly make biofertilizers a viable enterprise for ultimate customer satisfaction. Keeping these in view, programmes on development of liquid biofertilizers and biofertilizers based Integrated Nutrient management packages for plantation crops and medicinal plants have been generated. In addition, biofertilizers strains developed through transgenosis will be evaluated in contained conditions.

More than 300 independent transgenics lines in cotton (Coker 310FR) carrying the cry1Ac gene for attaining resistance to Helicoverpa armigera have been developed. In most of the transgenics the cry1Ac gene is under the control of the double enhancer CaMV 35S promoter, while in some it is driven by either the FMV double enhancer or the MMV double enhancer promoters developed in the laboratory. One of the problems observed in developing these transgenics was that a large number of the initial transformants showed abnormal phenotype and would not set seeds under green house conditions. Analysis of junction sequences by Southern hybridizations revealed that only a small population of the initial transgenics developed had a single copy of transgene. Currently the group is in the process of assessing insect resistance vis-à-vis expression of the cry1ac protein in the BC1/T1 progeny of 22 of the T0 lines which have either one or two copies of the transgene. These lines showed no abnormality in their phenotype and set seeds properly when grown in field under containment net houses. Improvement has also been made in the transformation protocol of cotton which allows the use of imidazolinone as a selection agent instead of kanamycin by using a double mutant acetolactate synthase gene as marker. These modifications have

The report will be a document to highlight the importance and the genesis of the programme, scientific hypotheses behind the main networks (e.g.

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Research and Development

transformants of Anabaena sp. strain PCC7120 with higher heterocyst frequency and nitrogenase activity, construction of Anabaena sp. strain PCC7120 constitutively expressing groES-El with increased thermotolerance.

BGA transformations, MPS transformation of PGPRs, Rhizobial transformations for rhizosphere competence etc.) and the significant achievements against completed projects supported with essential information (abstracts of data, figures, diagrams etc.).

The hyphal fusion of mycorhhiza programme, interand intra-species hyphal fusion of the AMF by the standardized protocol of co-culturing and injury repair mechanism, characterization of the fusants (progeny spores) employing morpho-taxonomic markers, possible nuclei based recombinant variation in physiological markers (glomalin production, heavy metal accumulation) among the fusants and some improvements in recombinant strains with regard to such properties have been achieved. The attempts to develop molecular markers employing RFLP and marker based distinction between parents and the presumptive fusant strains will be undertaken during 2007.

Some of the projects under development of transgenic biofertilisers programme have been able to develop a few transformed strains of the organisms. Further assessment of the scientific and technological merits of the transformed strains and prepare a plan of action for their testing for the transformed properties by carrying out uniform centralized trials in an institution are being carried out. For the purpose, the identified centres who have developed the strains will form a team incorporating one member from the investigating UAS Dharwad. The centres who have been identified to have prospective transformed strains BARC for BGA, University of Hyderabad for Azotobacter, UAS Dharwad for Azospirillum and University of Baroda for Pseudomonas. The action plan as to be prepared by BARC in this regard will be finalized in the beginning of 2007-2008 and one external expert will be deputed to oversee and evaluate the conduct of the trials and the results thereof.

The cloning of gdh gene and transformation of Azotobacter strain for gdh mediated MPS phenotype was successful. Results of in vitro studies showed gain of MPS phenotype in the strains with simultaneous reduction in nitrogen fixation ability. UAS Dharwad centre could clone pqq synthase and gadh genes from S. marcescens and K. pneumoniae, transformed and expressed gadh gene in Azosprillum and Pseudomonas. Demonstration of MPS phenotype in the transformed strains and results of plant growth promotion with a few of the transformed strains was demonstrated.

Under a previous project JNU developed some genetically engineered strains of Azotobacter. Against an ad-hoc project under the network, Division of Microbiology, IARI tested the performance of these strains by container grown studies for 2 years. Results of the study have shown improved growth promotional efficiency of these strains in wheat. The strain is fit for further and larger testing against a few more crops (e.g. rice) which will be undertaken during kharif 2007 along with other strains.

MS University achieved in cloning and expression of ppc gene under lac promoter in E.coli ppc mutant. The system was used to successfully transform P. fluorescens strains for over expression of the ppc gene. The transformed strains showed high MPS phenotype in buffered alkaline medium against TCP. Qualitative and quantitative changes in organic acid secretion profile of the transformed strains were demonstrated. Effect of these strains on plant growth promotion via P-solubilization could be studied during 2007.

Significant scientific contributions has been made by BARC in the development of strains of Cyanobacteria. The notable achievements are: standardization of electro-transformation protocol for selected cyanobacterial strains, construction a novel integrative expression vector (pFPN) for Anabaena, cloning hetR and groESL in shuttle vector, transformation, integration of pFPN and expression from psbA1 promoter from Anabaena, cloning of hetR, groESL and linA2 genes downstream to promoter sequence in pFPN, construction of hetR

DBT Annual Report 2006-07

The intra-and inter-species molecular phylogenetic differences among the widespread fluorescent Pseudomonas species strains in plant rhizospheres and development of molecular marker

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based identification of the strains were achieved by MKU, Madurai. Successful cloning of the target genes, construction of vectors for transformation and transformation of selected fluorescent Pseudomonas strains for ACC deaminase gene expression were demonstrated. The centre will continue with the line of investigation to bring the hypotheses of improved rhizocompetence of the transformed strains through over-expression of the ACC-demainase and catalase genes.

technologies of bio-control agents to control major pests and weeds of economically important crops, vegetables and other important plantation crops. A multicentric programme on control of storage pests was initiated during this period. Various ongoing and completed projects were reviewed for their progress and achievements in the three Task Force meetings. 15 new projects have been funded in different aspects of biological control. Salient achievements of the few projects are as follows:

The other unit of MS University procured cloned cross utilizing receptor genes (fhuA and fegA) from external sources and subcloned the same in an expression vector. It used the cloned constructs to transform strains and reported improved siderophore crossutilization phenotype of the transformed strains. Some of the transconjugants of a strain for fegA showed improved plant growth performance as compared to the parent strain.

Microbial Pesticides and Natural Enemies Indigenous entomopathogenic nematodes (EPN) are being used for the management insect pests of rice at DRR, Hyderabad. Three EPN isolates viz. Steinernema thermophilum, S. asiaticum and Rhabditis (Oscheius) sp. have shown entomopathogenic ability to infect and multiply on Corcyra cephalonica and Galleria mellonella. The recovery of Oscheius sp. and S. thermophilum from G. mellonella was much higher than C. cephalonica. Both EPNs survived up to 50 days in storage. Bioefficacy studies against rice yellow stem borer revealed that both nematodes caused 92-98% mortality of the egg masses. S. thermophilum caused faster mortality rate against larvae of both the pathogens.

Attempts have been made to clone the pqq gene from Pseudomonas striata following the genomic library and sub-cloning approach at IARI with some success achieved for development of cloned constructs. The main objective of the programme of developemt N. muscorum transformants with mps phenotype through gdh expression under expression of pqq will be undertaken during 2007. However, in another trial at IARI it was successfully brought out the potential of the genetically modified (nif+) strains with regard to growth and yield promotion of wheat by elaborate container grown studies. The results interpreted from N-response curves showed that inoculation with the modified strains resulted in higher N-accretion by wheat plants as compared to that with the wild type parent strains, both in presence and absence of low levels of externally applied chemical N-fertilizer. Evidence has been provided for differential survival rates of the transformed strains in soil, in some cases at par with the wild types.

Two novel heat tolerant EPN viz. Steinernema masoodi and S. seemae have been identified at IIPR, Kanpur. Three field trials to establish the efficacy of S. 9 masoodi indicated that at a dose of 1.5 10 infective juveniles/ha at IIPR recorded 83% larval mortality of H. armigera with an increase of 53.9% in yield over control. In another trial, survival of S. masoodi on pigeon pea after foliar application was conducted and evening spray was found to be quite effective with respect to EPN survival. A similar study was carried o u t a t R A U , U d a i p u r. St e i n e r n e m a a n d Heterorhabditis collected from different agro climatic regions were mass multiplied both in vivo and in vitro and rearing of Corcyra cephelonica and Galleria mellonella was also done on artificial diet. Bioefficacy of promising populations of Steinernema and Heterorhabditis against H. armigera under pot condition on tomato is in progress.

IGEB developed strains by site directed mutagenesis of host specific Rhizobium strains which showed atypical nodulation properties with non-host legumes. A few mutant strains showed strong salinity and acidity tolerance. Biopesticides and Crop Management

Under a collaborative project, arbuscular mycorrhizal fungi (AMF)and EPN cruisers interaction is being evaluated on the reproduction and

R&D efforts were continued to develop production

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larvae at high temperature of 400C and found to be effective against other insect pests viz. Anomis flava, Spodoptera litura, Pectinophora gossypiella, Sylepta derrogata and Earias sp. at ten to fifteen infective juveniles per host larva. A new bacterial formulation was also developed from bacterial symbionts against sucking pests of cotton and was found effective under field conditions. This formulation was also effective for control of Bracon sp.

development of root knot nematode, Meloidogyne incognita on tomato at TERI, New Delhi and PDBC, Bangalore. Seven EPN isolates from the collected from hills were identified as Heterorhabditis sp. Pathogenicity of nematode isolates against Galleria mellonella was studied and LC50 was calculated for different EPN isolates. Progeny production of EPN isolates in G. mellonella was studied and two best isolates were selected. Application of EPN prior to M. incognita infestation was found to reduce the root knot nematode population and also reduce the damage due to root knot infection on tomato plants.

At PDBC, Bangalore, genetically improved strain of Trichogramma chilonis Ishii resistant to multiple insecticide and high temperature has been developed. The strain showed cross tolerance to many other insecticides also. Genetic analysis of the strain for tolerance to endosulfan, monocrotophos and fenvalerate suggested that semi dominant and recessive genes are responsible for tolerance. Biochemical studies showed that difference in esterase isoenzyme composition is due to the role of esterases in insecticide resistant mechanism. Out of forty RAPD primers tested, fourteen primers were found useful in studying polymorphism. RT-PCR for hsp (heat shock proteins) gave three bands of the size 500bp, 400bp and 300bp.

At ICRI, Idukki, EPN are being identified for the management of cardamom root grub. Thirty one local EPN isolates were collected from the soil samples and so far two local isolates viz. Heterorhabditis indica (ICRI-18) and S.bicornutum (ICRI-35) have been identified. Efficacy of three virulent isolates viz. ICRI-18 (Heterorhabditis sp.), ICRI-90 (Steinernema sp.) and ICRI-81 (Heterorhabditis sp.) were confirmed against root grub by laboratory bio-assay. Significant reduction of root grub has been observed in the field conditions also and100% grub reduction was observed in some of the field trials.

At GBPUA&T, Pantnagar, also temperature and insecticides tolerant strains of T.chilonis and Chrysoperla are being developed . Native superior strains of these species were mass multiplied in the laboratory and subjected to selection pressure of insecticides and temperature to develop their tolerance. T.chilonis took seventy generations to develop tolerance against ¼ of field dose of both endosulfan and chlorpyriphos. Sixty two generations against imidacloprid and sixty six generations against cartap hydrochloride. T. chilonis and Chrysoperla carnea have developed tolerance up to 0 42 C temperature. C.carnea was reared at incremental doses of all four insecticides and found to have tolerance to the ¼ of the field dose of insecticides.

Genetic improvement of EPN for tolerance to environment and enhanced efficacy against Helicoverpa armigera, cotton bollworm, is being done at CICR, Nagpur. One isolate each of H. indica and S.riobrave was developed to tolerate high temperatures. Both isolates could infect H. armigera

At TNAU, Coimbatore a biopesticide formulation is being developed for the management of major pests and diseases of rice ecosystem. Four plant growth promoting rhizobacteria (PGPR) strains of Pseudomonas fluroescens viz. TNAU Pf-1, TDK-1, Py-15 and P. putida producing antibiotics like Diacetyl pholro glucinol (DAPG) and phenazine chitanase and ACC deaminase were found to be

Bacterial symbiont developed into formulation against sucking pests of cotton

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promising in bioefficacy and growth promotion activities. Field trials study confirmed that application of Pf-1 + TDK-1 + Py-15 bioformulation reduced the incidence of leaffolder, brown plant folder, stem borer etc. in rice plants.

had the additional effect of enhancing the plant growth. In the combination trials of mung bean, 80% substitution of the chemical control agent was achieved with the biocontrol agent. At University of Hyderabad, efforts are being made for the development of ecofriendly truncated peptides of harpin from P. syringae as broad spectrum biopesticides. So far, seven (four N terminally truncated and three C terminally) truncated peptides have been produced by cloning and expression of the truncated versions of the HarpinZ in to pET28a vector to produce truncated proteins. Four N terminal truncated peptides were produced in E. coli BL21 DE3, while the C-terminal truncated versions are currently being expressed.

At IISR, Calicut, fifty six isolates of endophytic bacteria were isolated from black pepper and characterized. Nursery evaluation of short-listed endophytes has confirmed the superiority of TC10, an unidentified bacteria, in suppressing Radopholus similis, irrespective of the black pepper variety. Two Pseudomonas isolates (BP 17 and BP 35) were also found to suppress R. similis significantly and found to be quite effective against Phytophthora capsici, a major pathogen of black pepper. Talc and chitin based formulations of these three promising endophytic bacteria were compared for their suitability and shelflife. Chitin based formulations supported 7 comparatively higher populations of bacteria (x 10 cfu/g) than talc-based, even after 90 days of storage. Study confirmed that chitin based formulations would be a viable option for management of two important soil-borne problems of black pepper viz. Phytophthora foot rot and burrowing nematodes (R. similis).

At KFRI, Peechi, control of teak defoliator outbreaks by seeding Baculovirus (HpNPV) in epicenter populations is being attempted. Out break patterns revealed that the sequence was similar to the predicted pattern of development from epicenter to epidemic phase. Modeling of virus transmission showed that third and fourth instars are not suitable for inoculation and manifestation of vertical transmission, as even very low dose of HpNPV caused total mortality in the parent generation itself.

A biological control strategy involving the conservation and augmentation of the two major predators viz. Dipha aphidivora and Microuns igorotus has been developed for the management of the Sugarcane Woolly Aphid, Ceratovacuna lanigera at PDBC, Bangalore. Augmentation of the predators were achieved either by redistribution of wooly aphids from high to low population density or by mass production in shade net over a 6 months old sugarcane crop already infected by the aphids. Experiments in farmer's field have shown that release of either 1000 larvae of Dipha or 2500 larvae of Micromus per hectare suppressed woolly aphid populations in 45 to 60 days.

At MPKV, Rahuri, in vitro production of nuclear polyhedrosis virus of Helicoverpa armigera and Spodoptera litura is being studied. Various cell lines viz. Sf-9, Sf-21, Su-893, Su-992 and Ha-197 were maintained. The wild type strains of SINPV were collected and maintained Sf-9 cell line was found to be best for multiplication of SINPV virus followed by Sf-21 and Su-893 cell lines. The Sf-9 cell line was also found susceptible for ACMNPV. About 3000 ml pure in vitro SINPV was produced in Sf-9 cell line. At Manipur University, Manipur, evaluation and augmentation of viral pesticide viz. Pieris brassicae granulosis virus (PbGv) for the control of cabbage butterfly is underway. Bioassay of virus was initiated rd against 3 instar larvae and preliminary studies indicated the utility of this virus for the control of cabbage butterfly.

At NCL, Pune, various formulations of the protease inhibitor as biocontrol agent against fungal pathogens were prepared. Talcum powder formulation was found most suitable to control wilt of pigeon pea. This formulation was used to coat pigeon pea seeds (ICPL 2376), which showed 45% disease control while drenching gave 60% control. A maximum disease control of 65% was obtained after additional drenching. Study confirmed that drenching boosters

The role of elicitor to combat Panama disease of banana is being studied at Sardar Patel University, Vallabh Vidyanagar. Study confirmed that elicitors not only protect plants from the Fusarium but also help in

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plant growth and development. Due to elicitors there is more lignin accumulation which helps in strenghtning the plant. Field trial data show that average production of elictor treated field is 28 Kgs/plant and that of untreated field is 22 Kgs/plant.

rearing of Pomegranate fruit borer, Deudorix isocrates successfully achieved at MPKV, Rahuri. Molecular Studies on Biocontrol Agents : Studies are underway to control sugarcane borer through transgenic endophytic Acetobacter diazotrophicus is being made at Pondicherry University. Cry1Ac gene from Bacillus thuringiensis was cloned using the shuttle vector pKT230, which produce cry toxin protein of 130 kDa in recombinant A. diazotrophicus. Preliminary bioassay studies employing artificial medium to study the effect of wild A. diazotrophicus and its transgenic and E. coli containing Cry1Ac gene in the control of early shoot borer revealed that the transgenic strains were able to drastically reduce the live weight of neonate larvae on 15th day post feeding but no larval mortality was observed.

Botanical Pesticides : At North Maharasthra University, Jalagaon, studies on the use of plant cyclotides are being conducted for the management of storage pests. Few indigenous plants belonging to family Rubiaceae and Violeaceae were screened and bioactive molecules were isolated, partially purified and characterized. The isolated protein from Rubia cordifolia has shown good antimicrobial activity in preliminary studies. Pheromones & Semiochemicals : In a collaborative project implemented at IICT, Hyderabad and MPKV, Rahuri indigenously synthesized pheromone components are being used under Integrated Pest Management. The pheromone blends of all the three species of bollworms were synthesized using viable synthetic routes with required purity and found to be economical. Pheromone application technology was demonstrated in cotton crop by mass trapping and mating disruption. All the three species of bollworms were effectively controlled by pheromone, which confirms the efficacy of synthesized pheromone blends. Pheromone dispensers were also developed and compared with the imported ones for their affordability and suitability and found to be economical.

In a multicentric project being implemented at ICGEB, and IARI, New Delhi, a 60 kDA native protein of Xenorhabdus nemetophilus was purified from cell lysate and oral toxicity was checked on H. armigera and S. litura neonates. The protein has shown potent oral toxicity on H. armigera with a LD50 value. However, it was not potent against S. litura. The recombinant 60Kda protein was also purified and checked for oral toxicity on H. armigera however, no oral toxicity on S. litura was recorded. A 4.5 kb DNA fragment containing the GroEL homolog was obtained by screening a genomic DNA library of X. nematophilus. Analysis of the 4.5 kb DNA fragment predicted an open reading frame of 1.7 kb, encoding a 60 kDa protein. Primary sequence comparison of the protein showed high homology with GroEL protein of E. coli and Photorhabdus luminescence. Gene specific primers were designed and a 1.7 kb gene was amplified by PCR. The amplified DNA was cloned for expression. The recombinant GroEL was expressed as a soluble protein, which is being used in the insecticidal assay

At IICT, Hyderabad, pheromones of Pomegranate fruit borer and fruit sucking moths of sweet orange were isolated, identified and synthesized. Several synthetic pheromone standards ranging from C12 to C16 carbon atoms both E & Z configuration were screened against male antenna for their bio-activity. Existence of female produced sex pheromone communication was confirmed in different bio-assay. Significant olfactory responses in male antenna against female gland extract are recorded using Electroantennogram (EAG). Studies on identification of bioactive fractions of pheromone glands extract are in progress. Preliminary GC-MS studies with female glands extract have given valuable leads for the characterization of bioactive fractions. Mass

DBT Annual Report 2006-07

Molecular studies on insecticidal toxins of indigenous P. luminescens and Xenorhabdus spp. are being conducted at PDBC, Bangalore. Study confirmed the presence of insecticidal toxin genes viz., TcdA1, TcdB1, TcdB2, TccC1 and TccC3 in 6 isolates of Photorhabdus luminescens and 4 isolates of Xenorhabdus spp. Insecticidal toxin gene TcdB1 of 4.5 kbs from P. luminescens was cloned in to TA

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cloning vector and transformed in to E. coli DH5 competent cells. The expression of the toxin moiety was examined through bioassay on Galleria mellonella and Plutella xylostella. The LD50 value of these cells was high compared to the original cells of the strain indicating that the native cells of Photorhabdus were more toxic compared to the transformed cells of E. coli.

of novel plant growth promoting bacterial strains, which was isolated from rhizospheric soil has been demonstrated against a range of phytopathogenic fungi under in vitro conditions. Some of the strains produce antibiotic Phenazine-1-Carboxylic Acid (PCA). The PCA gene ~ 1.4 kb (phz C and D) has been cloned and sequenced. The potential of strains as super-bioinoculant for plant growth promotion with parallel fungal growth suppression has been established both in pot culture and field studies in disease-conducive soil. Different combinations of bioinoculants were tested and consortium No. 19, consisting of P. aeruginosa - NJ15 + P. aeruginosa NJ101 + Rhizobium sp. -PS1 + P. putida - PS2 strains in equal proportions has proved to be the most efficient to increase the yield. Treatment with this combination, significantly decreased the disease incidence also.

At University of Pune, molecular characterization of antibiotic producing novel PGPR Acinetobacter species is being done and so far 37 strains have been isolated from rhizosphere of wheat and biotyped. These strains were tested for their ability to inhibit the growth of phytopathogenic bacteria and fungi. A. junii A7 and A.haemolyticus A19 demonstrated strong antibacterial and antifungal activity while A.haemolyticus A22 demonstrated only anti-fungal activity. Molecular identification of these three strains was conducted using rDNA sequencing. A.haemolyticus A19 produced antibiotic, which was inducible by co-cultivation method and 1H-NMR analysis revealed this antibiotic as pyrrolnitrin. Characterization of other strains in order to optimize antibiotic production is in progress.

At Pondicherry University, Pondicherry antagonistic Fluorescent pseudomonad isolates viz. W5, Pf6, Pf11 and Pf13 were isolated. Strain, W5 exhibited a broad-spectrum antifungal activity towards several phytopathogenic fungi that attack rice, groundnut, chilli, cotton, sugarcane, mango and banana. Parthenium Weed Management

A t L u c k n o w U n i v e r s i t y, s t u d i e s o n t h e characterization of antiviral resistance inducing protein gene of Clerodendrum inerme is being carried out. CIP-29 protein was purified from the leaves of C. inerme and polyclonal antibodies in rabbits were produced. The anti CIP-29 serum was tested and it was found to be specific. Cloning of the CIP-29 gene was initiated with the designing of primers for RTPCR. One set of forward and reverse primers yielded an approximate 950 bp length fragment, which is being sequenced.

A major multicentric programme on the management of Parthenium weed, which is an obnoxious weed, was launched. Salient achievements of few centers are as follows: Control of Parthenium through eco-friendly approach is being undertaken at NBRI, Lucknow. Effect of three tree species Jatropha curcas, Terminalia arjuna and Riccinus communis on the growth and control of Parthenium was studied and R. communis was found to suppress the growth of Parthenium maximally. The active fractions of the different parts of tree species were tested on seed germination. Bark fraction of T. arjuna at only 2.0% concentration inhibit 100% germination of seed. Leaf and bark extracted fraction of T. arjuna was developed and tested on P. hysterophorus and found to be effective at different stages.

At TNAU, Coimbatore, a chimeric gene, cry 2Ax1 of Bacillus thuringiensis was constructed by engineering C-terminal region of domain-III in Cry2Aa to improve toxicity of its protein against Helicoverpa armigera. The constructed chimeric cry2Ax1 gene was expressed in transformant of acrystalliferous Bt strain. Analysis of Cry2Aa, Cry2Ab, Cry2Ac and chimeric Cry2Ax1 proteins for toxicity against larvae of H. armigera showed about 22-fold higher activity in the chimeric Cry2Ax1 protein (in terms of LC50) than the Cry2Ab.

At RDU, Jabalpur, various Mycoherbicide are being developed for the control of Parthenium. Colletotrium gleosporioides, C. dematium, Alternaria alternate, Fusarium oxysporum and F. solani, have shown

At AMU, Aligarh, the concurrent antagonistic activity

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Research and Development

significant weed control at seedling stage particularly in seed function and germination. These mycoherbicidal agents have been successfully mass produced through solid substrate fermentation employing maize cob wastes and when applied to the seedling of the target caused more than 90% mortality. Cell free culture filtrates of these agents have also shown significant seed mortality. Purification and characterization of these byproducts are under progress.

and weeds. Salient achievements compiled from more than 124 completed projects have been included which will benefit scientists, entrepreneurs and farmers. The website will be launched shortly. Toxicological data generation In order to facilitate the commercialization of biopesticides, the department has taken a proactive step of the generation of toxicological data of potential biopesticides. In the first phase, 10 biopesticides have been taken up for generation of toxicological data both for primary cultures as well as for their formulations. Data have been generated for almost all the biopesticides by the two identified centers viz; ITRC, Lucknow and RRL, Jammu.

Large-scale demonstration on management of Parthenium through integrated approach is being done at NRCWS, Jabalpur. Few plant species were found competitive against Parthenium. Cassia tora was found to be the best plant as it replaces Parthenium infected site during rainy season. Introductory releases of bioagants, Mexican beetle, Zygogramma bicolorata at newer sites, confirm its establishment, which reflected its success in integrated management programme. Good quality

Patents Patents have been filed for the mass production technologies of various biocontrol agents viz. Nomuraea rileyi, Trichoderma virens, two effective bioformulations of B. bassiana, Verticillium lecanii and Myrotheceium verucarria. Two patents for the products viz. Biowiltex (Trichoderma harzianum), Bionem x (Pochonia chlamydoisporia) and Biocomp x (Pseudomonas fluorescens) have been filed in India and USA through DBT patent cell. Technologies transferred and products marketed Several mass production technologies of biocontrol agents/biopesticides have been developed and standardized. Mass production technology of Trichoderma viride (fermentation based) has been transferred to M/s Prathista Industries Ltd. in Nalgonda, Andhra Pradesh and M/s Haryana Biotech, Gurgaon. They have filed application for registration of their products and procured funds from the Technology Development Board (TDB). Registration has been granted to the product Biofungicide to M/s Prathista Industries Ltd. and the product has been launched in the market as “Protect”. Cost effective mass multiplication of Trichoderma and Pseudomonas and their mixed formulation on FYM and chicken manure have been developed at GBPUAT, Pantnagar and this technology has been transferred to the farmers of Uttrankhand and Uttar Pradesh as a recommendation from University as well as State Department of Agriculture.

A view of field visit of trainees doing practical during Parthenium Training Programme.

Parthenium compost was also prepared by pit method with low inputs. Parthenium compost along with 50% NPK gave higher yield than FYM and 50% NPK suggest its use in compost. Development of comprehensive website on biopesticide. A comprehensive website on “Bio-pesticides” has been developed which highlights the achievements made in major programmes supported by the Department on biological control of pests, disease

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species representing 133 trees, 20 shrubs, 98 herbs and vines, 65 grasses and 8 other life forms covering 67 families were recorded from 81 grids, covering 111 transects in the South Central zone. Overall, 23, 960 tree individuals representing 38 families with a range of 364-760 tree density were recorded. Eighty eight percent of all trees recorded in published literature and 8 new species were recorded from the forest region of kadapa hills. In the Northern zone, 143 plant species were recorded from 45 grids and 76 transects. The Northern Eastern zone recorded 180 plant species from 37 grids and 44 transects, while North-central zone recorded 235 plant species from 44 grids and 57 transects. Southern-Eastern Zone recorded 320 plant species from 51 grids and 70 transects.

Bioresource Development and Utilization National Bioresource Development Board Programmes under the Board continued on characterization of the biodiversity at both primary and secondary level, prospecting of bioresources for potential products, improvement of bioresources and capacity building/awareness generation. The Fourth meeting of the Board under the Chairmanship of Hon'ble Minister S&T and ES was held on 25th July 2006. During the year two meetings of the Steering Committee and four meetings of the Scientific Advisory Committee were held. In addition a number of idea generation meetings and brainstorming sessions were held to initiate programmes in new areas such as Zingibers, Honey Bee, DNA Barcoding, Microbial Prospecting etc. Some of the salient achievements of the ongoing programmes are as follows:

Jeeva Sampada a digitized inventory of Bioresources has been developed. The database of approx. 7 GB, packaged in nine CDs contains information on 39,000 species, with over 82,00,000 records. Over 400 scientists from over 150 centres across the country have worked together to complete this enormous task.

Bioresource Characterization and Digitized Inventorization: The programme on Biodiversity Characterization at Landscape Level using Satellite Remote Sensing and Geographic Information System executed by Indian Institute of Remote Sensing, Dehradun, NRSA in collaboration with other DOS centres, universities and NGOs, has generated geospatial biodiversity data on important biodiversity rich regions of the country. Under Phase II, Eastern Ghats, Central India and Mangrove regions have been mapped. 80% of the country's forest cover has been mapped. To complete the whole country a Phase III has been launched for Biodiversity characterization at landscape level using satellite remote sensing in parts of Deccan Peninsula, North West India and Himalayan Cold Desert.

For proper integration of all the databases - spatial and non-spatial, an Indian Bioresource Information Network (IBIN) has been launched, as a service and network system for all the digital databases on bioresources. The network comprises two interacting nodes, one for the predominantly non-spatial data sets and the other to serve predominantly spatial data sets, which would be on a common web-based portal. The purpose of such an effort is to facilitate the use of the existing digital databases by the diverse end users; promote interlinking of the diverse databases through a continuous interaction and promote a continuous growth of the databases and their utility in conservation. (The website address is www.ibin.co.in) This IBIN portal along with Jeeva Sampda was launched by Shri Kapil Sibal, the Hon'ble Minister of Science and Technology, and th Earth Sciences, GOI on 25 July 2006.

In the project on mapping and quantitative assessment of bioresources of Western Ghats about a third of the Western Ghats has already been sampled. Specimens of about 1000 species have been collected and new populations of at least four endangered species have been located, distribution maps have been prepared. About 4000 images have been compiled for 1000 plants from the field and fliers have been developed for 400 species.

Molecular characterization and conservation As many as 9860 rice landraces belonging to ten different groups based on their maturity and grain characteristics of CG Core Collections maintained at Indira Gandhi Agricultural University, Raipur were analyzed for the four nutritionally important traits, the grain protein, lysine, iron and zinc concentration. Top

In a similar project on Quantitative assessment and mapping of plant resources of Eastern Ghats, all the six teams have initiated the field work. 324 plant 57

Research and Development

A partial Na+/H+ clone from Porteresia coarctata (PcNHX) was isolated and complete sequence information for this gene was obtained at MSSRF, Chennai. The PcNHX- was transferred to tobacco via Agrobacterium-mediated transformation for gene validation. Of the seven lines obtained for the PcNHX construct, four were single copy insertions as revealed by Southern blot analysis. Northern analysis of the four lines indicated expression of the PcNHX gene in the tobacco plants.

three rice accessions with enhanced level of protein, lysine and micronutrients have been identified for all the ten groups. Wide genetic variations for all the four traits have been recorded. The rice accession CGR: 20390 was found to possess highest protein content (14.05%) and CGR: 18675 had the highest lysine concentrations (7.74 g/16gN). Protein and lysine concentration was found to be negatively correlated suggesting that with the increase of quantity, the quality of protein gets compensated. The protein and carbohydrate content was estimated in cereals like rice, maize, sorghum and pulses used by major tribes of Andhra Pradesh. A total of 52 rice, 32 maize and 60 Sorghum samples from tribal area were analyzed. Protein content varied between 5-9.71%, 8-13% and 7-11% respectively.

In another ongoing study at MSSRF, Chennai more than 4000 ESTs have been cloned and sequenced from Avicennia marina. Sequence analysis of these ESTs revealed homology to different categories of genes with different functionality. More than 90 full length genes have been isolated, out of which about 50% are with implications for abiotic stress tolerance. 36 full length genes have been analyzed for their expression profiles under abiotic stress conditions. Most of the genes have been tested in tobacco transgenic systems while currently ten genes are being used for generating transgenic rice systems.

In a collaborative research work between Centre for Ecological Sciences, Bangalore and CDFD, Hyderabad, 80 microsatellite markers from Ropalidia marginata genome have been isolated, thirty two microsatellite loci were polymorphic. The number of alleles per locus ranged from 2 to 11, these loci are being used to study the role of intra-colony genetic relatedness in social evolution and the pattern of queen succession in R. marginata. From the tropical silkworm Antheraea mylitta, 22 microsatellite markers have been isolated, seven polymorphic microsatellite markers were employed to study the genetic analysis of well defined mature grown ecotypes. Low genetic variation was observed among populations (11%), however, within population variability was 89%. A workshop on “Microsatellite Markers in Molecular Ecology” was also conducted at CDFD Hyderabad for young teachers and senior research students.

Salt stress analysis of the transgenics has shown that transgenics could tolerate upto 150 mm of NaCl concentration for about 7 days whereas the untrasformed control plants started showing signs of wilting from the 4th day itself. The salt stress analysis was carried out in two generations. The transgenic plants could withstand the drought stress and showed lesser amount of visible damage at the end of the drought stress in comparison to the untransformed control plants. Field trial of transgenic rice is being carried out at Kalpakkam in six plots. Transgenic plants were found to be of shorter height but the yield was higher as compared with control. The grain yield across the transgenic and control plants were found to be comparable.

Prospecting of genes and molecules for product development: Projects have been supported for prospecting of novel genes, molecules, enzymes etc. from plants, microbes, fungi, lichens for production of potential products of industrial importance. Novel genes/ promoters, transcription factors are also being identified so as to develop transgenics for biotic / abiotic stress and understand different metabolic engineering pathway(s) operative in a system.

A study has also been supported to fish out drought tolerant genes from Prosopis juliflora. A cDNA library of two-month-old leaf tissue after 25 days of water withdrawal has been constructed. Random EST sequencing of 1750 clones produced 1467 quality reads. The sequences have been deposited in the NCBI EST database. Two of the abundant genes coding for a non-specific lipid transfer protein and late embryogenesis abundant protein have been sequenced completely.

Prospecting of novel genes:

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Studies on gene prospecting by comparative proteomics in water stress legumes was supported at NCPGR, New Delhi. The extracellular matrix (ECM) proteome map of chickpea and the identification of 163 ECM-specific proteins has been completed. ESIMS/MS analysis has led to the identification of 134 drought related proteins (DRPs). Protein reference map of nucleus in chickpea from purified nuclear fraction has been developed. The largest percentage of the identified proteins has been found to be involved in signaling and gene regulation (36%), while 17% are found to be involved in DNA replication and transcription.

been obtained for two varieties using Agrobacterium mediated transformation. Bioactive Molecules A Novel superoxide dismutase (SOD) enzyme was isolated from Potentila and is being used for preparation of antioxidant cream and other products at IHBT, Palampur. A fully characterized heme peroxidase from Acorus calamus with antifungal activity has been purified at IFGTB, Coimbatore and is being tested for antifungal activity against Tricosporium vesiculosum. Further work on validation of peroxidase on fungal hyphae is being carried out in other plant species to understand its function during defence. The toxic nature of the enzyme which inhibited hyphal growth has been demonstrated against phytopathogens such as Macrophomina phaseolina, Fusarium moniliforme and T. vesiculosum. This study indicates that peroxidases may have a role to play in host defence by inhibiting the hyphal extension of invading pathogens.

Under a study supported at Bharath Vidya Peeth, Pune for enhanced Omega-3-Nutrition in Flax, several primers based on the Omega-3 desaturase were designed based on the sequences available. Many primers amplified different loci and several of these were cloned and sequenced. Two variants each of delta-9 desaturase and Omega-3 desaturase have been obtained. Omega 3-desaturase is being cloned in binary expression vector for plant transformation. The transformants are being analysed.

Secondary metabolites from lichens sps. like Rorcella montagnei, Dirinaria consimilis, Ramalina pollinaria have been screened at MSSRF, Chennai for presence of bioactive compounds. Two novel compounds have been identified and toxicity testing is being done.

Studies at TBGRI and RGCB, Trivandrum are continuing on isolation and characterization of gene(s) involved in the regulatory step(s) leading to the biosynthesis of hypericin using transcript profiling technology. Hypericin rich callus, shoot and cell suspension cultures of Hypericum sp were developed by optimization of nutrient media with respect to PGR's and carbon source. Possible enhancement in hypericin production through elicitor treatment is in progress. Differential expression of gene in NAA induced shoot and callus cultures is being screened by Suppression Subtractive Hybridization (SSH) analysis.

Studies have been supported at RGCB and TBGRI, Thiruvananthapuram on metabolic engineering of andrographolide accumulation. Agrobacterium mediated transformation protocol for Andrographis paniculata has been developed and transgenic calli overexpressing A.paniculata 1-deoxy-D-xylulose-5phosphate synthase (DXS) gene apdxs1 have been produced which on HPLC analysis show enhanced accumulation of andrographolides as compared to the control. Further work is on to develop a number of primary transformants from the generating calli transformed with apdxs1 and to do phytochemical analysis of the transgenic Andrographis plants for andrographoides.

At IHBT, Palampur a cold tolerant gene has been isolated and characterized from a plant of Lahul and Spiti region. Transgenic Arabidopsis plants using the Cu-Zn SOD gene have been developed and T1 generation has been obtained. Study of gene behavior and its expression is under progress. Seeds have been obtained from vacuum infiltered Arabidopsis plants for beta helix-loop-helix (bHLH) protein encoding transgenes. Selection process for getting positive plant transformants is in progress. Putative potato transformants for Cu-Zn SOD have

In a study supported at TERI, New Delhi, biopesticide formulation has been developed from plants of Myrtaceae family. The formulation prepared has been found to be effective not only against a bollworm but was also against jassid population -

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Research and Development

which has been taken up for field trials on crops affected by leaf streak disease at Rice Research Station, Tirur after being confirmed to be non-toxic. The occurrence of symptom in the new leaves was not observed and this study also prevented further spread of the disease to the adjacent field where the same rice variety was grown.

both adults and nymphs. First year multilocation trials of the biopesticide formulation developed has been under taken on cotton. The trials were undertaken at CICR, Nagpur and Coimbatore during Kharif 2005. The formulations were effective even 5 days after spraying in terms of %age reduction in square damage over control and significantly superior to control recording a higher yield. Multilocation trials on chickpea were also undertaken at Rajasthan Agricultural University, Jaipur and ICAR Centre at Sehore, Bhopal. All the treatments were found significantly better than control against H.armigera.

500 endophytic fungal isolates have been isolated from the inner bark segments of four medicinal plant species viz., Terminalia arjuna, Crataeva magna, Azadirachta indica and Holarrhena antidysenterica representing various habitats. Two thousand fermentation products of fungal endophytes prepared employing six media combinations, were screened for DPPH free radical scavenging assay and antimicrobial assay. One hundred isolates exhibiting more than 50% DPPH radical scavenging activity were selected and fermented on a large scale, which include species of Pestalotiopsis, Trichoderma, Myrothecium, Mycelia sterilia and unidentified sporulating fungi. These extracts were subjected to DPPH, lipid peroxidation, DNA protection and antihypertensive assay. In antibacterial assay, ten isolates were found positive against Gram-positive and Gram-negative bacteria by microplate assay as well as disc diffusion method. Endophytic fungal extracts from Aspergillus flavus columnaris, Pestalotiopsis spp. and some unidentified sporulating fungi showed positive antifungal activity against important plant pathogenic fungi.

Prospecting for Microbes Plant growth promoting microbial inoculants isolated at IHBT, Palampur are under field trial. Presently, field evaluation is in progress in cold desert of Himachal Pradesh. Eleven efficient PGPR isolates have been characterized on the basis of 16S rRNA gene sequencing. The sequence generated has been deposited with NCBI Gene Bank. At MSSRF, Chennai, novel salt tolerant nitrogen fixing and phosphate solubilising strains of bacteria already identified were further taken up for field trial. The 4 strains MSP-27, MSP-146, MSP-393, MSP573 were taken up for field trials for testing as biocontrol agents. Two strains - MSP393 and MP27 found to be efficient would be taken up for further testing. MSP-573 showed 33.4% of disease suppression under non-saline conditions. Comparative performance of phosphate solubilizers and nitrogen fixers was tested at lab/green house conditions. Field-testing of the selected strains of phosphate solubilizers and nitrogen fixers was found efficient under normal and saline conditions. MOU for formulations and third party testing was signed with Elbitec. Development of carrier based formulations for all the 16 strains have been achieved. Liquid formulations for Azospirillum MSA- 148 and Phosphobacteria PS-5 were also achieved but the liquid formulation for Swaminathania salitolerans could not be achieved. Extensive field trials have been conducted in 208 acres of paddy on a participatory approach with a consortium of biocontrol strains, phosphate solubilizers and nitrogen fixers. An efficient P. fluorescens strain has been identified

DBT Annual Report 2006-07

In another project on investigation of the microflora of insect of Western Ghats for potentially useful Bioactive molecules, about 30 insects specimens have been collected and identified morphologically from different parts of Western Ghats. Insects were identified by sequencing the mitochondrial 16S rRNA primers specific for insect taxa yielding 600 base pair amplicons. Bacterial isolates recovered from the insect gut were screened for protease, cellulase and lipase activities, and also for the presence of nanoparticles like gold and silver in presence of different salts. Region Specific Programmes launched specifically targeting bioresources of fragile ecosystems.

60

Eastern Ghats: Diversity of metallophiles in Eastern Ghats is being studied for exploration and exploitation in metal prospecting and bioremediation. Open cast and underground chromite mines in Sukinda valley of Jajpur district and Baula-Nuasahi belt of Bhadrak district have been surveyed and a total of 22 samples from mining environment were collected and analyzed for physico-chemical characteristics. The samples were more or less acidic to neutral in nature and contain significant amount of chromium (1.6 3%) and nickel (0.3 0.8%) in addition to cobalt, cadmium,

and zinc. Microbial density, diversity and activity of these metalliferous environmental samples were in general relatively low. Aspergillus niger SUK101, the most potent strain has been found to solubilize 52.8% nickel, 28.7% chromium and 20% iron from the mine overburden. Optimization of conditions for better nickel leaching is in progress. Western Ghats: In the project entitled “Evaluation of Viscaceae Members of Western Ghats Region for Prospecting of Ribosome Inactivating Proteins”, field surveys of Western ghats regions (Puna, Kolhapur, Belgaum-Amboli) were carried out. Different populations of Viscum spp. distributed in the region were mapped and diversity documented, distribution was found to be associated with the type of forest and host species. Using immuno-probe developed, different Viscum spp. were evaluated for the presence of ribosome inactivating proteins (RIPs). Domestication of potential ornamental plants from the wild bioresources of the Western Ghats is being attempted for Delphinium malabaricum, Impatiens pulcherrrima, Senecio bombayensis, S. belgaumensis, Pogostemon deccanesis, collected from various localities of the Western Ghats. Reproductive ecology and population enrichment of endemic and critically endangered plant species of Western Ghats is also being worked out. Considerable work has been carried out on reproductive ecology of two of the target species Dysoxylum malabaricum and Garcinia indica in the Uttara Kanda district of Karnataka. Himalayan Region: IHBT, Palampur, produced 65480 plants of Phyllostachys pubescens (Moso bamboo) and 82805 plants of other species of edible bamboo provided them to R&D institutions and State Forest Departments. An elite cultivar of Curcuma aromatica “Himhaldi” was released. A multipurpose herbal pain ointment, herbal toothpicks having antibacterial properties and an anti-inflammatory skin cream with anti-oxidant, venotonic and vascular protective properties were developed. Mapping of Solang nala in Kullu Dist. of Himachal Pradesh was completed and total 12 vegetation types were identified in the area. Complete genome of chilli vein mottle virus was amplified and cloned. Radioactive

Delphinium malabaricum a. cultivation in open area, b. flowering, c. cultivation in polyhouse, d. cultivation in pots, e. entire plant, f, g, h, i, j, range of flower colour

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Research and Development

Resource Specific Programmes:

c-DNA probes were developed using amplified region of coat protein gene for detection of group specific potyvirus. An important achievement was for Cryo-preservation of Podophyllum hexandrum and Aconitum heterophyllum seeds, which could withstand dehydration up to 5% level, and showed >90% survival even after 120 days of storage. Selected Acorus calamus accessions containing low beta-asarone were micro-propagated and transferred to field for evaluation. 12 training programmes benefiting 584 participants have been conducted during the year on different aspects of bioresource conservation and sustainable utilization.

Different priority resources have been identified for improvement through biotechnological interventions. Germplasm characterization, marker development, identification of genes, constructs, transcription factors, genetic transformation are some of the important activities being pursued. Sugarcane: Under the Sugarcane genome project, general cDNA libraries of different tissues (leaf whorl, mature leaf, stem and root) from sugarcane variety CoS767 were prepared in plasmid and phage vectors. In all, 34367 clones were sequenced from 5' end to generate EST sequence data. Good quality sequences (25384) having PHRED score >20 and sequence longer than 299 bp were submitted to the GenBank. Besides the general libraries, various subtractive libraries were also made. About 1440 sequences were generated from a subtracted library made from red-rot challenged and normal sugarcane stem, out of which 1069 good quality sequences were submitted to the GenBank. Some stressrelated and disease resistance genes, identified from the general as well as red-rot subtracted library, were tested for expression. A few disease-related genes did not show expression altogether in the tolerant variety, while some other genes were found to be upregulated or down-regulated. Various stress-related genes were induced to significant levels under dehydration stress conditions. The EST sequences showing homology to some useful genes have been selected for further analyses and constructs are being prepared to transform sugarcane with potentially useful genes.

A Mountain Bioresource Complex (MBC) has been established in Kumaon and Garhwal region of Uttarakhand as a community approach to apply the knowledge regarding new tools of biotechnology to untapped and exploit sustainably the under utilized resources. A novel method of composting has been developed which is unique for its low cost and reliability. 250 such composting units have been erected in village around MBC. A total of eight training programms were organised in villages to popularize the post harvest technology involving eighty women. Cultivation of spices such as turmeric, ginger, foneculum, etc. has been promoted and on a technology developed for extracting oil from turmeric leaves, 150 women were trained. In addition, school children are being trained on utility and conservation of important bioresources. Desert Region: At Birla Institute of Scientific Research, Jaipur the genetic and functional diversity of rhizobacteria of clusterbean (Cyamopsis tetragonoloba) and other arid plants is being studied. The isolates were highly diverse in effectiveness e.g. nodulation, nitrogen fixation, plant growth of cluster bean, and also in salt and temperature tolerance. Seventy-nine rhizobacterial isolates showed biocontrol activity against Macrophomina phaseolina and Fusarium oxysporum. Two isolates showed maximum salt tolerance (5% NaCI) among rhizobacterial isolates showing biocontrol activity. Partial 16S rDNA sequencing indicated that out of 79 isolates having antifungal activity many belonged to B. subtilis, B. cereus and B. thuringiensis and two were Pseudomonas spp.

DBT Annual Report 2006-07

In another programme, two genomic libraries including one enriched for six different microsatellite motifs and one cDNA library from popular sugarcane varieties were constructed for the identification of microsatellites. For use in gene mapping experiments, primer sequence information on 500 markers were provided to the two network partners. To evaluate the efficiency of the designed STMS markers, a subset of 50 markers were synthesized and amplified in five sugarcane species clones, eight varieties and three related genera for polymorphism survey. In addition, these STMS markers were amplified in a set of genotypes of five cereal species

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namely barley, wheat, rice, maize and Sorghum for studying their conservation and cross-transferability.

Maharashtra, UP, Karnataka and Tamilnadu and screened with a phytoplasma specific oligonucleotide primers corresponding to two regions - 16SrRNA and 16S-23S rRNA intergenic region. Antibodies against SecA and SecE proteins were developed and are being used to screen the field samples at Vasantdada Sugar Institute in order to develop the diagnostic tools based on ELISA to detect the early stage of infection. A PCR based diagnostic kit for red rot and smut diseased of sugarcane has been developed at IISR, Lucknow and is undergoing validation. It was concluded that the designed primer pair is specific to red rot pathogen and can amplify total genomic DNA of wide variety of isolated or pathotypes causing incipient infection in sugarcane stalks. Further to validation of specificity of synthesized primer pair to smut pathogen (Ustilago scitaminea) in three smut affected varieties of sugarcane, infected stalks from sugarcane varieties from Coimbatore, Pune and Lucknow were sampled.

For analyzing SNPS, twenty-two unigenes predicted to be involved in sugar pathway were amplified in a set of 13 sugarcane genotypes and sequenced. The presence of SNPs was revealed in four genes namely, sucrose synthase, sucrose phosphate synthase, soluble acid invertase and sucrose transporter. Polymorphism survey was extended to 25 genotypes belonging to different species, varieties, related genera of sugarcane and five cereals, and validated through CAPS analysis. 50 primers were designed for “R” gene analogues based on sequences from NCBI database and Sugarcane EST database (SUCEST). The chitinase gene (PR protein) a major gene involved in defence mechanism in crop plants, resistance protein (R30), metallothionein like protein with possible hypersensitive response regulation, receptor protein kinase (RPK) involved in cell signaling, reversibly glycosylated protein (RGP) involved in cell wall biosynthesis and transcription factor (SSHR5) were differentially expressed in cane tissues challenged with C. falcatum. Coronatine insensitive gene (RcoiII) involved in salicylic acid pathway regulation and basal antifungal peptide (BAF) with antifungal activity were the transcripts that were differentially expressed in cell lines.

Over expression of Cry endotoxins from B. thuringiensis in E.coli to test the efficacy against sugarcane borers is being done at PAU, Ludhiana. Cry 1Ac, Cry 1Aa3, Cry1IA5, Cry 1F were procured from IARI, New Delhi. DNA of Cry 1Ac and Cry 1IA5 has been isolated and restriction analysis of constructs has been done using different restriction enzymes (Bam HI and Hind III). Sugarcane borers collected from different places in the state are being reared on natural diet in the laboratory for insect bioassays using different Cry proteins.

Molecular Characterization of Interspecific Hybrids of Sugarcane Using Genomic In Situ Hybridization (GISH) is being attempted at G.B. Pant University of Agriculture & Technology, Pantnagar. The chromosome count (utilizing the root tips) of 20 interspecific hybrids indicates that the somatic chromosome numbers varies from 90 to 112. Some of the interspecific hybrids were utilized in in situ hybridization experiments using two repetitive DNA clones as FISH probes. The physical localization of these two clones is helping to develop FISH landmarks on 10 to 14 pairs of chromosomes of these interspecific hybrids.

Tea: U n d e r a n e t w o r k p r o g r a m m e o n “Characterization and improvement of tea through biotechnological tools” involving IHBT, Palampur, GBPHIED, Almora, Tocklai Experimental Station, Jorhat, Bose Institute, Kolkata, University of Delhi, Delhi, TERI, New Delhi, UPASI, Valparai, molecular and morphological characterization has been completed for 150 accessions. The study has been extended to around 2000 accessions, including garden selections. Genomic DNA has been isolated from a total of 500 tea clones (350 of TRA, 100- IHBT, and 50- UPASI). Pre-amplification was successfully done in 462 tea clones by TERI, and was provided to all the collaborative laboratories for running AFLP gels. So far, AFLP profiles have been generated

Efforts are on to develop a diagnostic tool for Sugarcane Grassy Shoot (SCGS) disease at ICGEB, New Delhi and Vasantdata Sugarcane Institute, Pune. A total of 339 samples with symptoms of phytoplasma were collected from various fields of

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Research and Development

across all the 462 clones with allotted six primer pairs by the three institutes namely TERI, DU, and IHBT.

quality of raw material used) and have reduced the period of hydrolysis by about 40%. The protocols and cost benefit analysis of the new method(s) are being evaluated to translate the findings in an industrial set up at pilot level.

Studies are continuing for development of transgenics resistant to blister blight and dormancy. Expression and validation of trait specific gene(s) isolated from tea in model plants was done and construct was developed for tumor suppressor gene, and transferred into Agrobacterium strain GV3101 using the helper strain PRK-2013. Agrobacterium has been checked for harbouring the desired insert. The work is in progress to transfer the gene into tea and for development of construct with two other genes.

Biological, chemical and molecular characterization of Lac insect host plant relationship is being studied. The two strains of lac insect i.e. rangeeni and kusmi were inoculated on five different host plant species viz. Acacia auriculiformis (Akashmani), Albizia lucida (Galwang), Flemingia semialata (Semialata), Schleichera oleosa (Kusum), Ziziphus mauritiana (Ber) and Butea monosperma (Palas) for the study of biological parameters. Bio-control and bio-rational approaches for management of lac insect predators are being evaluated. Wide variations in different parameters were observed when lac insects were reared on different host plants as well as between the two strains of the lac insects on the same host plant. In another study, molecular fingerprinting is being used for genetic characterization of races and species of lac insects, DNA isolation protocol has been standardized for single lac insects. Oligos have been synthesized and further work is in progress for development of microsatellite markers.

Coffee: As a follow up of the earlier work done, a Network programme on Coffee has been launched f o r d e v e l o p m e n t o f m a r k e r s , E S Ts a n d transformation for disease resistance. India is also a partner to the International Coffee Genome Network. Lac: Innovative laboratory processes are being developed at IIT, New Delhi for value added products from Lac. Existing processes for two export products from lac have been modified while lab processes have been developed for two new value added products (methyl alueritate and 16-hydroxy-9hexadecenoic acid) with a potential to generate better revenue in the flavors, fragrance and cosmetic industry. The modifications have enabled increase in yield of alueritic acid from 14-17.3% to 19-23% while purity increased from 92 to 96% (depending upon the

DBT Annual Report 2006-07

Network Programme on Bamboo: Under the Network Programme for the Establishment of Demonstration of Bamboo plantations in different location across the country, nearly 380 ha has been covered as per table given below.

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Dendrocalamus strictus D. asper Bambusa bambos Phyllostachys stocksii

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Research and Development

R & D programme have also been supported for developing and standardizing protocols for tissue culture micropropagation of different bamboo species as follows:-

Species

Institute

Bambusa balcooa

FRI, Dehradun

B.nutans

IHBT, Palampur IFGTB,Coimbatore TFRI, Jabalpur

B.pallida munro

IWST, Bangalore

B. tulda

TFRI, Jabalpur

D. hamiltonii

IHBT, Palampur

Dendrocalamus giganteus

IHBT, Palampur IFGTB, Coimbatore KFRI, Thrissur

Melocanna bambusoids

FRI, Dehradun

Phyllostachys bamusides sieb. E. jucc.

A

IWST, Bangalore

B

A. Multiple shoots of Dendrocalamus hamiltonii in vitro; 8. Rooted plants of D. asper in vitro; C

D

B. Seedings of Bambusa bambos growing in the polyhouse, C. Denderocalamus asper plants (TCPs) growing in the green house; D & E. Representative photographs of Setting up of demonstration plots by UBFDB, Dehradun and UFA, Haldwani in various forest divisions in Uttranchal;

E

F&G. Field performance of Tissue culture raised plants D. Hamiltonii (1 year old) Dehradun Forest Division.

F

DBT Annual Report 2006-07

G

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Bangalore. The Park was inaugurated on 25th November 2006, by Shri Kapil Sibal, Hon'ble Minister for Science, Technology and Earth Sciences, Government of India. The project was executed by the Zoo Authority of Karnataka in collaboration with the University of Agricultural Sciences (UAS), Bangalore and Ashoka Trust for Research in Ecology and the Environment, Bangalore. The butterfly park is spread over an area of 18 acres which includes a ten acre host-plant garden and 7.5 acres of the park which is open to visitors. The park comprises a butterfly garden which leads to a butterfly conservatory spread oven an area of 10,500 sq feet, under a polycarbonate roof. The visitors to the conservatory can see a 15 to 30 species of butterflies depending on the season. The butterfly conservatory leads to a museum spread over an area of 3000 sq feet that houses dioramas, live-exhibits, specimens and inter-active computer kiosks. The museum also has a multi-media centre attached to it which will be used for educational programmes.

In addition Transgenic Bamboo development at IHBT, Palampur is being attempted. D. hamiltonii plants produced through bombardment of somatic embryos with SOD gene were selected on the antibiotic hygromycin, these transformants are being multiplied for molecular characterization. Development of molecular markers for characterization of Bamboo germplasm is being done at TERI, New Delhi and IHBT, Palampur. Standardization of several molecular marker techniques for bamboo germplasm (AFLP, 4-slect AFLP, TE-AFLP and SAMPL) have been done. Analysis of genetic diversity of 48 accessions comprising 7 species using AFLP markers has been done. High level of marker polymorphism between different bamboo species (is 98.6%) was observed. At IHBT, Palampur genomic DNA of 58 bamboo accessions have been isolated and checked for its quality and quantification. RAPD fingerprinting of 40 bamboo accessions has been completed with 40 decamer primers. AFLP fingerprinting of 58 accessions of bamboo were completed with eight primer combinations and AFLP fingerprinting with remaining ECO-RI/Mse I primer combinations is in progress.

The UAS, Bangalore has achieved the following milestones during the project period. n

Developed captive breeding methods for 42 species of butterflies of peninsular India

New Initiative on Zingiberaceae: n

Recognizing the importance of ginger family (Zingiberaceae) an important bioresource with considerable economic potential, the department organized a brainstorming on “Zingiberaceae Biotech intervention for improved planting material and product development”. Programmes have been supported during this year in the identified thrust areas: development of improved planting material, role of gene environment interaction, biochemical and molecular characterization in relation to commercially useful traits, prospecting for selected secondary metabolites and domestication of some underutilized species of ornamental value.

n

Carried out DNA finger-printing 25 species of butterflies Developed methods for DNA bar-coding of butterflies

Butterfly Park: The country's first Butterfly Park has been established at Bannerghatta Biological Park,

A view of butterfly park

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Research and Development

At MSSRF, Wayanad about 100 students in the age group 6-16 years including school dropouts have been made aware of the importance of the bioresources. The activity has generated a high demand from various segments including schools, NGO's, farmers groups, nature clubs, and parentteacher associations for starting similar programme in their locations. Children attending this programme have started awareness programme in their locality about water-soil conservation and importance of organic farming. Under the project entitled “Involving India's high school / junior college community in inventorying and monitoring of biodiversity” supported at Indian Institute of Science, Bangalore, a number of schools and panchayats have been identified in Karnataka, Maharashtra, Madhya Pradesh and Tamil Nadu to implement the project. Participating students have completed data collection. Information collected is being entered into the software called PeBInfo (People's Biodiversity Information).

Inauguration of Butterfly Park by Shri Kapil Sibal, Hon.'ble Minister of Science & Technology and Earth Sciences.

n

n

Pathanga Bharathi - A database CD on butterflies of India has been developed Pathanga Suchya - An interactive CD for identification of common butterflies of peninsular India has been developed.

Twenty one Vacation training programme on Bioresources for school children were conducted benefiting about 600 children.

Capacity Building Programmes: A Kerala Forest Research Institute (KFRI) sub-centre at Nilambur established a Bioresources Nature Trail in an area of 5ha. The landscaping of the Nature trail is completed for the conservation and separate theme areas have been set up for the lower groups of plants such as algae and bryophytes, pteridophytes, plants found in specialized ecological niche such as xerophytes (cacti and succulents) and hydrophytes (aquatic plants), beneficial plants (medicinal plants), ornamental and aesthetic plants (orchids) with special reference to endemic and rare, endangered and threatened species of Kerala. Propagules of over 700 species of plants have been collected and introduced in the thematic areas of the nature trail. A gymnosperm garden with five native species and certain exotic species, which are of academic interest is being established in the Nature Trail. Thallpohyte and Bryophyte specimens are also displayed in a specially designed shade house with mist and drip irrigation facilities. The trail is being also equipped to provide the know-how for conservation of Kerala's biodiversity for the visitors.

DBT Annual Report 2006-07

Under the programme for the Visually Challenged, workshops were organized at MSSSRF, Chennai for teachers and heads of various blind schools. A Braille embosser was installed along with softwares for conversion of text to Braille and vice versa. An audio system with walkman has been set up at the garden for the students to listen to the additional resources developed in audio format. A capacity building programme for college and university lecturers on Bioresources and Biotechnology in Sustainable Development has also been supported. Under the programme training to the College teachers was imparted on thematic areas such as: Thematic areas of the Workshop include: (i) Relevance of Theme in Science and Society; (ii) Ecological Bioresources and Biotechnology; (iii) Economic Bioresources and Biotechnology; and (iv) Socio-Economic, Legal and Ethical Issues Linked to Bioresources.

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Rural Bioresource Complex:

accommodate 100 poultry birds. Till date, 843 families are direct beneficiaries and 2297 are indirect beneficiaries.

Five Rural Bioresource Complexes have been set up at University of Agricultural Sciences, Bangalore; Haryana Agricultural University, Hisar; G.B. Pant University of Agriculture and Technology, Pant Nagar and Marathwada Agricultural University, Parbhani and Orissa University of Agriculture & Technology, Orissa with the basic objective of economic empowerment of the target group through income generation and better employment opportunities along with entrepreneurship development, social benefits and environmental sustainability.

Medicinal and Aromatic Plants A programme on biotechnological intervention on medicinal and aromatic plants was continued for conservation, characterization, micropropagation, production of secondary metabolites, development of standardized herbal products, isolation and characterization of novel therapeutic agents, genomics and metabolic engineering. A network project on developing standardized herbal product(s) for bovine mastitis has been initiated. The salient achievements of the programme during the year are as follows:

In the Rural Bioresource Complex at UAS, Bangalore, inaugurated by Dr. V. L. Chopra, Member Planning Commission. 17 types of interventions were identified and 15 of them are being promoted in the RBRC project area at Tubagere in Doddaballapur taluk of Bangalore District involving the 6,067 families. The major interventions promoted were seed productions in Ragi and Groundnut, commercial production of Pop corn, Baby corn, Tissue culture banana, Rose, Watermelon, Drumstick, Mulberry, Improved fish farming and Value added products in ragi In order to effectively implement the interventions 42 training programmes and two study tours were organized. Market linkages are established to sell the produce of growers at a profitable price.

National Gene Bank for Medicinal and Aromatic Plants The four gene banks have been further strengthened with emphasis on collection, conservation and characterization: Tropical Botanic Garden and Research Institute, Thiruvananthapuram A total of 370 accessions belonging to 96 species were collected from Kerala, Karnataka, Tamil Nadu, Goa, Andhra Pradesh and Andaman Islands and introduced in to field gene bank. Morphological characterization based on qualitative and quantitative characters of 51 accessions belonging to 7 species and anatomical characterization of 10 accessions of two species were carried out. Seeds of 19 target species were collected and deposited to the seed bank. Seven accessions belonging to three species were added in to the in vitro bank. Zygotic embryo cryopreservation was standardized in Coscinium fenestratum. Molecular characterization of 24 accessions of Costus speciosus and six accessions of Trichopus zeylanicus using RAPD and ISSR markers has been completed.

At MAU, Parbhani, base line survey has been completed. Seed production programme has been taken on 271.6 hectare areas covering 678 beneficiaries directly. The intervention on pomegranate cultivation (Bhagva variety) was completed on 75 acres area benefiting 75 farmers. The goat keeping intervention was extended to 40 landless beneficiaries by providing them most sturdy Osmanabadi goats after vaccination and deworming, 25% of the total beneficiaries have reported birth of single or twins among two-three goats. For Poultry farming (Vanraja Variety), a low cost completely prefabricated poultry shed was designed and developed and is now under mass construction on the site of 40 landless beneficiaries. The shed is designed to

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Research and Development

species conserved in this mode. Seed bank has been enriched by 96 accessions and now comprises of 2286 accessions belonging to 418 species. Morphological characterization of various accessions of Matricaria chamomilla (62), Foeniculum vulgare (38), Withania somnifera (121) and Papaver somniferum (350) has been carried out. Thirty four accessions of W. somnifera have been chemically evaluated for Withaferin-A contents.

Germlasing of NAPs conversed in gene bank at TBGRI, Thiruvenanthpuram. 1. Abrus precatorius- an accession from Andaman Island 2. Rauvolfia serpentina- a potential accession from Tamilnadu 3. Accessions of Bacopa monnieri

Representative cultures of some new germ plasm accessions of MAPs been added to tissue bank at CIMAP. 1- Chlorophytum arundinaceum; 2Aloe vera; 3- Centella aisatica; 4- Acorus calamus; 5- Indigofera sp.; 6Chrysanthemum cineraraefolium; 7- Berginia sp; 8- Hypericum himalaicus; 9- Swertia chirayita; 10- Paris polyphylla; 11- Salvia scalarea; 12- Gentiana kurrooa; 13- lavendula officinalis.

1. 2.

Cryopreserved zygotic embryo of Coscinium fenestratum showing germination and development Shoot tips of Kaempferia galanga cryopreserved through vitrification showing recovery and development

DBT Annual Report 2006-07

Regional Research Laboratory, Jammu Field gene bank has been enriched by addition of 27

70

accessions of 15 high altitude species collected from Himachal Pradesh and Uttaranchal. About 3000 plants of Picrorhiza kurrooa have been provided to the field conservatory at Srinagar. Fifteen accessions have been added to the seed bank. In vitro repository has been enriched by adding various accessions of Swertia chirayita spp (5), Picrorhiza kurrooa (5) and Angelica archanglica (1). Somatic embryogenesis has been developed in Aconitum heterophyllum. Morphological characterization of various accessions of Argyrolobium roseus (7), Podophyllum hexandrum (2), Picrorhiza kurrooa (2), Jatropha curcas (3), Aloe vera (20) and Silybum marianum (3) have been carried out

different geographical locations were chemically analysed for withanolide and gymnemic acid content , respectively at The Energy and Resources Institute, New Delhi. Withaferin-A content varied in leaves from 0.22 to 3.38% and in roots from 0.002 to 0.75% in W. somnifera. In 18 accessions of G. sylvestre, the gymnemic acid content varied from 0.23 to 2.53%. Eleven different populations of Nothapodytes nimmoniana from the Western Ghats were chemically profiled for camptothecin content jointly at University of Agricultural Sciences, Bangalore and University of Agricultural Sciences, Dharwad. Out of 148 individuals assayed, 23 yielded more than 1% camptothecin. These high yielding lines are being multiplied clonally for re-introduction in to their natural habitat. SSR markers are being developed to assess the genetic variability of populations and also to identify molecular marker tags for camptothecin.

Ex-situ conservation and characterization A total of 1686 accessions belonging to 146 species used in Ayurveda are being maintained in the field gene bank at Arya Vaidya Sala (AVS), Kottakal. Seed samples of 44 species have been stored in the seed bank. An image library has been developed which has 2294 plant images belonging to 811 species. Morphological and phytochemical characterization of various accessions of Adhatoda zeylanica, Coleus aromaticus and Hemidesmus indicus have been carried out. At National Bureau of Plant Genetic Resources, New Delhi, in vitro repository of a total 30 accessions of Allium tuberosum, A. chinense, Bacopa monnieri and Centella asiatica has been established. Cryopreservation protocols of in vitro shoot bases in Allium tuberosum using encapsulation-dehydration and vitrification technique have been established. In vitro bulblets in A. chinense and encapsulated explants in A. tuberosum and B. monnieri in cryovials were successfully maintained without nutrient medium.

Micropropagation Multi-location field trials of tissue culture plantlets and rooted cuttings of elite patchouli (Pogostemon cablin) continued at five institutions; Kelkar's Scientific Research Centre (SRC), Mumbai; Konkan Krishi Vidyapeeth (KKV), Dapoli; NRC on Medicinal and Aromatic Plants, Anand; Central Plantation Crops Research Insititute (CPCRI), Kasaragod and University of Agricultural Sciences (UAS), Dharwad. Agrotechniques were standardized for cultivation of patchouli in Gujarat, Konkan and Karnataka region. A total area of 30 ha has been covered in different locations with the patchouli crop in the farmers' field. The biomass obtained from the nodal centres was analyzed for oil yield and profile. The oil obtained from different experimental trials have been found to be similar to the parent plant with respect to the composition. Based on the results, it is concluded that cultivation of patchouli can be undertaken in regions with high humidity as a single crop or in dry regions as an intercrop with other crops. The design of the mist bioreactor system for generation of patchouli plantlets has been further improved at Kelkar's SRC, Mumbai. The growth performance and oil profile of bioreactor-generated plantlets of

A rapid and highly reproduciable protocol for in vitro propagation of Picrorhiza scrophulariflor,an endangered medicinal plant of Eastern Himalayas has been developed at North-Bengal Agricultural University, Cooch Behar. About 350 in vitro raised plantlets have been hardened with 90% survival rate. A total of 162 accessions of Withania somnifera and 18 accessions of Gymnema sylvestre collected from

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Research and Development

patchouli were found to be similar to those of tissue culture and rooted cutting plants.

Herbal Formulation Pre-clinical studies on a herbal preparation from the stem bark of Terminalia arjuna for left ventricular dysfunction continued jointly at AIIMS, New Delhi and B. V. Patel PERD Centre, Ahmedabad. The 50% effective dose (ED50) value of the standardized extract was determined as 169.8 mg/kg in rat models. Preventive effect of the standardized extract of T. arjuna on the development of heart failure was also studied in rat models. No significant change in the left ventricular wall thickness (both systolic and diastolic) on echocardiography and left ventricular dysfunction was observed in extract treated group as compared to normal rats. Ninety-day toxicity study of aqueous extract of T. arjuna has been initiated.

Evaluation of field performance of tissue culture plants vs open pollinated seedlings of large cardamom (Amomum subulatum) planted over an area of 55 ha in farmers' field in Sikkim and Darjeeling district of West Bengal continued at the Regional Research Station, Indian Cardamom Research Institute, Gangtok. A substantial increase of growth and yield were recorded in tissue culture plantlets as compared to open pollinated seedlings. The clone SBLC-47 A (Varlangey) planted during 1998 season recorded maximum yield of 540 kg/ha. Fifteen farmers' training programmes and group meetings were organized during the year on various aspects of large cardamom production.

Two compounds-Daidzein and Genistein 8-C glucoside isolated from root extract of a legume have shown considerably high anti type-2 diabetes activity in vitro jointly at Viswa Bharati, Santi-Niketan and the IICB, Kolkata. Work on developing a standardized herbal formulation from a common weed having antiEntamoeba histolytica activity continued jointly at Bose Institute, Kolkata and IICB, Kolkata. Two separate collections of the plant have been analysed by activity-guided purification. Active principles were identified to be saturated fatty acids, their esters and an ester of the unsaturated fatty acid linolenic acid. At C.U. Shah College of Pharmacy, Mumbai, efficacy and safety studies of root extract of Cyperus rotundus and Aloe vera gel for anti-atherosclerosis activity have been taken up. Significant hypolipidaemic activity has been recorded in hydroalcoholic and alcoholic extracts of C. rotundus in hamsters. Aqueous, hydro-alcoholic as well as alcoholic extract of C. rotundus were found to be non-toxic in acute toxicity studies. Studies have been initiated recently to develop a standardized anti-diabetic herbal formulation from Annona squamosa jointly at Bombay College of Pharmacy, Mumbai and BYL Nair Charitable Hospital, Mumbai. A network project on development of standardized herbal product for bovine mastitis has been initiated recently involving five institutions: Maharashtra Animal and Fishery Sciences University, Nagpur; Central Institute of

Evaluation of the performance of elite tissue culture plantlets vis-à-vis stem cuttings of vanilla (Vanilla planifolia) in farmers' field over an area of 20 ha in Tripura state has been initiated jointly by the Tripura Biotechnology Council, Agartala and the Indian Cardamom Research Institute (Spices Board), Myladumpara. An area of 5 ha has been planted during 2006 planting season with tissue culture plantlets and stem cuttings of vanilla (in 80:20 ratio). Production of secondary metabolites Cell-cultures of Commiphora wightii were grown in 2L stirred tank and 6-L airlift bioreactor towards developing technology for guggulsterone production at M. L. Sukhadia University, Udaipur. Various empirical approaches were used for guggulsterone enhancement in embryogenic and non-embryogenic callus and cell suspension in C. wightii. At the Indian Institute of Technology, New Delhi, out of several root lines of Azadirachta indica developed, high yielding hairy root cell-lines have been selected on the basis of growth index and azadirachtin content. Effect of various elicitors, precursors, growth regulators and permeabilizing agents was studied on hairy root growth and azadirachtin production. Development and selection of a suitable bioreactor configuration for mass production of hairy roots has been carried out.

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72

Medicinal and Aromatic Plants, Lucknow; Indian Veterinary Research Institute, Izatnagar; Tamilnadu Veterinary and Animal Sciences University, Chennai and G. B. Pant University of Agriculture and Technology, Pantnagar. Isolation and Characterization of Novel Therapeutic Agents

Leishmania donovani infected BALB/e mice liver cells control (left); After treatment with the pectic substance from bael fruit for 10 days (IICB, Kolkata)

Five compounds (K-051, K-052, K-054, K-080) have been identified from plant extracts showing promising osteogenic (bone forming) activity at the CDRI, Lucknow. An Indian patent has been filed on anti-osteoporosis activity of Butea species. Work on in vivo efficacy of pure compounds showing promising osteogenic activity and molecular mechanism of osteogenic action is planned to be initiated. A plant extract has been identified which inhibit the differentiation of pre-osteoclastic cells to osteoclasts at Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram. It is also found to stimulate collagen synthesis in osteoblasts.

Leishmania donovani infected BALB/e mice spleen cells control (left); After treatment with the pectic substance from bael fruit for 10 days (IICB, Kolkata)

showed marked inhibition of acetyl cholinesterase effect and cognitive enhancing activity. A set of p 300 histone acetyl transferase (HAT) specific inhibitors have been synthesized and characterized from garcinol for cancer therapy and diagnostics purposes at JNCAR, Bangalore. A polyhydroxylated aromatic compound has been identified as a novel specific inhibitor of histone methyl transferase (HMTase) which was found to be toxic to cancerous cells but no effect on the normal cells. Hydroethanolic extracts of Citrus sinensis, Emblica officinalis and Ocimum sanctum have been found to inhibit farnesyl transferase enzyme activity at Jamia Hamdard, New Delhi.

Ethanol extract of Phyllanthus rheedii inhibited Hepatitis-B (HBs Ag level) up to 68% at RGCB, Thiruvananthapuram. At TBGRI, Thiruvananthapuram, ursolic acid was identified as the marker compound in Ixora coccinia and flavonoids and coumarins in Rhinacanthus nasuta extracts showing hepatoprotective activity. Potential anti-bacterial activity has been recorded in leaf extracts of Callistemon rigidus against standard strain of Staphylococcus aureus NCTC-65-71 at Thapar Institute of Engineering and Technology, Patiala. Bio-activity guided fractionation of the active extract has also been carried out. Purified pectic polysaccharide from Aegle marmelos (Bael) have shown significant in vivo anti-leishmanial activity using BALBc mice models infected with Leishmania donovani at IICB, Kolkata.

Genomics and Metabolic Engineering Efforts have been initiated to construct a high precision genetic linkage map of Catharanthus roseus at NCPGR, New Delhi. The Inflorescence architecture (ia) locus has been mapped on the linkage group 2 (LG2) of C. roseus using bulk segregant anlaysis of a population of recombinant inbred lines (RILs) and a series of RAPD, ISSR, SSR and sequence specific molecular DNA markers.

Root extract of Clitorea ternatea and taraxerol showed significant inhibition of acetyl cholinesterase activity and cognitive enhancing activity at Jadavpur University, Kolkata. Similarly, rhizome extract of Acorus calamus and β-asarone isolated from it also

73

Research and Development

With a view to isolate the taxol biosynthetic genes, a cDNA library from the endophytic fungus isolated from Taxus celabica was constructed at Indian Institute of Science, Bangalore. Presence of taxadiene synthase and taxadiene 13 hydroxylase was confirmed by DNA sequencing and it would be used in the screening of the Alternaria cDNA library to isolate the corresponding cDNA clones. Work has been continued to elucidate the novel metabolic route to a phenolic fragrance 2-hydroxy-4methoxybenzaldehyde formation in root organs of Hemidesmus indicus at Indian Institute of Technology, Kharagpur. Two novel enzymatic routes have been demonstrated.

pathway in genotype Sampada of Papaver somniferum (poppy) have been cloned at CIMAP, Lucknow. Plant Biotechnology During the year, support continued for programmes under forestry, horticulture and plantation crops; basic research; solanaceae genome and molecular taxonomy. Three meetings of the Task Force were held during the year and two meetings of the Scientific Advisory Committee on micropropagation research and technology development. In addition a number of idea generation meetings and brainstorming discussions were organized for initiating new programmes during the 11th Plan. A new initiative launched during the year was on host pathogen interaction. During the year another major new initiative has been on improvement of vegetable crops using biotechnology approaches. Some of the salient achievements of the ongoing programmes are as follows.

RAPD and minisatellite profiles of sandalwood (Santalum album) populations of the Southern regions of India have been generated at Vittal Mallya Scientific Research Foundation, Bangalore. Marked genetic variability has been observed in different populations and within individuals of a population. The wood oil quality of representative samples was assessed by GC-MS for presence of various sesquiterpenes. cDNA libraries from leaf and wood tissues have been generated to isolate complete ORFs of terpene synthases from sandalwood.

Forestry, Horticulture & Plantation Crops: In the area of forestry, studies continued on developing micropropagation protocols for important trees, germplasm characterization, conservation and other related aspects of forestry improvement. Micropropagation protocols are being developed for Grewia optiva, Buchanania lanzan, Red sanders, Sandal wood and Eucalyptus hybrid. Field evaluation

Work on cloning and characterization of regulatory elements of genes involved in picrosides biosynthesis in Picrorhiza kurrooa has been initiated at Institute of Himalayan Bioresource Technology, Palampur. Upstreem sequences of 1-deoxy-Dxylulose-5-phosphate reductoisomerase (DXR) and 3-hydroxy-3-methylglutaryl Co-A reductase (HMGR) have been cloned.

studies for two important eucalyptus hybrids have been taken up at 6 locations, and data is being collected.

Genomic analysis of sesquiterpene biosynthesis regulation in Artemisia annua metabolome has been initiated at CIMAP, Lucknow. The full-length amorpha 4, 11diene synthase (ads) gene from A. annua genotype CIM-Arogya have been cloned. Work on expression of genes controlling trichome number (from Arabidopsis) in A. annua has been recently initiated at University of Hyderabad, Hyderabad.

In a study supported at TFRI, Jabalpur, teak populations belonging to Andhra Pradesh, Karnataka, Kerala, Madhya Pradesh, Maharashtra, Orissa, Rajasthan and Tamil Nadu, were assessed for their genetic diversity using ISSR and AFLP molecular markers. The polymorphic loci amplified were 43 from the 29 populations by ISSR markers and 226 from ten populations by AFLP markers. Studies were also supported for genetic engineering of Leucaena leucocephala and Populus for

Four genes of the isoquinoline alkaloid biosynthetic

DBT Annual Report 2006-07

74

producing low lignin varieties. A full length clone of OMT ( ~ 1.1 Kb) was isolated by screening of cDNA library of Leucaena leucocephala and sequenced. On blast analysis, the sequence exhibited homology to reported OMT sequences in the data bank indicating authenticity of our clone. Leucaena leucpcephala was transformed with an antisense OMT gene construct from aspen and analysis of the transformant was done. The integration of a heterologous antisense OMT gene construct in transformed plants led to a maximum of 60% reduction in OMT activity relative to control. The evaluation of total lignin content by Klason method revealed a maximum of 28% reduction.

viroids and greening bacteria is being developed. PCR detection of greening bacterium and Citrus Yellow Mosaic Virus in Citrus tissues was standardized using a simplified template preparation protocol. Both the greening bacterium and CMBV were detected through PCR when the eluted liquid from the spotted membrane was used. The eluted liquid was found comparable in detection efficacy to a multi-step laboratory method or a commercial kit for nucleic acid preparation. The protocol is simple, inexpensive, rapid, and applicable to large-scale survey of citrus trees. Studies are also continuing on molecular characterization and detection of citrus yellow mosaic virus.

In the area of horticulture, studies were focused on development of protocols for generation of superior planting material, molecular characterization of germplasm and development of improved varieties for increased shelf life and disease resistance. Under the apple network programme, protocols for micropropagation of important root stocks were standardized and mass produced and nearly 10,000 root stocks have been planted with approximately 85 to 90% success. Protocol for root stock Merton 793 & M26 has also been standardized and approximately 15,000 plants have been produced, which would be field planted shortly. Germplasm assessment and molecular characterization for apple was successfully conducted during the year. 119 superior cultivars were collected from 13 orchards and morphological descriptors prepared for 80 cultivars with molecular profiles generated for 78 and cytological studies completed for 56 genotypes with a significant achievements of identification of a scab resistant cultivar. The microsatellite markers are being developed for specific traits.

A programme has been supported at IIHR, Bangalore and CISH, Lucknow for characterization of the germplasm of mango from Southern and Northern region. Based on microsatellite enrichment method, STMS primers have been developed. Using four mango species (Mangifera odorata, M. zeylanica, M. andamanica, M.camptosperma) and eleven cultivars (Muvandan, Kurukkan, Alphonso, Raspuri, Totapuri, Langra, Dashehari, Neelum, Kesar, Padari, Bhutto Bomaby) genescan analysis was done for 30 STMS primers with the help of automated DNA sequencer. Many of these primers amplified all the species used here indicating cross species compatibility. 13 ISSR primers were used to screen the 48 accessions of mango, among these 11 primers yielded distinct polymorphic products, cumulatively amplifying 160 bands ISSR 5 yielded 14 polymorphic bands and 7 monomorphic bands.Based on similarity matrix, dendogram using UPGMA tree was constructed, which has efficiently clustered the accession from these two regions. Expression of Bougainvillea antiviral protein gene is being studied at IIHR, Bangalore to develop virus resistant tomato plants. The cDNA encoding Bougainvillea anti viral protein was cloned into pMALc2X expression vector. After confirmation for the presence of the insert, the recombinant vector was transformed into E.coli. The transformants were selected by blue white colour. The transformants growth was slower when induced with IPTG which is

Large scale production and demonstration of disease free planting material of Citrus (Nagpur mandrin) is being done through shoot tip grafting. During the year nearly 35,000 bud grafts were released to the farmers and these are now being evaluated in the farmer's field. Disease free planting stock has been raised in the nursery for multiplication during the current year. A multiplex diagnostic kit for detection of viruses,

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Research and Development

attributed to the ribosome inactivating property of the Bougainvillea antiviral protein. The recombinant protein expressed in E.coli. was tested by SDSPAGE. Construction of suitable plant transformation vector is in progress.

translational promoter-GUS fusions were introduced into Arabidopsis. Expression of GUS was observed consistently in most tissues after ethylene (2ppm, 4h) treatment. Transformants of Arabidopsis expressing RbXTH2 constitutively showed significant changes in root length and plant height. These are being studied in detail. In gladiolus, genes encoding SAM synthetase, chalcone synthase, alpha mannosidase, XTH and several unknown protein were shown to be differentially regulated during the course of petal senescence.

A study was supported at NBRI, Lucknow for identification and characterization of fruit-specific and ripening related gene promoters from banana. A PCR select subtractive library was prepared using mRNA from unripe and ripe banana fruit. Nucleotide sequence analysis of these clones has identified several ripening related genes that are involved in ethylene biosynthesis, cell wall hydrolysis, defence/stress and detoxification and transcription/translation machinery. Most of the genes show strong ethylene dependent expression in ripening fruits, and are inhibited by treatment with 1-MCP an ethylene inhibitor. Different promoters isolated in the above study were analysed in silico for the presence of common cis acting regulatory elements. Method for transient expression analysis using reporter gene in banana fruit slices has been standardized.

During the year, a major new initiative has been taken on improvement of vegetable crops through biotechnological approaches. Programmes have been supported on development of transgenic brinjal for insect resistant. In this programme, a novel approach of pyramiding of genes cry 2A, cry1Ac, vip3A and cry1f has been adopted. Transgenic onion for resistant to purple blotch is also been studied. A project on molecular approaches for repression of cold induced sweetening in potato has also been supported at CPRI, Shimla Under the plantation crop area, one of the major achievement during the year has been completion of the field demonstration of tissue culture raised black pepper in 100 ha of area in the states of Karnataka, Kerala, Andaman & Nicobar Islands and North East. The tissue culture raised plants have performed better than the conventionally raised material and more number of laterals thereby giving as higher yield have been reported. Over 400 farmers have been benefited.

A study has also been supported at NBRI, Lucknow to identify and characterize genes involved in petal abscission in rose and senescence in gladioli. Partial promoters of two rose xyloglucan transglucosylase/ hydrolase genes RbXTH1 and RbXTH2, which are known to be differentially expressed during ethylene induced petal abscission, were isolated and Stigmatal hair A

B

Stamen

C

Basic Research The thrust of the programmes supported under basic research is to study the signal transduction pathway in identified plants to understand the mechanism involved in responding to external stimuli and the process of adaptation to these changed environment conditions. A number of projects have been supported broadly under three categories: signal transduction, root differentiation and flowering.

AZ

D E F GUS expression in transgenic Arabidopsis plants containing the RbXTH2 promoter (in translational fusion with GUS in pBI101) A -D ethylene treated (0.5 ppm , 4h); E – ethylene untreated; F – 1-MCP treated (followed by ethylene treatment) AZ- abscission zone

DBT Annual Report 2006-07

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Signal Transduction

noted in the presence of ABA treatments indicating the noted oscillation of phosphorylation of this kinase to be an upstream event.

In a study supported at JNU to study signal transduction machineries in plant under osmotic stress, complete physiological characterization of genotypes of Brassica campestris, B. nigra, B.oleracea, B. juncea, B. napus and B. carinata for their tolerance has been achieved under laboratory conditions. Primers have been designed to isolate the middle portion of the hybrid type Histidine kinase from B. juncea (CS52) which has resulted in cloning and sequencing of G80 bp fragment. Novel transcription factors have also been cloned from three B. juncea varieties specific to salinity tolerance. RNA has been isolated and primers were designed using Atmyb2 conserved domain. The PCR-product was amplified and cDNA is being cloned. An efficient plant regeneration and transformation system has been optimized in selected commercial cultivars of B. juncea. A study has been supported at ICGEB to clone and characterize stress responsive promoters from Brassica sps. Putative promoter regions for stress related genes like SoS2, Histidine kinase (HKI), Myb and MyC like factors have been cloned. The amplified fragments for putative promoters have been cloned, which show 50-80% homology. Further work is going on to assess the true stress inducibility of these promoter sequences.

Based on in silico analysis, 46-stress responsive transcription factors were identified from Arabidopsis highly stress responsive functional genes have also been identified and their promoter analyzed. No significant difference was observed for presence of transcription binding sites of stress responsive transcription factors belonging to 10 families. Functional involvement of a light signaling component (ZBF1) in root development in Arabidopsis. ZBF1 (Z-box binding factor 1), which codes for a bHLH protein has recently been cloned at NCPGR, New Delhi. The function of A novel transcription factor of light signaling on root development has been investigated. Mutations in ZBF1 results in elongated roots as compared to wild type plants. At NBRI, the expression of three Ethylene Responsive Factors (ERFs) was found ten days after watering was stopped. Increased transcript accumulation was also observed after a cold shock of 4 hours for LeERF5 and LeERF6 while expression of all three ERFs was repressed after a heat shock at 42°C for one hour. In order to functionally analyse these genes, they were expressed in a bacterial expression system in the vector pET28. The proteins isolated are currently being analysed. Transgenic tomato plants that overexpress the three ERFs have also been raised and are being studied for morphological and physiological changes.

Scientists at NCPGR, New Delhi has been able to clone 8 members of mitogen activated protein kinase family (MAPK) from rice. From the 8 clones, four full length genes have been obtained. They have further been cloned in PGEX vector having GST protein fusion for heterologous expression in bacterial system. MAP kinase induced by heat has been identified and work is on to transform rice with different constructs.To understand the mechanism operative during early signaling events mediating auxin dependent abiotic stress tolerance in groundnut at Calcutta University, CDPK has been characterized from Arachis hypogea showing 88% homology to a stress responsive CDPK from Arabidopsis. Result indicated that Dephsphorylation of AhCPK2 is not a response to water loss per se but to a change of water potential. No such change was

Floral Development To understand how floral organ development and regulation of size & shape of floral organs and leaves take place at molecular level, studies have been conducted at IISc, Bangalore on Arabidopsis. 17,000 EMS-mutagenized M2-seeds were screened and based on phenotypes, mutants have been categorized. To check the DNA binding properties of wild type TCPY, consensus binding site of TCP4 was determined using random binding site selection

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Research and Development

(RBSS) assay.

Tomato Sequencing Project”. India which is one of the partner has been assigned ~12 Mb euchromatic region of chromosome 5 for sequencing. The Indian initiative on tomato genome sequencing is being taken up by 3 centres with UDSC and NCPGR sequencing the short arm, and NRCPB sequencing the long arm. In all, 38 BAC clones, covering approximately 3.8 Mb regions have been mapped to chromosome 5. Status

Studies have been supported during the year on Host Pathogen Interaction concentrating on the following areas :- Molecular basis of determinants of host-pathogen specificity; Functional characterization of R/ Avr genes, Early events in pathogenesis initiation with respect to adhesion, penetration mechanisms, hydrolytic enzymes etc., Study of signal transduction cascade involved in early events both in host as well as pathogen, Gene expression profiling of plant(s) and pathogen(s) during pathogenesis, Innate immunity / non-host resistance; Systemic Acquired Resistance; Induced Systemic Resistance etc., Mechanism of Pathogenicity, Genetic diversity of important plant pathogens., Genome sequencing and functional genomics of important pathogens. These studies have been supported to understand and develop the complex interactions between plant pathogen and host plants.

Phase II Library Phase II Library Phase II Phase II Library Library Phase II Library Phase II Library Phase II Library

LE_HBa0040C21 LE_HBa0025A19 LE_HBa0131D04 LE_HBa0100I06 LE_HBa0006N20 LE_HBa0108A18 LE_HBa0019C24 LE_HBa0141A12 LE_HBa0239D11 LE_HBa0013H09 LE_HBa0251J13 LE_HBa0028M20 LE_HBa0245E05 LE_HBa0076P16

Marker CT101 C2-At1g60440 T1252 C2-At1g60200 cLET-8-B23 T0564 cLED-8-G3

cM 0 0 3 7

Euchromatic Region

10 11 15.5

T1592

16

TG432

21

BS4

Telomeric Region

22

T1360 cLEX-13-G5 T1746 T1777

73 79 84 105

T1584

108

TG69

111

CT130

115

UDSC & NCPGR

Heterochromatic Region

Centromeric Region

Heterochromatic Region

NRCPB

Euchromatic Region

TG185

119

TG597 CT138

119 119

Telomeric Region

Sequencing status of tomato chromosome 5

Annotation of fifteen BAC clones using FGENESH revealed a gene density of one gene per ~5 kb sequence. Some locus specific variations in gene density were also observed. Studies have also been supported to understand the molecular basis of root development in tomato. Root specific subtractive library from tomato using Suppression Subtraction Hybridization (SSH, Clontech) has been construccted. 34 root specific EST's have been generated. Another 500 clones are in the process of sequencing. Subtractive library for temporal specific genes (8-day root and 40-day root) have also been constructed and are further being screened.

SOL Genome

TILLING (Targeting induced local lesions in genome) is an advanced selective molecular breeding technology that allows improvement of crop plants for which the genome sequences are known or in progress. At University of Hyderabad, a study has been initiated where TILLING technology is being

An international consortium of ten countries, including USA, Korea, China, UK, India, Netherlands, France, Japan, Spain and Italy aims at sequencing the euchromatic region of the tomatog genome under the auspices of “The International

DBT Annual Report 2006-07

SL_MboI0050C14 LE_HBa0191B01 LE_HBa0106O06 LE_HBa0051A13 SL_MboI0005B15 LE_HBa0261K11 SL_EcoRI0086I08 LE_HBa0042B19 LE_HBa0189E17 LE_HBa0179E24 SL_MboI0037H06 SL_MboI0095J08 LE_HBa0074A13 LE_HBa0058L13 LE_HBa0195M17 LE_HBa0165L04 LE_HBa0051A18 LE_HBa0027B05 SL_EcoRI0053P22 SL_EcoRI0073I01 LE_HBa0169M21 LE_HBa0334K22 LE_HBa0138J03 LE_HBa0166A02

Long Arm

Host Pathogen Interaction

Clones selected

Phase I Phase III Phase II Phase II Phase II Phase III Phase I Phase III Phase I Phase II Phase III Phase I Phase II Phase III Phase II Library Phase II Phase II Phase III Library Phase I Phase I Library Phase II

Short Arm

Study was supported at IISc, Bangalore on functional analysis of meiosis in Arabidopsis using candidate gene approach. So far generation of AML RNAi constructs has been completed. The corresponding portion of the OML5 gene has been PCR amplified, cloned, and authenticated. A 3.6 kb region of the AML3 has been amplified and construction of the pAML2::GUS fusion in the reporter gene vector pBI101 has been completed. Transformation of Arabidopsis with AML RNAi construct has been done and multiple T1 transgenic plants have been obtained. Detailed characterization of phenotypes in 70 T1 plants screened has been completed. In 11 out of 70 T1 plants sterility was observed due to defects in male and female gametogenesis.

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applied for improvement of tomato in term of quality and shelf life. Nearly 12,000 mutant lines of M82 cultivar and 6000 mutant lines of Arka Vikas cultivar have been planted in the field. Fifteen major characters of tomato plants were targeted for the analysis at different stages of the plant development and a database for the distinct phenotype features has been created. Currently experiments are underway to detect the mutant lines with gene modifications for improved shelf-life and carotenoid contents. Under Functional genomics, three aspects were supported on (i) disease resistance, (ii) nutritional quality (iii) shelf life.

shown by the genotypes H24 and Hawai 7998 for TLCV was further confirmed through graft test as well as through ELISA test at KAU and PCR assay at ICGEB, New Delhi. As for the wilt, 11 genotypes were found to be resistant. Development of mapping populations for mapping the resistance gene(s) is underway. At IARI, New Delhi, samples of diverse begomoviruses infecting tomato have been collected from different states and subjected to PCR analysis. Those samples which were positive have been subjected to amplification with primers, that can differentiate the four begomovirus species viz., Tomato leaf curl New Delhi virus, Tomato leaf curl Gujarat virus, Tomato leaf curl Bangalore virus and Tomato leaf curl Karnataka virus. Based on the analysis, the virus that is expected to be present in the samples collected is further being identified.

Disease Resistance For developing mapping population and identification of molecular markers linked to Tomato Leaf Curl Virus (TLCV) resistance in Tomato (Lycopersicon esculentum Mill), 45 tomato lines including four wild accessions with reported resistance to TLCV were screened under field conditions, 39 were resistant and 3 tolerant. Eight lines were completely free for TLCV incidence when the susceptible Pusa Ruby exhibited 100% TLCV incidence. A total of 15 hybrids were produced involving six resistant parents and five susceptible lines for studying inheritance of resistance to ToLCV. Five F2's of crosses of three wild species viz; Lycopersicon hirsutum (Acc. LA-1777), Lycopersicon hirsutum f. glabratum (B-6013), Lycopersicon peruvianum & Lycopersicon chilense and two resistant cultivars with the susceptible cultivar have been raised as mapping populations. Study of parental polymorphism using 94 SSR primers, revealed 44 were polymorphic between ToLCV resistant parent IIHR-2101 and the susceptible parent 15 SBSB. Genotyping of the mapping populations has been initiated using the polymorphic markers.

Nutritional Quality To improve nutritional quality in tomato a study has been supported at IARI, New Delhi & IVRI, Varanasi to analyse carotenoid biosynthesis pathway genes. Lycopene - -cyclase (CRTL- B) from tomato (Lycoperiscon hirsutum) which regulates lycopene biosynthesis, has been cloned. The promoter is being characterized further. IIVR, Varanasi has collected tomato genetic stock for assessing variability for lycopene content. Protocol for estimation of lycopene by HPLC is being standardized. To understand the genetics and the pathway of lycopene, a crossing programme involving genotypes having maximum and minimum lycopene content has been initiated. Shelf Life In an ongoing study on post harvest biotechnology for improved shelf life of tomato at IARI, New Delhi, S. adenosyl-mithione decarboxylase (Sam-DC) has been overexpressed with the introduction of an EXPI cDNA driven by LeACS4 promoter into L. esculantum cv. Pusa Uphar through Agrobacterium mediated transformation using colytedon as explant. Transgenic plants at T1 stage have been raised successfully to maturity in phytotron and they have produced flowers and fruits. The fruits harvested

For isolation and characterization of genes encoding for disease resistance (TLCV and bacterial wilt), 60 genotypes have been screened against the diseases. Over three seasons 27 genotypes have been found to be resistant to TLCV. The resistance

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Research and Development

showed sign of spoilage even after 30 days of harvest whereas wild type fruits were rotten completely. Detailed post-harvest ripening data is being collected.

(Meghalaya), Eastern UP, Western Himalayas (Uttranchal) for morphometric and molecular taxonomic studies. Further work on DNA profiling of Atlantia accessions using RAPD and microsatellite markers is in progress. Besides, essential oils have been extracted from five accessions of four different varieties of Citrus using solvent extraction and hydrodistillation method. The chemical constituents of oil were analysed using different chromatographic techniques. Identification of essential oil constituents from other accessions of Citrus viz. C. jhambhiri, C. karma and Kaithari nimboo is in progress.

Transgenic tomato plants expressing LeMADS-RIN gene under 35S promoter were produced with the aim of producing tomato with delayed ripening as well as prepare material to find out genes controlled by LeMADS-RIN transcription factor at UDSC, New Delhi. However, though these lines showed substantial reduction of transcript level, all the antisense transgenic lines were found to have no significant delay in fruit ripening. This suggested that stronger suppression of LeMADS-RIN gene may be required for delay in ripening. Therefore, 3 RNAi vectors designed to suppress transcript level of LeMADS-RIN gene have been constructed and used for transformation of tomato plants. These vectors are likely to have stronger suppression of LeMADSRIn gene, but the degree of suppression may vary. Transformation experiments have been initiated with all three vectors.

At the University of Calcutta, Kolkata accessions of Phyllanthus from different parts of West Bengal have been collected and RAPD analysis is being carried out to segregate the populations. In another study at the same institute, DNA fingerprinting studies have been done for different species of Amaranthus and some members of Chenopodiaceae. Dendogram analysis showed that members of Amaranthaceae and Chenopodiaceae form broadly two clusters. Further work is going on to identify AmA gene on the chromosome and comparative study is on to see variation in AmA gene in some special of Amaranthus.

Six different ethylene responsive factors have been cloned from tomato cDNA using degenerate primers and full-length sequences of three have been obtained. Besides, transcript pattern for three genes during the course of ripening and in different stress conditions have been obtained. Sense and antisense constructs for three ESTs have been prepared.

A study has been supported at CIMAP, Lucknow to segregate different species of Phyllanthus using molecular tools. The fragments in the RAPD profiles generated by MAP-9 primer were cloned for Phyllanthus amarus, P. fraternus, P. debilis and P. urinaria. These fragments are being sequenced to generate SCAR (Sequence Characterized Amplified Regions) markers for differentiation of these species. The genetic relationship among Phyllanthus accessions of various species has been determined. Polymorphism rates were high indicating a substantial amount of molecular variation and potential genetic diversity. Subsequent analysis yielded eight groups out of which three major clusters are of P. debilis, P. amarus and P. urinaria. Least variation was detected among the P. amarus accessions, close to the group of P. amarus falls the cluster of P. fraternus. On the basis of molecular techniques (AFLP), it is possible to predict that four

Molecular Taxonomy Molecular Taxonomy was identified as R&D priority for those taxa which could not be segregated morphologically at family, genera, species and subspecies level. Some of the achievements reported during the year are as follows: Studies have also been initiated on genetic diversity assessment in wild species of Citrus and Atlantia at NBRI, Lucknow. Sixty eight accessions of three species of Atlantia (A. monophylla, A. racemosa; and A wightii) were collected from different locations of the Western Ghats of Maharashtra, Tamil Nadu and Kerala and 20 accessions of Citrus from Khasi hills

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herbaceous species (P. amarus, P. fraternus, P. debilis, P. urinaria) are more closely related to each other than other species of the same genus. P. amarus showed most similarity with P.fraternus following P.debilis and P.urinaria.

as follows: Animal Health Care: Vaccines and Diagnostics : The technology of large scale production of recombinant protective antigen (PA) based vaccine for anthrax was developed at JNU, New Delhi and transferred to industry. Phase I / II human clinical trials of recombinant vaccine has been successfully completed. Towards developing another anthrax vaccine, lethal factor (LF) and edema factor (EF) mutants of anthrax were constructed, expressed, purified and characterized. Novel mutants of EF and LF were found to be nontoxic in vitro which provided better protective efficacy in vivo and safe to be used as vaccine component.

Systematic and molecular taxonomic studies have been conduced in Asiatic Vigna and Macrotyloma at NBPGR, New Delhi. So far a total of 193 samples of Vigna and Macrotyloma have been collected from different national and international sources. Analysis of sequence variation in rDNA regions has been completed using PCR amplification of ITS1, ITS2, 26S & 18S regions. Further work on cloning and sequencing of the amplified region is in progress. In an another study at NBPGR, 153 germplasm accessions of Solanum melongena; 25 accessions of S. insanum and 6 accessions of S. incanum have been collected and herbarium specimens have also been deposited at Botanical Survey of India. AFLP analysis was done for 48 accessions comprising S. melongena, S. incanum, S. viarum, S. virginianum, S. sysibriifolium, S. gilo, S. violaceum, S. acculeatissimum, S. macrocarpon and intermediate types. The AFLP data differentiated species belonging to the egg plant complex (S. melongena, S. insanium, S. incanum). The 'intermediate' accessions occupied positions among the S. melongena, S. insanum and S. incanum accessions indicating that they might be natural hybrids of these species.

Efforts were made to develop DNA vaccine against rabies through a multicentric project implemented at CBT, JNU, New Delhi and IVRI, Izatnagar. The RNA from rabies virus virulent strain infected mouse brain was isolated and G gene was amplified and cloned in m a m m a l i a n e x p r e s s i o n v e c t o r. T h e immunofluorescence and immunoperoxidase tests were standardized for detection of the expression of rabies virus G gene in cell culture. DNA vaccine constructs using the glycoprotein gene of rabies virus ERA strain have been developed and their potency are being studied. Molecular analysis of Indian buffalopox virus (BPXV) isolates recovered from outbreaks in both cows and buffaloes was carried out at IVRI, Mukteshwer. Sequence analysis of structural and non structural genes of BPXV showed more than 96% sequence identity (over 96%) with vaccinia virus (VACV) at the nucleotide and amino levels. PCR and PCR-RFLP based diagnostics for BPXV were devised and evaluated successfully for their diagnostic efficacy. An attenuated BPXV vaccine was developed and experimental trials in buffalo calves confirmed its safety and protection against challenge infection. Field trial of the vaccine is underway.

Animal Biotechnology R&D support was continued for the development of novel animal vaccines and diagnostics, characterization of indigenous breeds, development of newer reproductive techniques, utilization of animal byproducts etc. A new multicentric programme on various aspects of animal nutrition was also initiated. During this period, 36 new projects were considered for financial support and so far 26 projects have been funded. 14 completed and 44 ongoing projects were reviewed for their outcome and successful progress. Currently, 85 projects are under implementation. Significant achievements are

At GBPUAT, Pantnagar, molecular characterization of fowl adenoviruses associated with Hydropericardium syndrome (HPS) and Inclusion

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body hepatitis (IBH) in domestic fowl were studied with an aim to develop a cell culture based vaccine. Both the viruses were successfully isolated in chicken embryo liver (CEL) and chicken kidney (CK) cell lines. Cytopathic effects (CPE) produced by HPS virus were characterized, which was evident from the first passage itself. All three HPS isolates were serotyped. Viral DNA of all 3 HPS isolates extracted from infected CEL revealed the presence of 1223 bp and 1190 bp PCR products.

self replicating gene vaccine for foot and mouth disease virus(FMDV). Two vectors viz. one for cellular response carries a CMV promoter and polyadenylation signal and another vector for humoral immune response contains eEF1 promoter, Sindbis virus polymerase gene and secretory and anchoring signals were constructed. The linked polyvalent protein genes of FMDV serotype A, O and Asia 1 were cloned into the vectors and the presence of the insert was confirmed by restriction enzyme digestion. The constructed DNA vaccine was injected into guinea pigs and the immune response was compared with the naked DNA vaccine. This approach of constructing self replicating DNA vaccine for humoral response is the first report in FMD. At NII, New Delhi, attempts were made to develop recombinant ε-toxin and DNA based vaccine against Clostridium perfringens. Gene encoding epsilon toxin of C.perfringens type D has been cloned in pQE60 expression vector. High level expression and single step purification of recombinant epsilon toxin have been achieved. Toxicity of recombinant epsilon toxin was tested in MDCK cells and has been found to be toxic. The gene for the toxin has been cloned in order to produce epsilon toxin independently or in fusion with GFP aimed at the development of a DNA based vaccine. Recombinant constructs have been confirmed by DNA sequencing and authenticated by the fluorescence analysis of GFP in CHO-K1 cells transfected with the recombinant constructs.

In an effort to develop a candidate vaccine against Haemonchus contortus, at IVRI, Izatnagar, a 66 kDa antigen (p66) secreted by adult worms was characterized as monocyte inhibiting factor. Challenge experiment in goats showed reduction in worm burden that ranged between 34% (lowest reduction) to 70% (highest protection). The protection reported to be mediated by antibody as immunized animals had elevated levels of anti-p66 antibodies whereas no cell mediated immune response was observed. Characterization of Haemonchus CalR indicates that it may act as an anti-coagulant. A 55 kDa protein that inhibits neutrophil and monocytes functions also appeared to down regulate T lymphocyte but not B cells.

At IVRI, Izatnagar, a recombinant Brucella antigen (L7/L12 ribosomal protein) was developed and found to provide considerable protection to immunized mice. To increase the efficiency of this DNA vaccine, two additional immunodominant antigens viz. Cu-Zn Superoxide dismutase (SOD) and P39 antigen were also cloned and expressed. DNA vaccine constructs of these genes have been made and three antigens (L7/L12, SOD and P39) were fused to generate a chimeric antigen. Recombinant proteins as well as the DNA vaccine constructs were purified and produced in large scale. Antisera were also raised against the recombinant antigens and their protective efficacy tested in laboratory animals.

At IVRI, Bangalore, attempts were made to develop a

In a joint programme implemented at MVC, Chennai

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and IISc, Bangalore, a recombinant baculovirus expressing the nucleocapsid protein of peste des petits virus(PPRV) was generated and used for detection of PPRV-specific antibodies in infected animals. Diagnostic methods were developed for PPRV antigen and antibody detection. The lateral flow method for qualitative detection of PPRV antibody was found to be ideal for use as a fieldbased kit. For quantitative estimation of PPRV antibodies, single serum dilution ELISA using precoated plates was developed using self-designed modules. An antigen competition ELISA was also developed as a 'lab-based' second tier test for PPRV antigen detection. Some of these tests have been validated at IVRI, Mukteswar and efforts are underway to transfer the technology.

flagrans produced profuse chlamydospores that 0 germinated easily at 26 C and could survive passage through the guts of both monogastric and polygastric animals. Efforts were made to develop diagnostics for leptospira, at IVRI, Izatnagar and TANUVAS, Chennai. An immunodominant surface outer membrane lipoprotein, LipL41, was targeted as an antigen for serodiagnosis using ELISA. The protein gene from various serovars was amplified by PCR using a set of designed primer and the gene from Canicola serovar was cloned and expressed in E. coli DH5 cells. The purified recombinant protein was found to be seroreactive using western blotting and ELISA. The 41 kDa protein and the 32 kDa recombinant protein were used for serodiagnosis of leptospirosis in various animal species. As compared to the conventional microscopic agglutination test, the recombinant proteins based ELISAs were found to possess 100% sensitivity.

Phage display technique was successfully used as an alternative to hybridoma to produce mono-specific antibodies against recombinant gag antigen of Bovine immunodeficiency Virus (BIV) at HSADL, Bhopal. Five anti-gag recombinant antibodies (RAbs) from two gag specific hybridoma clones were developed from gag-immunized mouse. One of the high reacting anti-gag RAb was successfully used in competitive inhibition ELISA. RAb based ELISA showed high sensitivity with reference to positive serum in comparison to MAb based ELISA. Using mRNA from mouse immunized with recombinant NP antigen of Avian Influenza virus(AIV), five anti-NP RAbs were generated. Preliminary results with one of the anti-NP RAb has shown encouraging results for developing a RAb based competitive ELISA for detection of antibodies to nucleoprotein of AIV in chicken sera.

Molecular studies of pestivirus infections of small ruminant was done at HSADL, Bhopal. Prevalence rate of pestivirus antibodies was 26.1% in sheep and 29.1% in goats while it was 7% in sheep and 2.8% in goats for border disease virus (BDV) antibodies tested by ELISA. pestivirus antigen was detected in 11.8 % sheep and 8.7 % in goats providing serological evidence of infection first time in Indian small ruminants. A RT-PCR assay was standardized for specific detection of border disease virus and a nested PCR was developed for differentiation of BDV, bovine viral diarrhea virus (BVDV) 1 and BVDV 2 using 5' UTR primers. Animal Reproduction: Embryo Transfer (ET) Technology and related areas :

Biological control of parasitic gastroenteritis by nematode trapping fungi in livestock was studied at Veterinary College, Anjora. Two promising fungal isolates viz. Arthrobotrys oligospora and Duddingtonia flagrans were studied for their ability as bio-control agents using growth assay, predatory activity, germination potential and ability to survive passage through the guts of domestic livestock. A. oligospora was better than D. flagrans in terms of growth and predatory activity. However, only D.

Isolation, characterization and in vitro development of buffalo preantral follicles for embryo development was attempted at IVRI, Izatnagar. Mechanical methods of isolation were found to be better as compared to the enzymatic isolation as more number of follicles of different sizes were recovered in short period. Buffalo preantral follicles were cultured with

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goat caput epididymis has been attempted to study its role in the sperm maturation function. A 66 kDa protein with high affinity for binding both testosterone and estradiol was isolated and purified. Thus a unique system exists where one receptor binds two hormones. A primary culture of caput epididymal epithelial cells showed the presence of plasma membrane localized binding sites for testosterone.

FSH, IGF-1 and cumulus granulosa monolayer in control medium during in vitro culture showed their better survival.

Embryo transfer technology in equines was standardized at EBS, Babugarh and an ET foal was born. A total of 58 flushings were made from 24 mares and 47 embryos were collected. At present, 2 mares of more than 250 days, 4 mares of more than 190 days and 2 mares of less than 190 days are held as pregnant. Attempts to preserve native Ongole breed by producing embryos through ET technology were made at Lam farm, Guntur. So far, 165 embryos were produced through non-surgical collection and 96 were found to be transferable embryos. Significant improvements in the yield of transferable embryos were recorded. A new protocol to induce synchronous ovulation and to enhance embryo quality was studied which focused on emergence of new follicular waves using steroid hormones prior to superovulation treatment. Signaling mechanisms controlling the follicular differentiation in rat were studied at Delhi University, Delhi. Protein-protein interactions involving SH3 domain of src tyrosine kinases was used to study novel proteins involved in proliferation and differentiation of granulosa cells. Differential gene expression profiling by DDRT-PCR during folliculogenesis helped to identify important genes. Cloning and expression studies have given lead for furtherance of the study for human cases of infertility and ovarian diseases. Transgenics and Cloning Spermato- transgenesis technique for the production of transgenic mice has been standardized at NII, New Delhi. EGFP and IGFBP6 mammalian genes having viral or mammalian promoters were injected in the

At RGCB, Thiruvanathapuram, purification and characterization of testosterone specific receptor of

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intra seminiferous tubular space of the testis and electroporated males were mated with the wild type females. The transgene was successfully propagated to F3 generation as evidenced by PCR as well as Southern blotting analysis of genomic DNA. In another study for the development of cloned embryos, enucleation technique in mice and buffalo has been standardized using various somatic cells. The cumulus cells were found to be best suitable for production of cloned embryos in both the species. Transgenic cloned mice were successfully produced r using granulosa cells from Neo mice and its presence in cloned embryos were analyzed by PCR. Non GFP as well as GFP expressing cloned buffalo embryos were also produced.

fragments from 167 (59%) primers pairs could be amplified and sequenced. To increase the efficiency of buffalo EST development, a database of primer pairs for 1960 potential buffalo ESTs was created. Out of these, 1023 ESTs have been validated. At present, more than one thousand DNA markers (microsatellite and ESTs) are available for creation of a radiation hybrid map of buffaloes. Characterization and mapping of fertility related hormone / hormone receptor genes in Buffalo was attempted at NDRI, Karnal. The full coding part of the buffalo FSHβ gene corresponds well to the GenBank reported sequence of buffalo. The mutation study revealed a total of eight substitutions of nucleotides in the FSHβ gene coding part. Out of these, five pertained to amino acid changes and three were silent mutations. The FSH receptor gene was sequenced and conserved motifs and Nglycosylation sites also corresponded with most of the other mammalian species. A total of nine nucleotides substitutions were observed in the coding part of FSH receptor gene and out of these six mutations were reported to be silent.

At IISc, Bangalore, EGFP-expression pattern was evaluated to study transgene expression during embryo development in mice during the entire period of oogenesis and spermatogenesis. In the EGFPtransgenic female, oocytes exhibited green fluorescence during the entire meiotic maturation process. EGFP trangene was expressed during fetal oogenesis and the expression of the transgene persisted in embryos, which inherited the transgene. EGFP-expression was detected in 100% oocytes and embryos.

At NBAGR, Karnal, cDNA library from buffalo mammary gland tissues (lactating & non-lactating) was screened for maximally expressed genes. Sequence information for 764 buffalo mammary gland ESTs from lactating and non-lactating stages was generated and charaterized. Out of these 222 EST sequences were submitted to GenBank/ dbEST data-base. Complete sequence characterization of two important buffalo mammary gland derived genes viz. Osteopontin and Beta-casein genes was carried out and compared with other livestock species. Buffalo specific cDNA probes with respect to mammary derived genes were generated to screen the redundancy and expression profile of individual genes in mammary gland cDNA library.

Buffalo Genomics: A multicentric programme on Buffalo Genomics was initiated with an emphasis on identification of genes of economic importance. Significant achievements are as follows : Radiation hybrid mapping panel and DNA markers for creation of buffalo genome map is being developed at CCMB, Hyderabad. So far, 81 radiation hybrid clones of buffaloes with the background of Chinese hamster genome were created. Besides this, 598 microsatellite markers were developed which would be used for mapping a radiation panel. An additional, 74 putative hybrid clones were isolated and are being tested for contents of buffalo genome. To develop expressed sequence tags(EST) markers for buffaloes, 284 cattle EST primers pairs on buffalo genomic DNA were tested. Out of these, unique

Genes regulating embryonic survival and maternal recognition of pregnancy in buffaloes were characterized at IVRI, Izatnagar. The complete coding sequence of Uterine Milk Protein (UTMP) cDNA of 1309 bp was cloned and characterized. The

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buffalo UTMP cDNA exhibited 94.5, 88 and 88.6% homology with cattle, sheep and goat respectively. Genomic sequence (>4 kb) of buffalo ghrelin gene was characterized and this is the first report in any non-human species. One of the 12 variants of buffalo interferon-tau (IFNT) was expressed in prokaryotic system. The identity of the recombinant buffalo interferon-tau protein was confirmed using western blot analysis. Expression profile of osteopontin (OPN) gene in uterine tissues of buffalo during different reproductive phases has also been analyzed using Real-time PCR.

Consequently, the gene frequency for A and B allele was calculated as 0.907 and 0.093 respectively. Monomorphic pattern was reported in BuLA-DRB3 and TCR-Zeta genes with different REs. At NBAGR, Karnal, identification of single nucleotide polymorphisms (SNPs) in quantitative trait loci was studied for diversity analysis of buffaloes. Quantitative trait loci for milk traits in cattle were selected for designing the primers for amplification of regions of selected genes in buffaloes. The primers were subjected to a panel of buffalo samples (drawn from 6 different breeds) for amplification and SNP studies. A total of 32 of the selected 36 genes were amplified in buffaloes and sequenced for detection of SNPs.

Molecular characterization of Satellite Tagged Transcribing Sequences (STTS) in buffalo genome was studied at NII, New Delhi. A total of 43 known and novel genes have been accessed showing differential expression employing minisatellite associated amplification (MASA) with cDNA from different tissues of buffalo. Characterization of two species of BamHI repeat showed that only one is localized in the centromeric regions of all the chromosomes whereas the other one remained confined to the centromeric regions of acrocentric chromosomes. Isolation of 2973 bp full length c-kit cDNA from testis and other tissues showed specific nucleotide changes resulting in novel truncated peptides. Phylogenetic analysis of these sequences showed unique tyrosine kinase domain in buffalo. Highest expression of c-kit in testis and its mRNA transcripts in sperm substantiate its predominant role in spermatogenesis besides other pleiotropic functions.

Animal Nutrition: A multicentric program on Animal nutrition was initiated during the current year and projects were supported on various aspects viz. to study genetic manipulation of rumen microbial ecosystem for improving productivity and protecting environment, Identification of genes for better feed conversion of agro-byproducts (lignin, oil cakes, oil meals, cellulose etc.), microbial feed additives (Probiotics) for enrichment of feed quality, use of fibrolytic enzymes in feed to improve digestibility and nutritive value and genetically modified silage bacteria. Effect of growth factors in augmenting reproductive efficiency in local and crossbred pigs of Assam was studied at College of Veterinary Science, Guwahati. Lactobacilli strain, isolated from the fecal samples of healthy piglets, were characterized and found to have antimicrobial activity against enteric pathogens. Further studies on Lactobacilli as probiotics application with feed is in progress.

Characterization and mapping of selected genes known to control immune response in water buffaloes was done at IVRI, Izatnagar. Polymorphism studies of ITGB2 (CD18), BuLA-DRB3 and TCR-Zeta (CD3Z) genes known for their immunity were carried out in 152 Murrah and Bhadawari buffaloes by PCR-RFLP. Polymorphism in ITGB2 gene was reported in the 570 bp region of gene with Msp I RE digestion which revealed two genomic patterns. The genotypic frequency was observed to be 0.909 and 0.091 for AA and BB genotype respectively whereas AB genotype could not be found.

DBT Annual Report 2006-07

Molecular techniques for identification of meats of different animal species were developed at GBPUAT, Pantnagar. A simplex PCR technique was optimized for the identification of sheep, goat, cattle, pig, horse and chicken. This technique was found to be of immense value for the detection of adulterated raw meat samples. An attempt was also made to

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develop a unique technique for meat identification i.e. multiplex PCR, which efficiently differentiate six species in a single reaction as against multiple reaction in simplex PCR.

domesticated avian species were undertaken at CARI, Izatnagar. Red jungle fowl (RJF) showed genetic similarity with all the chicken breeds except Kadaknath (KN), with whom it showed least genetic similarity. Very few changes were observed in IL-2 as well as IFN- promoter sequence in RJF in comparison to chicken. Based on nt sequence comparisons for IL-2 as well as IFN- gene, RJF showed maximum genetic similarity of with chicken, followed by turkey and quail.

Animal Byproducts : Efforts were continued to develop biomaterial of bovine origin for reconstructive surgery in animals at IVRI, Izatnagar. A protocol for the production of completely acellular tissue metrics by specifically removing cellular components has been standardized. In-vitro cell cytotoxicty of the biomaterials extract was studied using chick embryo fibroblasts. None of the biomaterial had shown any cytopathic effect with in 48 hours of post addition of extract in the cell culture.

At IVRI, Izatnagar, PCR and nested PCR techniques have been standardized for developing diagnostics against chicken anemia virus(CAV). One of the CAV isolate was propagated for restriction endonuclease (RE) study. Preliminary study indicated the prevalence of CAV in difference states of the country on the basis of CAV DNA detection by PCR in field samples and PCR-RE analysis revealed variation among the CAVs circulating in the poultry flocks of the country.

Structural and functional aspects of the 3 D Scaffold of bovine origin for cardiomyocyte culture were studied at SRMC and CLRI, Chennai. Normal bovine heart tissue was decellularized and its criss cross trabecular architecture resemble to honey comb with cavities varying in size. Fibrillar Type I/III collagen found in the heart provides structural scaffolding for cardiomyocytes and coronary vessels. The 3D sponge fabricated from bovine collagen Type I/III has a hexagonal appearance. Hence it can be expected to mimic the extracellular matrix of the heart for cardiomyocyte culture invitro. Poultry : Various aspects on disease resistance, genomics and diagnostics were pursued. Three generations of six immunodivergent lines of chicken were produced to study disease resistance at CARI, Izatnagar. Highest genetic distance (0.453) among these lines was found between high SRBC and high index lines. AFLP analysis of these lines with two primer sets revealed total 42 recordable bands ranging from 23 bp to 456 bp. The phylogenetic tree based on AFLP and RAPD-PCR showed linewise clustering. Genotype of IFN- promoter (-318G) was found to be associated with immune response to NDV.

Immunofluorescent infected with CAV at 48 hr PI. FA stain x 400

Aquaculture & Marine Biotechnology R&D Efforts continued for increasing productivity, development of new vaccines and diagnostics, molecular characterization of fish stock, cell culture, molecular signaling, development, bioactive molecules and immunostimulants etc.

Comparative genomic studies on wild and

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Biosurfactant

for Penaeus monodon and Fenneropenaeus indicus showed that the raceway technology found suitable for successful survival of over 80%. Sustainable water quality in raceways was possible with the addition of phytoplankton (Chaetoceros calcitrans) and yeast based fermented products. Prototype developed was successfully adopted for sustaining water quality management and raceways operations for high stocking densities using bioremediaiton approach in aquaculture. A training course on raceway-based shrimp farming technology was conducted to disseminate the technology to shrimp farmers. The protocol developed on raceway technology was under transfer to a private entrepreneur, M/s. Pancham Aquaculture Pvt. Ltd., Mumbai.

Work on screening of marine Acinetobacter genospecies for production of biosurfactant/s, purification and antimicrobial activity was continued at Pune University. The Acinetobacter strain was confirmed and DNA transformation was carried out. Bioemulsifier production by A. haemolyticus grown on cheaper substrates viz. molasses and whey reported the best medium for the bioemulsifier production. Highest yield of bioemulsifier was obtained with A. junii with 2.5 gm/lit. Marine Acinetobacter haemolyticus was found to produce antimicrobial compound and bioemulsifier and showed wide spectrum activity against Gram positive and Gram negative bacteria and also inhibited sporulation in Tramentia serialis, Tramentia spp, Alternaria solani, and Ustilago medis and human dermatophyte Microsporum audouinii.

Marine Enzymes Over expression of engineered superoxide dismutase enzyme in marine cyanobacteria for bioremediation was undertaken at Bharathidasan University, Tiruchirappalli. Fifteen species were screened for their ability to degrade azo dye, Orange G. Spectral studies showed a 14% reduction in colour in sulphur deficient condition. SOD profiles of these species revealed the presence of Ni-SOD. Fe/Mn SOD of Thermosynechococcus elongatus BP-1 was cloned in a pET vector system. Work on marine cyanobacterial plasmid for vector construction and plasmid profile was undertaken. Characterization of superoxide dismutases (SODs) from halophiles was pursued at Institute of Life Sciences, Bhubaneshwar to understand possible role of the enzyme in salt tolerance. The aquatic plants, N. gramenia and Chlorella, showed greater tendency to increase the activity levels of SOD than catalase, while the terrestrial plant, S. maritima showed greater increase in the activity of catalase than SOD. Significant increase in enzyme activity in test plants upon challenge with salt eloquently indicated the possible involvement of enzymes in salt tolerance.

Biosafety and Water Quality Studies were continued on occurrence of human pathogenic viruses in coastal marine waters and their detection using molecular techniques at the College of Fisheries, UAS, Mangalore. From the oyster, clam and water samples analyzed, fecal coliforms were isolated. 88% of shellfish and 84% water samples showed the presence of coliphages. Enteroviruses were detected in 32.29% shellfish samples tested and seasonal variation was observed in the occurrence of enteric viruses. The samples tested were negative for Norwalk like viruses and rotaviruses. The study revealed the possible use of coliphages as fecal indicators for detecting enteric viruses. Raceway for Shrimp Production A project on the development of a prototype for raceway based shrimp production technology was continued at the Fisheries College and Research Institute, Tuticorin. Nursery raceway trials conducted

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Bioactive Molecules

Epizootic Ulcerative Syndrome was identified and its role in virulence and pathogenesis was studied. 21kb plasmid appears to be an important virulence marker for A. hydrophila. Immunization studies with plasmid cured isolates lead to significant increase in serum immunoglobulin levels and conferred significant protection when challenged with wild type A. hydrophila as well as A. sobria and A. salmonicida. Transformation of the plasmid helped the bacteria to regain the virulence attributes.

Various aspects on development of bioactive molecules from marine organisms were continued and over 7000 marine bacteria were screened for antibacterial activity. Eighteen isolates showed antibacterial activity against both gram positive and gram negative bacteria. Sand anemones from Karnataka coast were found to have anticancer activity. Purification, characterization and synthesis of bioactive molecules produced by marine Pseudomonas were undertaken for screening antiviral and anticancer agents. Batteries of bacteria isolated from the deep sea water were collected from Bay of Bengal, of the Andaman Coast and a few of them showed the potential of producing anti-cancer / anti-inflammatory bioactive molecule(s). Production of highly unsaturated marine lipids, ester and specific bioactive analogues from fish for medical applications was undertaken at Indian Institute of Chemical Biology, Kolkata. Cell culture and animal model for the testing of the highly enriched EPA and DHA were developed using an animal model for in vivo studies and studies on kidney damage, diabetes development and cataract formation were conducted using different concentrations of phospholipids enriched with EPA/ DHA. Purified phospholipid fractions were reported to stimulate sperm motility and increase in flagellar movement. Phospholipid at 50mg/ ml level caused the highest degree of motility activation. Information of mangroves having medicinal values was collected from Sundarban Estuary in a collaborative project funded at IICB, Vivekananda Institute of Biotechnology and Vishwa Bharti University. A survey was conducted on the traditional uses of the mangrove plants in different ailments.

Alternate fish feed supplement Applicability of cell-bound phytase of yeast Pichia anomala in improving growth of freshwater fishes, rohu and magur, was pursued. A protocol was standardized for yeast cultivation and the growth conditions were optimized. When the fingerlings of Labeo rohita (rohu) were fed with fish feed supplemented with yeast phytase, specific growth rate (SGR) and feed conversion ratio (FCR) were better than the control. A decrease in phosphate and ammonia excretion was observed, suggesting better utilization of phosphate and protein in the fish fed with yeast phytase. Supplementation of cane molasses medium with urea was found to be better for achieving good growth and phytase production. Growth conditions were optimized for yeast cultivation using air-lift fermenter leading to higher biomass and phytase production. Neuro-peptide Synthesis Cone snails from Indian coasts were collected and venoms characterized using mass spectrometry. Peptides obtained from crude venoms were studied for structural determination using 'de novo' sequencing by mass spectrometry. Novel conus peptide sequence derived were further confirmed by synthesizing the corresponding peptide and comparing the fragmentation pattern of natural and synthetic peptides. Construction of cDNA libraries for conus venom was undertaken. Three putative

Plasmid immune response A collaborative project was implemented at CCMB, Viswa Bharti University and North Bengal University. Plasmid gene involved in the pathogenesis of

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conotoxins along with the posttranslational modifications were identified from Conus achatinus sp. Experiments were undertaken to identify the biological activity of conus peptides and three novel toxins were found to target voltage-gated ion channels. The 3D-structure has been determined using NMR resulting a cysteine knot motif.

also identified, cloned, sequenced and expressed in E. coli. The sequence has been deposited in GenBank. Recombinant proteins were found to be immunogenic in Labeo rohita and induced protective immune response. On challenge, the immunized fish showed relative percentage survival of over 60 indicating promise in development of commercial vaccine.

Bioreactor

Bacteriophage therapy in improvement of

A microbial analysis of marine sediments was undertaken to develop an anaerobic consortium for wastewater treatment, at RRL, Thiruvananthapuram. Qualitative and quantitative analysis of bacteria, archaea, protozoa and micro-metazoa in anaerobic marine sediment were done. Fluorescent labeled 16S/23S rRNA targeted synthetic nucleotides were used for analyzing the bacteria and archaea. Protocol for whole cell Fluorescent In Situ Hybridization (FISH) technique was standardized. Different batch experiments were conducted to evaluate the activity of protozoa and micro-metazoa under anaerobic and organic rich saline conditions. The organic and nutrient load in shrimp processing wastewater was quantified and a Laboratory scale UASB type bioreactor for wastewater treatment was studied. This is expected to lead to development of a microbial based treatment system for seafood industrial discharge.

Expression of recombinant ompTS protein. Lane 1- Molecular wt marker (Pmw m); Lane 2- Non recombinant M15 E. coli cells without IPTG; Lane 3 Non recombinant M15 E. coli cells with IPTG; Lane 4 Recombinant M15 E. coli without IPTG; Lane 5 Recombinant M15 E. coli with 1 mM IPTG; Lane 6- Purified ompTS protein.

Bacteriophage shrimp larvae therapy in improvement of shrimp larvae Luminous bacterial disease is a leading cause of Luminous bacterial disease is a leading cause shrimp larval mortalities in hatcheries. of shrimp larval mortalities in hatcheries. Development of resistance to antibiotics by Development of resistance to antibiotics by luminous luminous bacterial disease is a major hurdle in bacterial disease is a major hurdle in tackling the tackling ofthe problem of disease. luminous bacterial problem luminous bacterial At College of disease. At College several of Fisheries, Mangalore, Fisheries, Mangalore, lytic bacteriophages several luminous lytic bacteriophages against luminous against Vibrio harveyi have been isolated Vibriocharacterized harveyi have isolated and and at been both morphological and molecular atlevel. lytic abilityand of characterized both The morphological these bacteriophages has been over molecular level. The lytic tested abilityagainst of these bacteriophages has been tested against over

Vaccine development for fish Aeromonad septicaemia is an important disease problem affecting warm water aquaculture. Due to antigenic heterogeneity among motile aeromonads, vaccine development has been a problem. Conserved outer membrane proteins of aeromonads were evaluated as candidates for vaccine development. Gene coding for the outer membrane protein ompTS was cloned, sequenced and expressed in Escherichia coli. A new gene coding for outer membrane protein of A. hydrophila, omp48 was

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200 isolates obtained from different parts of the world including Asia, Australia and South America. Using bacteriophages having a broad s p e c t r u m o f a c t i v i t y, t e c h n o l o g y f o r bacteriophage therapy of luminous bacterial

disease in shrimp larvae has been developed. In hatchery trials, bacteriophage therapy led to over 80% survival in larvae while antibiotic therapy led to 40% and untreated control showed less than 20% survival .

Electron microscopic images of lytic bacteriophages (Phages A-c and E-F belong to siphoviridae and phage D belonds to myoviridae).

Hatchery trial of bacteriophage therapy- survival of Penaeus monodon larvae infected with luminous bacteria under various conditions

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Oligonucleotide probe for monitoring vibrio counts in hatcheries

Genbank. Molecular characterization of antimicrobial peptides in shrimp and their enhanced production was undertaken, which showed broad antimicrobial activity. Studies on novel drugs targeted at Vibrios from marine microorganisms were undertaken and an antivibrio compound produced by Pseudomonas was identified as pyocyanin. A bioprocess technology for large-scale production of marine yeast Candida sake as single cell protein was programmed and executed. An isolate was identified as the source of alkaline protease for industrial applications. The crude enzyme was partially purified and found to have blood stain removal property from fabrics in the absence of any detergent. A training programme in Marine Biotechnology was initiated.

Vibrio sp including luminous V. harveyi, V. vulnificus and V. parahaemolyticus are major bacterial pathogens of shrimp larvae. Monitoring Vibrio counts in hatchery water and larval samples would be an important aspect in health management. To overcome the problem and get a realistic estimate of Vibrio counts, an oligonucleotide probe binding to a region of gyrB gene of all Vibrio spp has been designed. When labeled with alkaline phosphatase, this label could be used for enumeration of Vibrio spp in hatchery environments by colony hybridization technique.

Genetic marker for population of cultured animals would be important for genetic selection of populations with commercially important traits like fast growth and disease resistance. Using microsatellite enrichment techniques, two DNA microsatellites were identified in genome of freshwater prawn, Macrobrachium rosenberii . The sequences of these microsatellites, (CA)13 repeats, (GA)38 and (GA)30 repeats have been deposited in GenBank with accession number DQ793216 and DQ 793215. Primers binding to flanking region of these microsatellites were designed and evaluated in population of M. rosenbergii from Kerala and Karnataka. The discriminative ability of microsatellite was very good with heterogeneity index of 0.27 ± 0.32 and 0.92 ± 0.29 respectively. Shannon index for these microsatellites were 0.6161 ± 0.2310 and 0.6619 ± 0.2276 respectively. Organ Development Aspects of in vitro organ development and differentiation were undertaken through regeneration and stem cell biology to search for novel biomolecules from hitherto untapped sources that will be useful in the differentiation and regeneration of vertebrate organs. Preliminary studies showed that perivitelline fluid (PVF) of Indian Horse Shoe Crab influences early vertebrate development. Chick embryo explants cultured in vitro were used as a

Colonies of Vibrio spp hybridizing with alkaline phosphatase labeled oilgonucleotide probe.

Genetic characterization of marine organisms Studies on genotypic characterization of antagonistic and detritus degrading bacteria based on their 16S rDNA sequence ere carried out and partial sequences of 6 organisms were deposited in

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unidentified cells.

model. The seventh fraction of the PVF contained cardiac development promoting activity. Sequencing and identification of active protein molecules was done. Due to possible relationship between cardiac development and angiogenesis, preliminary experiments to assess the angiogenic potential of PVF were carried out. Transcriptional studies were carried out by quantitative real-time PCR for the markers Noggin (Signalling molecule, antagonistic to BMP2, recently shown to be involved in cardiomyogenic differentiation). There was overall increase in the expression of these markers in chick embryo of the 5th stage of development. At 7th Stage of development there was a decrease in their overall expression and this trend was reversed in stage 10th of development of chick embryo. Perivitelline fluid of the eggs of the horse shoe crab was fractionated and 7th fraction was found to possess early cardiac development promoting activity.

Extensive CPE with multiple vacuolation (arrow) in SISK cells infected with nodavirus

Shrimp Genomics Expression of immune function associated proteins from shrimp Penaeus monodon was studied at College of Fisheries Mangalore, Central Institute of Brackishwater Aquaculture Chennai, Anna University Chennai and National Institute of Oceanography Goa. The gene coding for a c-type lysozyme from P. monodon was amplified by reverse transcriptase PCR after the animals were challenged with luminous bacterial pathogen, Vibrio harveyi. The amplified DNA fragment was cloned in pEXP5NT/TOPO expression vector and recombinant plasmids were transformed into competent BL21 Escherichia coli cells. Lysozyme was over expressed by induction with 1 mM IPTG. The recombinant protein was found to be active against both gram positive bacteria (Micrococcus luteus) and garm negative bacteria (Vibrio harveyi). The commonly used lysozyme standard from hens egg white is active only against gram negative bacteria.

Cell Lines from Seabass A project was continued to develop cell lines from sea bass (Lates calcarifer). Kidney and spleen cell lines from sea bass were established and designated as SISK and SISS. These lines have been deposited in NCCS, Pune. The established cell lines have been used for isolation of nodavirus which is responsible for viral nervous necrosis (VNN) in sea bass larvae. The nodavirus was isolated from infected sea bass (Lates calcarifer) larvae during a massive outbreak in sea bass hatcheries using these cell lines. The typical cytopathic effect of nodavirus was characterized by multiple vacuolations was observed in SISK. Embryonic stem cell cultures were initiated from blastula stage embryo of sea bass. The sea bass embryonic cells were small, round or polygonal with a big nuclei and sparse cytoplasm in shape. Differentiation of embryonic cells into variety of cell types was induced by all-tansretinoic acid and differentiated into various cell types, including neuron-like cells, oligodentritic, muscle cells and

A similar collaborative project was undertaken at Cochin University of Science and Technology on development and application of CMG family recombinant hormones, their antagonists and RNAi technique for induced maturation and spawning of Penaeus monodon. Sequence data available with the genbank was utilized to design primers to get ORF amplified. The amplified product showed a 98%

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similarity with the already reported Penaeus monodon Crustacean Hyperglycemic Hormone 1 precursor gene complete cds. Primers for amplifying CDS of GIH gene were designed from the conserved region of the available GIH gene sequence of Lobsters and Metapenaeus sp. Total RNA isolation from the eyestalk of shrimp was standardized and the method yielded sufficient amount of good quality total RNA for further purification and analysis.

proteins from Anopheles and Drosophila and not with the classical lysozymes. At the Central Sericultural Germplasm Resource Centre (CSGRC), Hosur work has been carried out to identify variable regions existing in the diapause (NB4D2) and non-diapause (Pure Mysore) silkworm (B. mori) races and to corelate with diapause gene expression. High homology percentage between the diapause and the non-diapause indicates that there is no difference in the genomic organization of the diapause gene in both silkworm races. Expression pattern of the diapause related genes is being studied.

Patents a. PAT/4.9.2/06052: Biocontrol of luminous bacteria in shrimp hatcheries using bacteriophages

A network project involving five institutions: CDFD, Hyderabad; Seribiotech Research Laboratory, Bangalore; Central Sericultural Research and Training Institute, Mysore; Andhra Pradesh State Sericulture Research and Development Institute (APSSR&DI), Hindupur; and Karnataka State Sericulture Research and Development Institute, Bangalore continued with a view to identify baculovirus resistant strains and DNA markers linked to baculovirus resistance in silkworm (Bombyx mori). Screening of silkworm germplasm for baculovirus resistance has resulted in identification of three strains each of bivoltine and multivoltine. Out of these, most resistant strain has been used as a donor parent to cross with the most susceptible strain to raise a mapping population, which is being used to map the markers that are polymorphic between parental strains. In a collaborative project at the University of Delhi, Delhi and Central Muga Eri Research & Training Institute, Jorhat, morphometric data were collected, compiled for 14 populations of muga silkworm (Antheraea assama) and maintained at Jorhat, Assam. Seventeen microsatellite primers provided by CDFD, Hyderabad and other repetitive sequence derived primers were successfully tested by PCR with A. assama DNA. A distinct polymorphic band of 1.4 kbp size was successfully cloned and sequenced from A. assama. Work is underway to screen population with more molecular markers and statistically analyze data for population genetic diversity.

b. PAT/4.9.15/06053: Oligonucleotide probe for detection and enumeration of Vibrio spp. in aquaculture systems Technologies transferred - Technology for Biocontrol of luminous bacteria in shrimp hatcheries has been transferred to Mangalore Biotech Laboratory Seribiotechnology A programme on application of biotechnology for improving the productivity and enhancing the quality of silk alongwith the improvement of host-plants continued during the year. A brainstorming session on application of biotechnology for tasar culture was organized in collaboration with Central Silk Board (CSB) during November 17-18, 2006 at Central Tasar Researh and Training Institute, Ranchi. Significant achievements during the year are summarized below:Development of better races of silkworm for increased productivity Lysozyme-like proteins (LLPs) identified in Bombyx mori and Antheraea mylitta exhibited a broadspectrum anti-bacterial activity jointly at Centre for DNA Fingerprinting and Diagnostics (CDFD), Hyderabad and National Institute of Immunology (NII), New Delhi. Phylogenetic analysis clustered LLPs with some uncharacterized lysozyme-like

DBT Annual Report 2006-07

Studies have been continued on genetic analysis of

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quantitative trait loci (QTL) for cocoon weight, cocoon shell weight and identification of DNA markers linked to the QTLs in the silkworm (B. mori) jointly at Andhra Pradesh State Sericulture Research and Development Institute, Hindupur and CDFD, Hyderabad. Two silkworm lines with lower (Nistari) and higher (CSR2) quantitative traits for cocoon weight and cocoon shell weight were selected as initial parents for raising F-1 generation. Using the F1 population, eight back crosses (four each as direct crosses and reciprocal croses) were made to develop mapping population with linkage disequilibrium. In addition, two F-2 generations were also developed for raising bulked segregant population. From each cross, individual cocoons (both male and female) were assessed for the desired traits. A network project on phylogeography of tropical tasar (Antheraea mylitta) and muga silkworm (A. assama) has been continued involving three institutions: Seribiotech Research Laboratory, Bangalore, Central Tasar Research and Training Institute, Ranchi and Central Muga Eri Research and Training Institute, Jorhat. Microsatellite analysis carried out in muga silkworm populations indicated genetic variability in hill populations in comparison with the plain area populations. Tasar silkworm populations exhibited intra-and inter- population vaiability when amplified with SSR anchored marker system.

Development of control measures for major diseases of silkworm At Central Sericultural Research and Training Institute, Mysore, a Bombyx mori gene that code for antiviral protein has been partially characterized. The partial sequence of gene (644 bp) encoding the protein showed homology with NADPH oxidoreductase and xanthine dehydrogenase proteins. Silkworm larvae fed on mulberry leaves supplemented with Lactobacillus plantarum, a probiotic showed significant increase in weigh of larva, cocoon and pupa; including shell ratio and pupation rate as compared to control groups at the BAIF Development Research Foundation, Pune. Feeding of silkworm larvae (III moult) with L. plantarum and Sporobolomyces roseus and then treating with Bm NPV showed significant increase in larval weight and survival percentage as compared to controls. Effect of probiotics varied among different silkworm races when treated with Bm NPV. Studies have also been initiated on epidemiology, spatial and temporal dynamics of diseases of muga silkworm (A. assama) alongwith the identification and characterization of their causal agents jointly at Assam Agricultural University, Jorhat and Central Muga Eri Research and Training Institute, Jorhat. Silkworm Genome Programme Under the International Consortium on Lepidopteran genome project, the muga silkworm (Antheraea assama) transcriptome has been investigated by generating over 35,000 ESTs which resulted in the identification of over 8200 unique putative genes at CDFD, Hyderabad. Functional annotation of these revealed several genes that are involved in silk production, circadian rhythm, sex determination, immune response and also several novel tissue specific genes. Digital differential display showed over 1000 transcripts expressing only in testes. Similarly, ESTs that code for fibroin and sericin were also identified. Preliminary analysis of fibroin and sericin shows that silk proteins of A. assama are quite distinct from those of Bombycids. Comparative

Cocoon of different ecotypes of tropical tasar silkworm (Antheraea mylitta)

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studies on expressed sequences of the B. mori and wild silkmoths with the genome sequences of insects and model organisms are underway to uncover Lepidoptera specific genes. A database is being developed to catalogue all the ESTs developed in the present study. At Indian Institute of Technology, Kaharagpur, two cDNA libraries from Antheraea mylitta were prepared in lambda ZApII vector from the mRNA. Clones were excised as plasmids from these libraries, sequenced and analyzed. Clustering and assembly of 2500 sequences showed presence of 660 unique sequences (400 contigs and 260 singletone). Analysis of microsatellites within nonredundant sequences showed that pentanuclotide and trinucleotide repeats represents more in the transcribed sequences.

the cross “Mysore Local” and “V-1” using 250+RAPD primers and 45 in-house developed mulberry specific microsat markers for construction of linkage map in mulberry jointly at Centre for Cell and Molecular Biology, Hyderabad and Central Sericultural Research and Training Institute, Mysore. Two sets of primers based on unique SNPs have been developed which are found to be diagnostic of good rooting ability and have potential to be used as MAS marker. Molecular 'ID' of 18 elite mulberry varieties have been developed. A new trait-specific (alkaline tolerance) mapping population was developed by crossing Sujanpur-5 (tolerant) and V-1 (sensitive) varieties. A set of putative genotypes have been identified for developing mapping populations for two other traits of interest (photosynthetic efficiency and moisture retention) using RAPD-PCR based screening of two sets each of 18 diverse mulberry genotypes. A total of 67 mulberry accessions have been conserved in vitro and 238 accessions have been successfully cryostored using dehydration and stepwise freezing protocols under a joint project at the Central Sericultural Germplasm Resource Centre, Hosur and the National Bureau of Plant Genetic Resources, New Delhi. The species represented are Morus indica, M. alba, M. rotundiloba, M. australis, M. multicaulis, M. bombycis, M. cathayana, M. nigra, M. laevigata, M. macroura, M tiliaefolia, M. serrata and M. sinensis. Genetic stability analysis using molecular markers ascertained the similarity between the fresh and cryo-regenerated samples. Transgenic mulberry were generated using barley late embryogenesis abundant proteins (HVA I gene) under a constitutive rice actin promoter via Agrobacterium mediated transformation at University of Delhi, South Campus, New Delhi. The transgenic plants showed better cellular membrane stability, photosynthetic yield, less photooxidative damage and better water use efficiency as compared to the non-transgenics under both salinity and drought stress conditions. Among the lines analysed for stress tolerance, transgenic line ST8 was found to be relatively salt tolerant whereas ST-30 and ST-31 were found to be drought tolerant. Transgenic lines ST-11 and ST-6 respond well under both salinity and

Anterior silkgland

Middle silkgland

Posterior silkgland

B. mori

A. assama

Architecture of Bombyx mori and Antheraea assama silkglands. Silkglands of Muga are morphologically different from those of B. mori.

Improvement of Host Plants DNA polymorphism data was generated for the segregating populations (160+progeny plants) from

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drought stress. A project has been recently initiated on field evaluation of mulberry transgenics (with HVA I gene) jointly at University of Delhi, South Campus, New Delhi and Central Sericultural Research and Training Institute, Mysore. Work on identification of QTLs associated with drought tolerant traits such as water use efficiency (WUE) and root traits in mulberry has been continued jointly at the University of Agricultural Sciences, Bangalore and Central Sericultural Research and Training Institute, Mysore. Significant variability was observed for WUE and other physiological parameters in the segregating population. A strong corelationship was observed between root biomass and the predicted transpiration among the mulberry germplasm accessions. SSR markers are being used to screen the root mapping population (200 individual) for polymorphism. Three crosses were made between high root and high WUE genotypes to identify desirable lines having both WUE and root QTLs by marker assisted selection. In another collaborative project at both these institutions, efforts have been continued towards cloning and characterization of epicuticular wax synthesizing genes from mulberry with a view to reduce transpiration and post-harvest water loss. A few epicuticular wax (EW) related gene fragments having homology with Arabidopsis have been cloned. The cloned genes and other related EW genes from Arabidopsis are being used as a probe to examine the pattern of EW gene expression by northern blot in contrasting mulberry accessions.

at the respective participating centres. Good genetic variability has been recorded in the selected mulberry germplasm for resistance to three biotic agents. The DNA polymorphism data on 144 selected genotypes of mulberry have been generated using 200 RAPD decamer primers and 50 inhouse developed mulberry specific microsatellite markers. The same is being scored for undertaking diversity analysis to infer the genetic base and genetic interrelationships of the identified genepool. Basic research in modern Biology Support to R&D projects having fundamental questions was continued to provide new vistas to the knowledge required for understanding the intricacies involved in applied research. An initiative was taken to bring biologists, mathematicians, physicists and engineers to develop a new and fast emerging area of systems biology. During the period, the department received 70 proposals and sanctioned 52 projects across various disciplines. On-going projects resulted in publication of several research papers in high impact journals. Significant achievements of some of the projects are presented belowDrug Discovery Studies are being carried out at NIPER, Mohali to improve the oral bio-availability of cyclosporine and to reduce nephrotoxicity associated with the commercial formulation (Sandimmune Neoral®). The relative bio-availability of cyclosporine from the nanoparticulate formulation is 119.19% as compared to Sandimmune Neoral®. Additionally, cyclosporine nanoparticles exhibited significantly lower ® nephrotoxicity as compared to Sandimmune Neoral in rats which was indicated by reduced blood urea nitrogen (BUN), serum creatinine (SC), as well as histological examination of kidneys.

In a network project being implemented at the Centre for Cellular and Molecular Biology (CCMB), Hyderabad, Central Sericultural Research & Training Institute, Mysore, Karnataka State Sericulture Research and Development Institute, Bangalore; Central Sericultural Research & Training Institute, Berhampore and Central Sericultural Germplasm Resource Centre, Hosur, work on identification of DNA markers associated with disease (powdery mildew) and pests (tukra and nematode) resistance in mulberry has been continued. One round of screening of the identified germplasm for disease response to three biotic agents has been completed

Hypoxia, a major factor influencing the extent of cell injury in myocardial ischemia and infarction, is a powerful regulator of gene expression for multiple regulatory proteins, including cytokines and growth

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factors. Scientists at Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum used an in vitro cell culture model to evaluate the response of adult rat cardiac fibroblasts to hypoxia.

motif, the signal peptide that directs them to their target membrane. SecB is a soluble cytosolic chaperone component of the sec export pathway which binds to the newly synthesized precursor proteins thus preventing their premature aggregation and folding and then targets them to the translocation machinery on the membrane. PreMBP is the precursor form of maltose binding protein (MBP) with a 26-residue signal sequence. Studies carried out at IISc, Bangalore indicates that both MBP and preMBP are more prone to aggregation under crowded conditions with preMBP showing a greater extent of aggregation.

Uracil DNA glycosylases (UDGs) excise uracil from DNA and initiate the base excision repair pathway. Of the known UDGs, Ung proteins belong to a category of highly conserved and efficient enzymes, and shown to be crucial in mycobacteria. Studies carried out at IISc Bangalore revealed that UdgB plays a significant role in mycobacteria. The ability of MTB to survive and adapt to changing environmental conditions appear to be regulated largely by two-component signaling systems (TCS). Of the eleven TCSs that MTB possesses, the PhoPPhoR system is the only one disruption of which has been shown to drastically affect the bacillus's replication in cellular and animal models. Scientists at IMTECH., Chandigarh have shown that phoP promoter activity is negatively autoregulated by PhoP through sequence-specific interaction(s) involving 3 direct repeat subunits with a 9-bp consensus binding sequence.

The tumor suppressor protein p53 regulates transcription of many genes, which mediate cell cycle arrest, apoptosis, DNA repair and other cellular responses. Studies being done at CCMB, Hyderabad has shown that caspase-1 activator Ipaf is a p53-inducible gene involved in apoptosis because blocking of Ipaf function by RNA interference or by a dominant negative mutant reduced p53-induced apoptosis. To determine the molecular components involved in p53-dependent apoptotic pathway, they have analysed the role of Ipaf and caspase-1. Since the mechanism by which caspase-1 executes apoptosis under these conditions is not known, the investigators have also determined the sequence of events that lead to apoptosis upon caspase-1 activation by Ipaf. The results show that Ipaf is involved in p53-dependent apoptosis (induced by PTP-S2) and that caspase-1 activated by Ipaf primarily functions upstream of mitochondrial events to mediate p53-dependent apoptosis.

One of the most clinically significant mechanisms of azoles resistance in the pathogenic fungi, Candida albicans is an over expression of the drug efflux pumps encoding genes CDR1 and CDR2 belonging to the ABC (ATP-Binding Cassette) and CaMDR1 belonging to MFS (Major Facilitator Superfamily) transporters. Among the ABC transporters, high level of expression of CDR1 invariably contributes to an increased efflux of fluconazole and thus corroborates its direct involvement in drug efflux. The study conducted at JNU, New Delhi suggests that Asp327 of N-terminal NBD has acquired a new role to act as a catalytic base in ATP hydrolysis, a role normally conserved for Glu present adjacent to the conserved Asp in the Walker B motif of all the nonfungal transporters.

SMARCAL1 belong to the SWI/SNF family of DNAdependent ATPases. The SWI/SNF protein family has been shown to be involved in all DNA metabolic processes- DNA replication, DNA repair, transcription, and DNA recombination. Scientists at School of Life Sciences, JNU carried out experiments to delineate methods to explore the physiological role of SMARCAL1. The conditions for over-expression and solubilization of the protein have been standardized.

Proteomics Many proteins in Escherichia coli are initially synthesized in their precursor form with a signalling

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Scientist at IISc., Bangalore carried out experiments for elucidating the significance of glycosylation for the immunomodulatory activity of glycodelin. Despite the similarities in overall folding status of glycodelin and LG, the study revealed that the stabilizing contacts in the folded conformations of these proteins are different.

The malarial parasite Plasmodium falciparum synthesizes fatty acids by the type II mechanism. In this cycle, the dehydration of the βhydroxyacyl acyl carrier protein is catalyzed by FabZ. Scientists at IISc., Bangalore purified FabZ and crystallized using the hanging-drop vapour-diffusion and microbatch techniques. The crystal structure PfFabZ has been determined by molecular replacement method. PfFabZ has a hot-dog fold and has been found to exist as a homodimer (d-PfFabZ) in the crystals of the present study in contrast to the reported hexameric form (h-PfFabZ) which is a trimer of dimers crystallized in a different condition. The existence of the inactive state of the enzyme and the pH triggered conformational switching between active and inactive states were revealed by this crystallographic analysis and provides to new strategies in the design of novel antimalarials.

Scientists at CDRI, Lucknow used NMR spectroscopy to solve structure of Mycobacterium tuberculosis, Escherichia coli, and Homo sapiens peptidyl-tRNA hydrolase. The peptidyl-tRNA hydrolase enzyme is essential for the viability of bacteria. The ORF Rv1014c of Mycobacterium tuberculosis H37Rv, labeled as mtpth gene, was cloned in pET 28b vector and over-expressed in Escherichia coli BL21 (DE3) cells. A structural model based on E. coli Pth crystal structure, was generated.

Oligomerization of PfFabZ

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Molecular Immunology

A study was implemented at NCCS, Pune to delineate the role of insulin in regulating the expression of IGFBP-1 in the context of active hypoxia inducible factor (Hif1). The hypothesis is that insulin plays a dual role in the regulation of Insulin-Like Growth Factor Binding Protein 1 (IGFBP-1). IGFBP-1 gene expression is positively regulated by hypoxia and negatively by insulin. The scientists have cloned the 1.5 kb upstream sequence of IGFBP-1 promoter in the luciferase reporter gene and have generated various deletion fragments to analyse promoter activity in the presence of insulin and iron chelators. They have also cloned the 300 bp intron1 fragment containing a putative Hif1 site in the luciferase reporter vector to analyse the role of Hif 1 in insulin mediated activation of IGFBP1.

Studies were carried out at IISc, Bangalore to understand the trafficking of salmonella in dendritic cells and its implication in antigen presentation. The data elucidates a very potential role of intracellular tolls like TLR9 in the pathogenesis of salmonella and might be modulating the trafficking and antigen presentation of salmonella. Studies are underway to elucidate how TLR9 can modulate the trafficking and presentation of salmonella in dendritic cells. The Toll-like receptors (TLRs) represent a group of single-pass transmembrane receptors conserved from Drosophila to mammals, expressed on sentinel cells, which are central to the innate immune response and responsible for sensing microbial products to trigger an antimicrobial response. The studies carried out at Bose Institute, Kolkata revealed how a virulence factor of the pathogenic M. tuberculosis is able to trigger signaling processes which dampen TLR-dependent proinflammatory signals. They also raise the possibility of using ESAT6 or peptides derived from it as potential modulators which may dampen prolonged undesirable inflammatory signalling associated with conditions such as sepsis.

A novel non-apoptotic cell death has been recently identified which is designated as paraptosis. The hallmark features of paraptotic cell death are cytoplasmic vacuolization, mitochondrial swelling, and absence of caspase activation and oligonucleosomal DNA fragmentation. Absence of caspases makes Dictyostelium discoideum a good model system to study the role of Poly (ADP-ribose) polymerase (PARP) in caspase independent cell death mechanism. Studies done at JNU, New Delhi suggest that Dictyostelium discoideum under oxidative stress exhibits PARP mediated caspase independent paraptotic cell death.

Gene Cloning and Biochemical Characterization Studies were carried out JNU, New Delhi for understanding the initial steps in the Glycosylphosphatidylinositol (GPI) biosynthesis pathway in Candida albicans GPI anchored proteins in C. albicans are essential for morphogenesis, virulence, and adhesion of the fungal cells to their host cells.

Computer Modelling, Simulation and Optimization A project is being implemented at IIT, Kharagpur to understand the structure and interaction between Rv0600c/Rv0601c/ Rv0602c proteins from M. tuberculosis. In silico molecular model of Rv0600c showed a typical kinase catalytic domain, with a well conserved ATP binding site. The histidine kinases were cloned into E.coli expression vector pQE30, overexpressed and purified. Their secondary structure was analysed by circular dichroism and that confirmed the molecular models generated.

DBT Annual Report 2006-07

Amino acid sequences for PIG-A of Sachromyces cerevisiae, Homo sapiens and putative Candida albicans were analysed. Candida albicans, and Sacchromyces cerevisiae possess 56% identity while Candida albicans and, Homo sapiens has 46% identity. Very long chain fatty acids (VLCFA) represent 30% of the total fatty acids in some tissues like retina, lens

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and brain- indicating that they might play a crucial role in normal functioning of these specialized tissues. Studies are being carried out at NIN, Hyderabad to characterize ELOVL4 with regard to physicochemical, structural and functional properties of the protein. Trans fatty acid not only decreased Elvol4 expression but also affected the structural integrity of retina in rats

nonpolymeric coupling agent. The design of such folate-nanoparticle conjugate has many combined advantages in view of its targeting functionality to tumor cells. Figure below shows the log normal distribution of folate conjugates along with particle size histograms. The effective hydrodynamic dimeter the synthesized folate conjugates was found to be 88 nm The attachment of folic acid was confirmed through Fourier Transform Infrared Spectroscopy (FTIR). Figure below indicates the confocal image showing the cellular uptake of folates in B96 Melonal S0 cancer cell line. This result shows that the targeted intra-cellular delivery of iron oxide-folate-FITC nanoparticles has taken place via a receptor mediated endocytosis.

Oxidative stress induced DNA damage in Intra Cytoplasmic Sperm Injection (ICSI) is being investigated at IIT Kharagpur. A total of 72 ICSI cases have been analysed to ascertain oxidative stress induced DNA damage in ICSI. Results indicate that a positive correlation exists between Reactive Oxygen Species (ROS) and sperm morphology and its DNA damage. Further, samples with higher ROS levels appear to be unfavorable for pregnancy outcome.

One of the important aspects of the forthcoming nanotechnology revolution is the use of DNA as a structural material. Scientists at University of Madras, Chennai are using biophysical techniques, chiefly Xray crystallography, but also computer modelling, UV spectroscopy and gel mobility, to study the structures of DNA junctions, such as three-way and four-way junctions, as well as unusual DNA packing modes that lead to novel microstructures.

Plant Molecular Biotechnology Studies are being carried out at ICGEB, New Delhi on Calcineurin B-like Protein (CBL) and CBLinteracting protein kinases (CIPK) paradigm: Cloning and characterization. The calcineurin mediated signal transduction pathway in response to salinity stress was analyzed. By using PCR approach followed by pea cDNA library screening, full-length cDNAs of both the CBL and CIPK have been cloned and also purified the encoded proteins of 26 and 58 kDa, respectively. The genomic clones of CBL and CIPK have also been cloned and found that CBL contains nine introns while CIPK is intronless. The CIPK protein has been characterized and shown to contain autophosphorylation activity. Nanobiotechnology Targeted delivery of magnetic nanoparticles is a highly desirable strategy for enhancing efficacy and reducing unintended side effects and toxicity. Receptor targets are especially suitable for such a strategy. Folic acid is generally recognized as an effective tumor targeting agent to conjugate with nanoparticles. Scientists at IIT, Kharagpur made an attempt to make folate-nanoparticle conjugate by grafting folic acid through some biocompatible

Confocal image of B96 cells showing intracellular upatake of iron oxide nanoparticles

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Medical Biotechnology

strain without loss of physiological viability ensuring genetic stability, have been worked out. A site for Phase III human clinical trials has been prepared with all necessary demographic patterns in a slum population of about 50,000 in Kolkata.

Health related biotechnology research has assumed a central place in the field of biotechnology. The department during the year has made concerted efforts to focus research activities in the area of vaccines & diagnostics, infectious disease biology, chronic disease biology, stem cell research and bioengineering. A new impetus has been given to newer diagnostic approaches, better therapeutics, vaccines, diagnostics, clinical proteomics, cancer biology, cardiothoracic, bioengineering, neurological disorders, RNAi, medical devices etc. that are aimed through generation of good infrastructure, trained manpower and high-class science. An active collaboration with the industrial partners is being sought in all product oriented projects. A major emphasis has been given towards development of newer vaccines dengue, chicken guinea, typhoid, HPV, and continued efforts were made to carry out clinical trials of rotaviral diarrhoea vaccine, malaria, typhoid, rabies etc. The various Task Forces of medical biotechnology met thrice during the year alongwith expert committees, inter-disciplinary research committees and more than 100 new projects have been recommended for support they reviewed the progress of ongoing projects. Follow up action has been initiated wherever leads are available.

During the year, proposals were invited from competent industries for licensing and/or contract manufacture of the clinical grade material under cGMP for conducting Phase III human clinical trials following ICH-GCP guidelines. Shantha Biotechnics, Hyderabad has been identified through a competitive bidding for the pre-filled and finished product on a contractual basis. Clear-cut “Product Development Plan” and “Clinical Development Plan” have been laid. Management Structures to monitor formulation, regulatory and clinical trial issues are being established outside DBT for ensuring quality and timelines. Typhoid Vaccine Development Technology Transferred to Industry Work towards development of Vi-Conjugate typhoid vaccine has been carried out at the All India Institute of Medical Sciences (AIIMS), New Delhi. The conjugate typhoid vaccine has been developed. The vaccine has shown promising results in animal studies. This vaccine may be effective even in children below two years of age and school going children. Therefore, it has the potential to prevent typhoid fever more effectively than Vi alone in children and adults, thereby the morbidity and mortality could be reduced to a greater extent. The technology has been transferred to M/s. USV Ltd., th Mumbai on 19 July 2006 through a License Agreement with Biotech Consortium India Ltd. (BCIL), New Delhi to upscale, manufacture (cGMP grade), preclinical toxicological studies and human clinical trials.

The significant progress are highlighted below : Vaccines & Diagnostics Cholera The oral, live, recombinant, non-residual cholera candidate vaccine strain VA1.3 developed through DBT's financial support using novel methods of strain isolation and genetic manipulations completed Phase 1 safety trials and Phase IIa efficacy trials after detailed pre-clinical animal toxicology studies. The vaccine has been patented in USA and India and all necessary regulatory approvals required for recombinant products as per EPA, 1986 obtained. Fermentation conditions as well as a semi-synthetic medium, which allows healthy growth of vaccine

DBT Annual Report 2006-07

Tuberculosis As a sequel to a “Brain storming” held in May 2005, the approach towards research on tuberculosis has been broadened and funding expanded during the year. The department invited proposals through open

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advertisement in the following areas: l

Collection and storage of aliquots of specimen i.e. sputum, serum, CSF, urine, tissue etc.

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Creation of centralized facilities and reagent bank

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International level repository/depository of Mycobacterium

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A central animal facility for breeding various inbred and knockout strains Creation/ setting up of a centralized vaccine/drug testing facility

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l

Containment facilities

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Preparation of clinical trial site/s

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Human Resource Development

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Industrial Collaboration

suspected patients was found in 89% of the TBM patients. The studies cumulatively identified two mycobacterial antigens, Rv3804c and Rv1886c (Ag85A and B respectively) in the CSF of TBM patients. Antibodies produced against the 30-kDa protein band reacted with the whole Ag85 complex, and the indivitual components of the Ag85 complex, (Ag 85A and Ag 85B). Ag85 complex expression in the CSF of TBM patients might provide prospective insights in the area of infection by M .tuberculosis. Immuno-modulator / Adjuvant Research The department has been supported a double blind, randomized, placebo-controlled multi-centric trials to see the safety and efficacy of an immuno-modulator , Mycobacterium w as an adjunct to chemotherapy in Category II of the pulmonary tuberculosis at seven centers i.e. AIIMS, New Delhi; LRS Institute of TB & Respiratory Diseases, New Delhi; Central JALMA Institute for Leprosy and other Mycobacterial Diseases, Agra; SMS Medical College, Jaipur; N.H.L. Municipal Medical College, Ahmedabad; RNT Medical College, Udaipur and Tuberculosis Research Centre, Chennai . Trials are at full swing at six centers.

While the focus of research remained on early diagnosis and vaccine development, the department entered into the area of drug development for tuberculosis using proteomics, genomics and bioinformatics approaches. About 90 R&D proposals were received from established and new research groups including from the leading industrial groups. About fifty projects have been funded at different places i.e JALMA, Agra, IIT, Kharagpur, IIT, Kanpur, University of Delhi, South Campus, NIPER, Mohali, as well as at Ranbaxy Labs. A “National Tuberculosis Research Initiative” in a strategic ten- year missionlike activity with in-built mechanism for uninterrupted funded and strict monitoring is in place. An Expert- cum-Working Group has been set up to monitor the progress of the mission as also to provide guidance on newer areas.

The first Interim Analysis (IA) is due shortly as ¼ of the patients have completed the treatment. IA template for the data entry and compilation has been firmed up. Data auditing, data cleaning and data capture processes are going on. An independent group under an eminent Bio-statistician has been set up for the purpose. About 40 patients of the randomized and 26 healthy control subjects at AIIMS, New Delhi have been studied for the immunophenotyping of various T cell subset(s) and their intracellular cytokine production profile. In the recruited patient cohort, enumeration of various T cell subsets has shown differential peripheral representation following vaccination. Though patients decoding have not been done, an over all enhancements in central and effector memory cells on follow up visits have been observed.

Work has progressed well in about 30 on-going projects on tuberculosis. In a study on TBM diagnosis At CIMS, Nagpur, a 30 kDa protein antigen markers unique to CSF samples collected from

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Research and Development

Mw as an adjuvant in other clinical indicationsThere are emerging indications that Mw may be clinically useful as an immunomodulator in other diseased indications including cancer. During the year, the Department invited proposals to explore the potential of Mw, both in pre-clinical and clinical conditions and to elucidate basic pathways of the action of Mw in appropriate animal models for cancer, leishmania , hepatitis, rabies etc.

10 4

1000

3.69%

CD25

800

FSC

600

400

88.6%

10

3

10

2

10

1

10

0

8.03%

200

0 0

200

400

600

800

62% 10

1000

0

26.3% 10 1

SSC

10

2

10

3

10 4

CD4

10

4

800

2.09%

0.58% 10

F o xP 3

# C e lls

600

62% 400

10

10

3

2

Dengue diagnostics and Vaccine/s

1

200

10

0 10 0

10 1

10 2

10 3

10

91.3%

0

10

4

0

6.05% 10

1

10

2

10

3

10

4

CD25

CD4

Taking into consideration the recurrent outbreaks of dengue in the country, R&D projects have been

+

Presentation of Fox-3 regulatory T cells tuberculosis patients vis-a vis healthy controls

DBT Annual Report 2006-07

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initiated for the development of dengue diagnostics and vaccines using approaches that could generate propriety leads.

New Delhi progressed well. Vero cell-derived, inactivated JEV vaccine was successfully transferred to M/s Panacea Biotech. This technology has since been validated, optimized using WHO-supplied Vero cells, and upscale to the fermenter level. A GMP facility for JE vaccine production is now in place at Panacea. Several reagents and quality assurance methods related to the vaccine production process have been developed and validated. However, methods for virus purification are being optimized for which Panacea, Delhi has procured a continuous-flow zonal ultracentrifuge which is likely to be installed by February 2007. The centrifuge will then be used for the large scale virus purification process development after which the material for pre-clinical toxicology and phase I trials would be produced.

A group at NIMHANS, Bangalore has initiated the development of dengue specific monoclonal antibodies for an assay towards development of a serological kit. Efforts are being made to replace the mouse brain antigens. Work has been initiated to make non-replicating subunit tetra-valent dengue vaccine based on a critical domain of the major dengue structural envelope protein that is involved in host receptor recognition and in the induction of robust protective immunity. The prototype strains of each of the four DEN serotypes have been incorporated into Pichia pastoris and a novel 'monovalent' and 'tetravalent' dengue antigen genes encoding the serotypespecific Pichia shuttle vector created. Efforts are now being made to express the recombinant domains III of all four dengue (DEN) virus serotypes (tetravalent) in P. pastoris, which can grow in simple defined medium and produce functional recombinant proteins in gram quantities per liter of culture. The “Proof of Principle” has been established to use tetravalent domain III based vaccine candidate in animal models. The same is being taken up on a fast track with an industrial partner i.e. Bharat Biotech International Ltd., Hyderabad.

The approach using Adeno based vectors as a backbone for JE DNA vaccine has shown good results. The “Proof of concept” in animal model has been established for using Adeno 5 as backbone for relevant DNA in JE infection. It was suggested to be seen whether there are pre-existing antibodies to Adeno 5 in infant/children that may hamper the protective response. In a study carried out, results show that children below 12 months age possess significantly lower anti-adenovirus antibodies as well as Ad5-neutralising antibodies, after which there is a marked increase in antibody titers. The findings are consistent with the reports from other parts of the world. Although alternate strategies are being explored to overcome the pre-existing Ad5 immunity in humans, the results, together with earlier finding that low levels of Ad5 antibodies do not interfere with the recombinant adenovirus vaccine uptake in mice suggest that Ad5-based vaccines or other therapeutics may still be effective if administered during the 6-12 months age bracket. This may pave a path for Adeno- based JE vaccine for that age group.

Parallely, the scientists at ICGEB are working on a hypothesis that a single tetravalent DNA virus-based vaccine may provide a means of circumventing viral interference. Japanese Encephalitis The Department continues to support translation research towards improvement of current vaccines; and development of newer approaches for the JE vaccine especially due to recurrent epidemics as also serious adverse events reported by the introduction of a Chinese vaccine. The project on the development of a tissue culture based vaccine (Indian strain adapted to Vero cells) carried out NII,

The study on molecular mechanisms of microglia/macrophages mediated neuroninflammation in Japanese Encephalitis carried out at NBRC, Manesar has demonstrated that microglial activation may be an important contributory factor in

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the pathogenesis of JE. It has been seen that JEV infection activates microglia both morphologically and functionally; infection leads to an elevation of proinflammatory mediators for which microglia are predominantly responsible and the cytotoxins released from activated microglia are instrumental in inducing neuronal death that accompanies JE. Avian Influenza (H5N1) Virus A brain-storming session was organized by the Department on the status of Avian Influenza Research in India: Past, Present and Future on September 9, 2006. Experts from India and USA participated and deliberated. The priority areas identified are genetic diversity, pathogenesis and zoonosis, diagnostics, vaccines. Accordingly, the Department has funded two R&D projects on development of a cost effective and easily up-scaleable multivalent vaccine using a novel yeast expressed virus like particles approach against the deadly Influenza A (H5N1) virus and molecular biology studies on the pathogenic strain of H5N1 virus (Indian isolate): protein-protein interaction based approach to study molecular evolution of the virus from its non-pathogenic strain at ICGEB, New Delhi.

A new multi-centric project entitled “Use of artemisinin and curcumin combination in treatment of uncomplicated P.falciparum malaria” was implemented at five institutions i.e. at National Institute of Malaria Research, Delhi; Indian Institute of Science, Bangalore; NIMR Field station, Rourkela; Ispat General Hospital, Rourkela and NIMR, Jabalpur.

Malaria The project implemented at ICGEB, NIMR, New Delhi and Ispat General Hospital, Rourkela the cell bank and technology for production of recombinant PfMSP-119 and PfF2 were transferred to Bharat Biotech International Limited (BBIL), Hyderabad for developing master cell bank and master working cell bank for characterization. The BBIL has successfully produced cGMP grade material of three consistent batches at 10L scale for human clinical trial and toxicological studies. Efforts have been made to scale up to 50-100L ferment.

Another project implemented at IISc., Bangalore on development of candidate anti-malarials based on new drug targets, analyzed Pfcrt mutation as a marker for chloroquine resistance in 200 P. falciparum infected patients. Analysis of Pfmdr1 gene as a marker for chloroquine resistance has revealed that only around 30% of the Indian samples show the N86Y mutation, although in Africa there is a good correlation between K76T-Pfcrt mutation and N86Ymdr1 mutation. The high prevalence of K76T- Pfcrt mutation in the Indian isolates of P.falciarum is a

Malaria vaccine trial site has been developed at Sundergarh District of Orissa to evaluate the malaria candidate vaccinogens PfMSP-119 and PfF2 through collection of clinical, entomological and molecular epidemiological /immunological indicators from the study. The group conducted longitudinal

DBT Annual Report 2006-07

entomological surveys in two indicator villages each from forest and plan area. A total of 11 anopheline species from forest area and 10 speciies from plain area were recorded. It was observed that Anopheles Culificacies was the most predominants species and accounted for 41.2 and 36.5% of the total anophelines in forest and plain area respectively. Further observed that An. Fluviatilis was restricted to only forest area and its prevalence rate was 1.1%. It was measured that the antibody levels against PfMSP119, EBA 175 and TRAP antigens in 222 (110 from forest and 112 from plain areas) and 248 (138 from forest and 110 from plain areas) blood samples collected during low and high transmission seasons, respectively. The results suggest that there was a boosting in antibody production against these molecules by natural infectious in these individuals. The level of antibodies in study groups appeared to be related to their exposures to the parasite during high transmission phase. Update on progress on the project was reviewed in the month of January, 2007 and decided to validate the data generated to initiate phase I/II clinical trial with malaria vaccinogens through national and international experts.

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cause for concern. The modeling studies have led to elucidation of differences between PfALAD and host enzyme at the active site and metal binding site. These results would be useful in designing molecules to specifically inhibit PfALAD. Further it was found that rifampicin inhibited the malaria parasite apicoplast RNA polymerase. Studies in mice infected with P. berghei, revealed that rifampicin at the optimum dose could give around 50% protection, the end point being parasite load and mortality. Further studies have been carried out to establish the production.

Towards identification and characterization of CD4 Independent HIV receptors on spermatozoa at NIRRH, Mumbai, human mannose receptor has been identified as CD4 independent HIV receptor present on spermatozoa. Its differential expression on the spermatozoa may determine the possible risk of sexual transmission of HIV. Results also indicate the need for revised strategy for prevention of sexual transmission of HIV. Towards identification and characterization of antiHIV compounds in Indian marine bivalves at NCCS, Pune, NIO, Goa & ICGEB, New Delhi, new anti-HIV compounds have been isolated, identified and characterized. Application for filing patent is under progress.

Infectious Disease Biology Two meetings of the Task Force were organized th during the year. Priority areas for 11 Plan period were identified. A total of 52 new project proposals were considered and 24 were recommended for support. Besides, advertisements for call on proposals were made in the areas of HIV/AIDS & microbicides, Chikungunya research and Virus Research Programmes. Some of the significant progress are highlighted below:

Studies on HIV-1 pathogenesis and the role of integrase in reverse transcription and nuclear transport of viral genome at CDFD, Hyderabad suggested that Integrase play a critical role in reverse transcription in addition to its role in integration. Understanding of its role in early viral life cycle may help to develop new effective antiviral molecules. Work on functional analysis of the NF-KB polymorphism in the terminal repeat of HIV-1 subtype-C viruses at JNCASR, Bangalore revealed that the NF-kB polymorphism unique for subtype C promoter (C-LTR) contributes significantly for gene expression regulation. A novel strategy has been developed to prepare Tat protein with higher efficiency more importantly by preserving the biological functions of the recombinant protein intact.

HIV/AIDS Studies on the molecular basis of CTL dysfunction in HIV infection at NCCS, Pune showed that CD40 and IL-12 in priming of CD4+ cells provide help in CD8+ cells in CTL maturation. Appropriate CD4+ T cell help is necessary for maturation of even fully expanded CD8+ T cells into functional CTLs. The importance of IFN-γ in inducing expression of CTL effector molecules, perforin and granzyme has been already demonstrated. Finding means of rescuing an impaired CTL functions may devise an immunotherapeutic strategy towards control HIV replication or to boost existing strategies. At the same institute studies on extracellular Tat Mediated inhibition of HIV-1 replication has led towards identification of a novel interaction of SMAR1 with the host protein TAP (HIV-1 Tat activating protein). SMAR1 peptide may be used to block the transcription complex formation between Tat-TAP and SMAR1.

Studies on development of a lentivirus (HIV-2, Indian Isolate) based high efficiency gene transfer vector were carried out at ACTREC, Mumbai. An indigenous gene transfer vector is in final stages of development with novel features of versatile multiple cloing site with expanded cloning capability. Core Immunology Lab Efforts were continued towards establishing an immunology core/clinical laboratory to evaluate HIV vaccine elicited immune responses at ICGEB, New

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Research and Development

Delhi. The protocol for 4-colour whole blood flow cytometry has been standardized. Forty HIV infected Indian patients and 15 normal individuals for T cell activation and coreceptor expression were analysed using the protocol. Indian HIV patients were found to have a marked decrease in percentage of activated CD4 T (CD4+HLA-DR+) cells expressing CCR5. The expression of CCR5 on CD14+ monocytes or CD8+ T cells is not down regulated. HIV infection induced T cell activation in Indian HIV patients alters both the percentage and expression of CCR5 on activated CD4 T cells. The protocol for IFN-γ Elispot has been standardized and used to test immodominant epitopes in 4 HIV infected patients. Attempts were made to isolate HIV-1 by co-culture methods from an Indian patient with a skin cancer and seropositive positive for HIV on routine testing as it was unique for two reasons: (a) it would be the earliest case from India, and (b) it showed long-term non-progression. These observations suggest that the virus may be a CXCR4 utilizing virus. This itself would be a unique observation as all Indian isolates have so far been reported to be CCR5-tropic.

interaction domain. It has been found that ORF6 may play a role in virus replication as observed in interaction between SARS-CoV accessory protein and nsp8. Work on reagents for Immunological detection of SARS associated Coronavirus (SARS-CoV) at DUSC, New Delhi led to development of a sandwitch ELISA using high affinity anti N MAb to capture intact N and purified anti-N polyclonal antibody to reveal captured N-protein. This assay detected 10 pg of Nprotein. The same will be evaluated for detecting Nprotein in SARS-CoV infected persons. At AIIMS, New Delhi a real time RT-PCR has been developed for SARS virus. HEV Studies were carried on development of nonradioactive antigen specific reporter release cytotoxicity assay and analysis of cytotoxicity against HEV proteins at AIIMS, New Delhi. Bicistronic eukaryotic HBV core/HEV reporter expression vectors were generated to develop and validate the non-radioactive antigen specific CTL assay using HBVcore as target antigen were checked in HepG2 cell line for the expression of both antigen and reporter proteins. The expression of the reporter proteins were transiently transfected in non-adherent mouse A20 cell line but transfection efficiency was found to be low. Bicistronic HBVcore/HEV (RdRp, ORF2, and ORF3) reporter pLXSN retroviral constructs were made and tested for expression in HepG2 cell line. These vectors were transfected into retroviral packaging cell line to generate viral particles and were able to infect human HepG2 and mouse NIH3T3 cell lines. Neomycin selectable HBVcore reporter retroviral vector containing packaging cell lines were generated to produce retroviral particles.

SARS Towards studies on functional analysis of SARS virus proteins for understanding pathogenesis at ICGEB, New Delhi, polyclonal antibodies to the X1 protein have been generated and tested. Protease protection study suggested that the topology of X1 is such that its N-terminus is towards the luman of the vesicle and C-terminus is towards the cytoplasm, thus exposing its cytoplasmic domain to trypsin. It was also found that X1 and Caveolin are present in the same biochemical fractions, supporting the case for their interaction. Studies on cloning and characterization of the SARS virus structural proteins and their biomolecular interactions at ICGEB, New Delhi indicated that s phase inhibitory activity of the N protein may have major significance during viral pathogenesis. Also, the 3a accessory protein of SARS-CoV is an RNA binding protein and specifically interacts with the 5' UTR of its viral RNA using a 92 amino-acid

DBT Annual Report 2006-07

HCV Studies on inhibition of HCV RNA translation and replication using small RNAs jointly at IISc., Bangalore and DUSC, New Delhi demonstrated the

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'proof of concept' that the transient expression of these si/sh RNAs in vivo. The results laid a solid footing to investigate the efficacy of these molecules using in vivo model using Sendai virus Virosome based delivery system.

Towards development of new generation diagnostics against HIV and malaria by using Quantum Dots at NCCS & NCL, Pune. Anti-EBA-175 antibodies were found to be useful for developing quantum dot based diagnostic kits.

Leishmania

Shigellosis

Studies on Leishmania target antigens from promastigotes and amastigotes on the basis of Th1 stimulatory proteins for immunoprophylactic activity against experimental Visceral Leishmaniasis at CDRI, Lucknow have led to identification of four subfractions having immunostimulatory properties. They were analyzed by MALDI-TOF-MS analysis.

Studies were carried out on molecular epidemiology and characterization of Shiga Toxin-producing Escherichia coli (STEC) at SKUAST-K, Srinagar. Isolation of stx2f variant of stx2 gene from bovines appears to be the first report in the world. Subtyping of eaeA gene into eaeA-δ, eaeA-e, eaeA-ζ, eaeA-η, eaeA-κ subtype analysis seems to be novel work from India. The group has been able to isolate the stx genes from human diarrhoeic samples in J&K.

Towards exploration of mechanism of drug unresponsiveness to SbV in field isolates of Leishmania donovani at CDRI, Lucknow, role of thiols in clinical resistance against sodium stibogluconate was established for the first time. No amplification of γGCS1 and ODC was observed in resistant isolates as compared to sensitive ones.

Wound healing Studies on the development and delivery of stress induced siderophore for wound healing at CLRI, Chennai have resulted into a bilayered membrane collagen scaffold. It was found to simulate with normal healing pattern, considering infection as a predisposing factor after injury. The designing of collagen based wound dressing to deliver the therapeutic agents locally in a finite dose and controlled fashion is a novel approach. The biphasic

STD Studies on virulence genes in Indian strains of Chlamydia trachomatis at Institute of Pathology and ACBR, New Delhi were conducted. Expression of pkn1, ompA and incA genes was studied in Indian isolates of C. trachomatis and expression of proteins for virulent genes pkn1 and ompA has been achieved. Candidiasis Studies on molecular aspects of Candidiasis at JNU, New Delhi has been carried out to determine the role of GlcNAc in Candida albicans in relation to morphogenic changes and pathogenicity. Identified several critical residues in Cdrlp which could be exploited further to develop rational inhibitors/blockers. Diagnostics

Morphological Features of TPHS Loaded Chitosan Microspheres Impregented Collagen Scaffold

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Research and Development

homolog of B. anthracis. Drug Delivery Studies on drug delivery system targeting of antileishmanial drugs to specific sites using lipid microspheres were carried out at Kakatiya University, Warangal & IICB, Kolkata. The pharmacokinetic parameters of Amphotericin B in rats and mice were influenced by the type of lipid microspheres formulation administered. Pegylated lipid microspheres exhibited higher mean residence time while mannose grafted and positively charged systems cleared quickly due to rapid tissue distribution. Tissue distribution studies revealed the facts that both the mannose grafted lipid microspheres and positively charged lipid microsphers are concentrated in liver and spleen where the leishmania parasite resides. This is in conformity with the results obtained in in vivo antileishmanial studies. The nephrotoxicity observed is due to high Amphotericin B levels in kidneys following fungizone administration. Amphotericin B distribution to kidney and brain has not been influenced much by the type of formulation administered. The pharmacokinetic parameters obtained both in rats and mice with piperine followed similar pattern. The clearance was very high for free piperine as compared to those obtained following the administration of formulations. The tissue distribution pattern of piperine following the IV administration of free piperine and lipid microspheres formulations is similar to that of Amphotericin B indicating the strong influence of the characters of the lipid microspheres on drug distribution.

Cross Sectional Morphology of the TPHSM Impregnated Collagen

delivery device is one such class of new generation wound dressing. Drug Development Investigations on programmed cell death in pathogenic bacteria: towards developing novel antibiotics to control infectious diseases were carried out at JNU, New Delhi. The organism under study, Bacillus anthracis, is a pathogen that causes deadly anthrax disease in cattle and humans. Through extensive database mining and BLAST search, a Toxin-Antitoxin module has been hypothesized in B. anthracis chromosomal DNA where it exists as an operon and was found to be 50% homologous to E. coli. The toxin is reported to belong to a family of PemK like toxins (116 amino acid residues) while the antitoxin (95 amino acid residues) is not well defined in the database. The genetic organization of toxinantitoxin loci led the investigation to infer the presence of second ORF upstream from the PemK

DBT Annual Report 2006-07

Acanthamoeba Studies continued on in vitro pathogenicity, molecular characterization and molecular diagnosis of Acanthamoeba infections at LVPEI & CCMB, Hyderabad. Results of the assay done on clinical samples from test and control subjects were found to have a sensitivity of >85.0% with some of the smear/culture positive samples failing to amplify the Acanthamoeba specific amplicons.

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Zinc as an immunomodulator

institutions and disciplines in the areas of HIV/AIDS and microbicides. The emphasis is to address major scientific challenges, which require a team approach. The idea is to augment the advanced scientific research and development through the implementation of shared scientific strategic plan, mobilization of adequate financial resources and greater collaboration among the HIV/AIDS researchers in the country. The strategic areas identified include understanding the pathogenesis of HIV/AIDS, designing novel vaccines and microbicides concepts, curtail HIV replication etc. In response, 133 Letters on Intent (LOIs) were received from various research institutions, universities, private companies and NGOs. The LOIs were considered by an Expert Committee and 14 projects have been implemented.

Studies on zinc as an immunomodulator in the treatment of possible serious bacterial infection in infants of more than 7 days up to 4 month of age are being carried out at AIIMS, New Delhi. Since it is a double blind randomized controlled trial results of the outcomes by groups are not available at this stage till the code is broken at the end of the study. In this study, the treatment failure rate is 15.7% which is lower than the treatment failure rate of 25% used for calculating the trial size. Eighteen infants died during the study and the predominant cause was septic shock. The overall case fatality rate of 4.5% has been consistent over the last one and half years of enrollment which is usual this kind of clinical settings and also reflects the better case management for enrolled infants in the study. Three hundred and eleven infants (about 78.5%) achieved overall recovery without change in initial antimicrobial therapy. Forty three infants were discharged after they had achieved clinical recovery but had not gained 10g/day for two consecutive days. An overall 6.5% rate of loss to follow up, is much lower than observed in the initial 6 months of enrollment, which reflects the good co-ordination of the research team. The isolation rates of pathogens in blood culture were 17.4% and out of these 43% were due to Staphylococcus (aureus, coagulase negative aureus and methicillin resistant aureus). DSMB was constituted before the enrollment of patients was started. The adverse event forms and the death summary of the infants are being constantly reviewed by the DSMBs as per the stipulated procedures.

Chikungunya Virus Research Programme The Department invited pre proposals in order to conduct focused research related to Chikungunya Virus keeping in view of the resurgence of Chikungunya virus infection in India and other countries around the Indian Ocean. The aim is to initiate a broad and comprehensive research programme related to the virus and disease aspects primarily based on team effort in the areas of structural biology and molecular mechanism of viral replication, rapid methods of virus detection (serologic and molecular tools), viral tropism and host factors & animal reservoirs, viral diversity and molecular epidemiology, immune correlates of infection or protection (establishment of links with access to clinical material), viral pathogenesis, development of experimental animal model systems, designing of new antivirals based on the molecular structure of the virus, development of novel vaccine platforms and immunogens, novel vector control strategies. A total of 20 pre-proposals were received and out of which 11 have been selected to develop as full proposals after screening by an Expert Committee. The full proposals will be examined further by an Expert Committee and implemented as per the recommendations.

New Initiatives: DBT-ICMR Collaborative Effort on HIV/AIDS and Microbicides The Department of Biotechnology and Indian Council of Medical Research jointly made a programme announcement inviting proposals/ concept papers from investigator driven research initiatives with interactive collaborating efforts across

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Virus Research Centers or Networks

candidates for Human Papilloma Virus has several components and progressed well during the year. The whole approach is based on identification of strains that could be uniquely responsible for considerable number of cervix cancer ( CaCx ) cases in Indian women. Scientists are making Virus Like Particles (VLPs), Capsomere Like Particles (CLPs), synthetic peptides and use these with or without novel antigen presenting systems. Companies have joined the development efforts under proper agreements.

The Department invited pre-proposals towards establishment of Virus Research Centers or Networks in order to develop comprehensive biomedical programme for virus research in India. Under this programme, Centres of Excellence capable of developing broad and comprehensive research programmes for virus research has been emphasized. This will primarily be investigator driven team effort to address diverse aspects of virus research ranging from basic structure, biology, replication, antigenic and genetic diversity, methods of detection, pathogenesis and vaccine development. The priority research areas include Respiratory viruses with emphasis on Avian Influenza, Enteric viruses, Hepatitis 'C' & 'E', Japanese Encephalitis, Dengue, Cytomegalovirus and other emerging viruses such as Chikungunya. A total of 46 pre proposals have been received.

Six centers located in different parts of the country. i.e. AIIMS, New Delhi; CMC, Vellore; KMIO, Bangalore; ACTREC, Mumbai; RCC, Trivandrum and CFI, Kolkata collected 125 samples each from the confirmed cases of CaCx and genotyping done to identify the presence of ulcerogenic and nonulcerogenic HPV strains using standard protocols. The genotyping of all 600 samples would be available and sequencing of L1, E6, E7 protein done. The results may provide distinct clue as to what strains could be important for designing vaccine candidate in Indian women so to give maximum protection against oncogenic strains. HPV genotyping has been done in collaboration with XCyton Diagnostics Ltd., Bangalore who have designed and developed a “Papilloma chip” for testing all strains in one go. The chip has been validated and a 100% concordance found between the results of the collaborating centers and that of the company.

Chronic Disease Biology The department has provided impetus to Chronic Diseases Biology (CDB) as the cases of cancer; cardiothoracic disorders, diabetes, neurology and joint diseases etc. have reported to be on the rise in developing countries. An independent Task Force has been constituted and areas such as joint diseases, cancer immunotherapy, vascular biology, stroke and reproductive biology identified for brain storming/ interactive meetings. It is proposed to set up a few Centers of Excellence in the area of CDB.

Further on, L1, E6 and E7 proteins of HPV 16 are being sequenced by Lab India Instruments Pvt. Ltd, Gurgaon to check if there is any strain variability in HPV 16.

Cancer The focus continues to be towards early diagnostic and prognostic markers. While there are about 90 on-going investigator driven projects adopting various intervention strategies i.e. new synthetic compounds, study of novel signaling pathways and their intervention strategies using novel proteins; cell based therapy etc., there are well-defined network projects that have shown good promise.

VLPs of HPV 16 have been prepared with average diameter ranging 35-50 nm. A dialogue between ACTREC and Nicolas Piramal India Limited, Mumbai (NPIL) was initiated towards co-development efforts and a possible technology transfer of rHPV-16 L1 VLP preparation and purification. As a preliminary feasibility study, ACTREC provided seed culture, which was grown to 5L fermentor at NPIL and VLPs purified. The protein yield (VLP) increased to >5 fold

Human Papilloma Virus Vaccine Project: The network programme on the development of vaccine

DBT Annual Report 2006-07

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in fermenter as compared to the flask. Animal studies have began with GLP produced VLPs with or without APCs as per the co-development agreement with M/s Nicolas Piramal Pvt Ltd., Mumbai. VLPs were provided in batches to ACTREC, Mumbai for assessing VLP induced immune response in mice using different routes. It is expected that animal studies would be completed soon. Work on therapeutic aspects is also be studied at AIIMS, New Delhi.

cervicitis” is being carried out at KMIO, Bangalore to assess the inherent immunity to HPV and to study the role of t-reg cells. Major progress had been achieved in terms of validation of the role of Notch signaling in human epithelial oncogenesis and defining the pathway that may serve as therapeutic targets. Curcumin Cancer Clinical trials: The department has taken an initiative to start clinical trial using a common herb, Curcumin for various cancers. As a follow up of a brainstorming in May, 2005, proposals were invited and funded to objectively study the effect of curcumin in Oral pre-cancerous lesions/OSMF; Head & Neck cancer; and cervical cancer. A herbal formulation, BASANTTM for clearance of early cervical lesions associated with HPV is also being investigated. As the bio-availability of curcumin is poor, different formulation that have shown enhanced bio-availability in animal models would be procured in bulk from a vendor/drug suppliers after ensuring quality control and physico-chemical uniformity. A comparative bio-availability studies on different formulation of Curcumin is being done at DIPSAR, New Delhi. Four groups involved in the trials are :

For developing immunological assays for assessing the immune responses to the indigenously developed HPV vaccine, C57BL/6 and Balb/c mice were immunized via intra-peritoneal route to study the lymphocyte responses to the vaccine. Studies are in progress to analyse different immunization schedules and analyse antigenic specific functions in control and immunised mice. Cohort Development by AIIMS, New Delhi is going on. The sample size for the study on HPV vaccine trial site preparation has been calculated to be 2000 women based on the assumption that there will be a 20% prevalence of HPV infection in the population of married women aged 16-24 years and that 50% of them will be positive for HPV 16/18. Govindpuri slums have been selected as the study site where a support services set up for examination of patients. Enumeration process has started, a database was created based on the enumeration forms and a list of all the eligible women in the age group of 16-24 years generated. Pre enrollment and enrollment forms, consent form and information Sheet as well as food frequency questionnaire were designed and identity cards printed for all recruited women. Samples for Zinc testing on serum are being stored at present. Sri Ram Institute in Delhi University has agreed to collaborate in processing the samples. Follow up has been done on 76 women so far. Cervical cancer and Toll-like receptors (TLR) and Notch induced oncogenesis: The study on “TLR expression profile in langerhans and Dendritic cells in invasive cervical cancer, cervical intraepithelial neoplasia and chronic

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Pre-cancer lesions and Oral Sub mucous Fibrosis- Co-ordinating centre - AIMS, Kochi ; Participating centers: Tata Memorial Hospital, Mumbai; RGCBT and RCC, Trivandrum; AIMS, Kochi; RDCH and CDRF,Chennai

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Head and neck cancer- Co-ordinating centre, AIIMS, New Delhi and Participating centers: AIIMS, New Delhi; HIMS, Dehradoon

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Cervical cancer: Co-ordinating centre- AIIMS, New Delhi and Participating centers: AIIMS, New Delhi; TMC, Mumbai.

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Basant for the Cervical Cancer- Early lesions associated with HPV infection (BASANT formulation, Co-ordinating centre - ICPO, Noida and Participating Centers: ICPO, Noida; TRF, New Delhi; TMC, Mumbai; CNCI, Kolkata.

Research and Development

As the clinical trials ae planned to be done following ICH-GCP guidelines, Contract Research Organization (CROs) have been engaged clinical trial and data management. CROs that have been firmed up are: l

Neeman Medical International, New Delhi,

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Manipal Acunova Ltd, Bangalore

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Catalyst Clinsys Services Pvt Ltd, New Delhi

initiating cancer stem cells (CSC) have been found within GBM. In a study to understand the signaling cascades involved in the proliferation and differentiation, CSC from GBM cell line U87MG have been generated by a group at NBRC, Manesar. It is now proposed to study intrinsic circuitries regulating gene programs involved in maintenance and differentiated.

A

Cell based immunotherapy Phase I studies on the development and clinical evaluation of Dendritic cell (D.C.) vaccine derived from autonomous mononuclear cells from peripheral blood of patients and primed with whole cell lysates from tumours of individual patients have been carried out at WIA Cancer Institute, Chennai. The data indicated that there was minimal or no toxicity or autoimmune reaction in the vaccinated patients whereas a DTH response was seen in 2/3 patients receiving primed DC and CD8 infiltration elevated in one patient indicating immune modulation. Though no objective tumor regression was seen, the studies were not intended to see the efficacy as it was carried out in extensively treated patients. Encouraged by the results, the Task Forces on “Vaccines and Diagnostics” and “Chronic Diseases Biology” suggested the need for setting up a full fledged “Centre for Cancer Immunotherapy” with several peripheral centers. A discussion meeting on “Center for Cancer Immunotherapy” was held at Cancer Institute (WIA), Chennai. Considering the high quality of on-going translational work, CI, Chennai is being considered as a Centre for Cancer Immunotherapy (CCI) with the peripheral units as Associated Centers for Cancer Immunotherapy (ACCI). Institutions likely to be linked as ACCI are NCCS, Pune; NII and AIIMS, New Delhi; ACTREC, Mumbai, TRF, New Delhi and RLS, Mumbai.

Cancer Stem Cells generated from gBM cell line U87MG

Neurosciences The Department is supporting R&D projects in the area of neurosciences that , includes projects on neuronal aging, cognitive malfunctioning in normal and diseased conditions, neuro-genetic disorders etc. The area has been identified as a priority for the next plan bringing together scientists, clinical partners and industries. The Department is proposing to set up “Centers of Excellence” on stroke, vascular biology, dementia and basic neuronbiology in close association with NBRC, Manesar; AIIMS, New Delhi; CMC, Vellore; NCBS, Bangalore; and NIMHANS, Bangalore It is proposed to hold a brainstorming on “Stroke” . Metabolic Disorders

Cancer Stem Cells: Glioblastoma multiformes (GBM) represents one of the most malignant brain tumors characterized by intense proliferation, widespread invasion and poor prognosis. Tumor

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B

An elevated level of homosysteine has been implicated as an independent risk factor in cardiovascular diseases as well as in a variety of

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other diseases like end-stage renal disease, hypothyroidism, neural tube defect, diabetes, Alzheimer's ec. Work is under progress to study the effect of hyper-homocysteine in CAD and stroke. It is emerging that the levels of homocysteine are significantly higher in subjects adhering to a vegetarian diet. Homocysteine levels are also associated with MTHFR C677T polymorphism. Insilico studies indicated that elevated levels of homocysteine probably result in endoplasmic reticulum stress. Similarly, in stroke patients with hyperhomocysteinemia, the probable cause was identified in about 61%

identified and implemented on various aspects of both embryonic and adult stem cells such as limbal, haematopoitic, embryonic, pancreatic, neural, cardiac stem cells; generation of human embryonic stem cell lines; use of banana lectins for stem cell preservation; haematopoitic stem cells (HSC) for haplo-identical HSC transplantation; use of limbal stem cells for ocular surface disorders, isolation & characterization of mesenchymal and liver stem cells; in vitro differentiation of human embryonic stem cells to neural and non- neural lineages; phenotypic and genotypic characterization of limbal stem cells, cultivated limbal epithelial cells; etc.

Joint Diseases

Major research leads include use of limbal stem cells to repair corneal surface disorders caused by limbal stem cell deficiencies. So far, more than 300 patients have been treated at LV Prasad Eye Institute (LVPEI), Hyderabad. A technology has been established at Christian Medical College (CMC), Vellore for collection, isolation and purification of HSCs for haploidentical haematopoietic stem cell transplantation. Banana lectins have been isolated and purified showing stem cell preservation activities by the scientists of NCCS, Pune and IISc, Bangalore. Indigenous human embryonic stem cell lines are being generated at few institutions in the country. In the programmes supported on spinal cord regeneration using stem cell transplantation and other novel techniques, olfactory ensheathing cells have been isolated and cultured from human as well as rat samples of olfactory mucoa and bone marrow stromal cells have been cultured and differentiated in to neuronal tissue. In the study supported on molecular characterization of human liver stem cells for use in the treatment of hepatic diseases, hepatic progenitors have been isolated from human fetal gestation (13-20 weeks) and characterized by hepatocytic lineage markers. The isolated and characterized hepatic progenitors were cultured and maintained as per the standard procedures.

A workshop on Joint Diseases was organized at SGPGIMS, Lucknow and suggestions made for biomarker studies for diagnosis and prognosis; understanding mechanisms of apoptosis and autoimmunity, clinical genetics, cohort development, bio-banks etc. It is proposed to create a “Center on Joint Diseases” to address crucial th scientific issues on the subject during early 11 plan. Stem Cell Research The potential of stem cell technology to develop therapy for many untreatable diseases through cellular replacement or tissue engineering is widely recognized. A highly interactive field of life sciences, it requires close interaction of basic researchers, clinicians and the industry for the overall growth and development. In view its potential therapeutic applications, major programmes on stem cell science was initiated in the country. After a wide consultation with the national and international experts, priority areas have been identified and categorized into basic research, translational research, institutional development, creation of facilities/infrastructure and human resource development. Both basic and clinical research are being promoted by the Department. The programmes have been

During the period, about twenty programmes have

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been implemented for both basic and translational research at various institutions and hospitals. The details are as follows:

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Clinical research is an integral part of stem cell science. Thorough clinical research for determining the safety and efficacy of stem cells in human is essential. A system to consider clinical research proposals has been established by constituting four separate committees (i) “Human Studies Committee” for evaluation and guidance for clinical research particularly for development of clinical research protocols; (ii) “National Bioethics Committee” to ascertain rigid ethical guidelines being followed while conducting research on human beings; (iii) “Task Force on Stem Cells and Regenerative Medicine” to evaluate basic research and also recommend funding for clinical research based on the evaluation of the above committees and (iv) “Programme Advisory Committee” to consider the proposals of Centre of Excellence and infrastructure. Following this system and based on the thorough review of literature, a multi-centric phase-I clinical study has been implemented at six hospitals (CMC, Vellore; SGPGIMS, Lucknow; PGIMER, Chandigarh; Research & Referal Hospital, Delhi; AFMC, Pune and AIIMS, Delhi) to determine the safety and efficacy of bone marrow mononuclear cells in acute myocardial infarction. The pilot study on acute stroke using bone marrow mononuclear cells has also been implemented initially at one center i.e. AIIMS, New Delhi. Based on the results of the pilot study, the main study would be designed. A multi-centric phase-I proposal on limb ischemia is under active consideration.

Programmes have been formulated to support long and short-term overseas training in niche areas of stem cell research. For close interaction amongst the researchers, a “Stem Cell Research Forum of India” has been created. The “Forum” will provide a platform to the scientists and clinicians for discussion on new developments and also facilitate sharing of the achievements. An Annual conference was also being organized by this “Forum” during January 29February 1, 2007 with the support of DBT to discuss various aspects of embryonic and adult stem cells with the international experts. Draft guidelines for stem cell research in the country have been formulated jointly by the DBT and ICMR. These were discussed jointly by both the Committees to finalize this document. The guidelines will be placed for public debate soon. As per the draft guidelines, stem cell research has been classified under permissible, restricted and prohibited categories Research pertaining to adult and umbilical cord blood stem cells would be classified as permissible. However, embryonic stem cells research falls under restricted category. Research pertaining to reproductive cloning, introducing animal embryos in human, etc. has been categorized as prohibited. Bioengineering The area of “Bioengineering” has been identified as a potential area of research by the Department during 10th plan. The broad goals of the programme are: to initiate advance research, human resource development, creation of facilities in the four key areas of bioengineering i.e. tissue engineering, biomaterials, biomedical sensors, medical devices, implants and bioinstruments. It was felt that there is a need to initiate mission mode programmes at institutions having adequate facilities in collaboration with the industry; to strengthen R&D activities for the development of biomaterials especially for drug delivery, cellular/molecular imaging technology; MEMS biosensor using multi-parameter approach; fabrication of medical devices and bio-instruments, development of implants, disposable biosensors at

A “CMC-DBT Centre for Stem Cell Research” at CMC, Vellore has been established to carry out basic and translational stem cell research. This Center is under construction and will be functional soon. Facilities have been created to handle stem cell at few hospitals in the country such as PGIMER, Chandigarh; SGPGIMS, Lucknow; LVPEI, Hyderabad and KEM Hostipal, Mumbai. A training proposal has been implemented jointly at NCBS and JNCSAR, Bangalore. Efforts were also made to bring clinicians and basic researchers together for close interaction by organizing a number of clinical r e s e a r c h w o r k s h o ps , e x t e n s i v e t r a i n i n g programmes, brainstorming sessions, etc.

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low cost for rapid diagnosis of diseases, etc. A number of workshops in the identified areas were organized at various institutions such as Sree Chitra Ti r u n a l I n s t i t u t e o f M e d i c a l s c i e n c e , Tiruvananthapuramon; IIT, Mumbai; Central Scientific Instruments Organization, Chandigarh and National Physical Laboratory (NPL), New Delhi. Brainstorming meetings were also organized on “Devices and equipments for maternal, new born and child health care” and “Indigenous production of surfactants for the treatment of pre-mature babies”. As an outcome, several network groups of clinicians and basic researchers have been formed. “Letter of Intent” was also invited in these areas through advertisement and placed before the expert committee for consideration.

with the national and international experts. Draft white paper has been prepared jointly with the international experts and the same is being finalized. Similarly, a new initiative has been taken for “Centre for Biodesign” in the country in collaboration with Stanford University, USA for the indigenous production of biomedical devices, implants and bioinstruments. Draft proposal on “Stanford-India Biodesign” programme is under consideration and was discussed in a meeting on January 24-25, 2007 with national and international experts. Others Masters Course / Fellowship in Translational Health and Clinical Sciences, Necessary steps have been initiated to start masters course/fellowship in translational health sciences and clinical sciences to create trained manpower in the area at selected medical schools.

During the period, thirty nine projects have been considered on tissue engineering, biomaterials and medical devices, bioinstrumentation and biomedical sensors. Out of which, nine projects have been implemented on polymeric scaffolds, bio-medical sensors, tissue engineering, low cost diapers for newborn infants, imprinted polymers as substrates for glucose, etc. The projects on cultured autologous chondrocyte, scaffolds for tissue engineering of blood vessels, multi-axial mechanical tests on soft tissues, chondrocyte culture on 3D collagen scaffold, etc. are under active consideration. Currently in the country, few medical institutions, IITs and industries are involved in the development of biomaterials for therapeutic applications, biomedical sensors and pilot scale indigenous production of medical devices, implants and bioinstrumentations. Based on wide consensus, the need has been felt to establish a dedicated multidisciplinary “Center” or the “Institute” academically connected with other related institutes, hospitals and industry in an integrated manner for application oriented progammes. In order to discuss existing models at global level and also various possible models in the country, discussion meetings have been organized by inviting national and international experts. Efforts are also being made for “Health Science and Technology Initiatives” in the country based on good existing models internationally. The concept was discussed in detail

DBT Annual Report 2006-07

Setting up of Clinical Research Training Centres Steps have also been initiated to set up 5-6 clinical trial research training centres to train graduates and post-graduates students of medical, veterinary and life sciences to design clinical trials implementation, data management analysis, regulator requirement,documentation, concept of bioequlence bioequence for pharma and bioproducts, assay validation for product evaluation, ethical, legal issues etc. Rapid Grant for Young Investigators In order to reduce the age at which scientists get their first Principal Investigator's grant and expand opportunities for young scientists, DBT invited proposals in various branches of biotechnology related to medical, agriculture, vetinerary, environment, marine, industrial, bioresources. Out of 144 proposals received, about 45 proposals were recommended for support and implemented. Human Genetics and Genome Analysis The Human Genetics & Genome Analysis of genetic

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programme is under implementation since 1990-91 with the objective to identify, map and characterize genes related to genetic disorders prevalent in India for diagnosis; prevention and management of genetic disorders and to develop new methods for the analysis and interpretations of genomic DNA sequences. Further, based on a strategic meeting held in August, 2000, several new major initiatives were taken based on the announcement of Human Genome Sequences under International Human Genome project and the information available in the public domain to identify genotypes associations with varied clinical symptoms of different diseases and disorders. These were aimed at better molecular diagnostics and preventive strategies including vaccines, new drug targets, which would lead to development of new drugs with customized use in patients/subjects.

and DNA extracted from 78 samples. The Conformation Sensitive Gel Electrophoresis (CSGE) has been standardized which can accommodate small and thick gels. Linkage markers are being used for carrier detection in females with a positive family history of Hemophilia A&B and the females with affected fetus are being advised to abort the fetus. In the multi-centric project on Methotrexate in Rheumatoid Arthritis: Pharmacogenenetics and Clinico-Immunoogical correlates implemented at AIIMS, R&R Hospital and UDSC, New Delhi, 292 cases of rheumatoid arthritis [AIIMS=210, R&R Hospital=82] were recruited and patients were classified into methotrexate responder and nonresponder phenotypes as per the criteria described in the project. The DNA was isolated from a total of 292 samples and 267 samples have been analysed. Two approaches were used for the genetic analysis i.e. (1) re-sequencing of selected genes to identify novel SNPs and validate published SNPs in a small number of control samples, (2) genotyping of published SNPs and novel ones identified from (1) above in responders and non responders to MTX. Further one novel SNP in exon 5 of RFC gene has observed and its analysis is in progress.

Significant achievements during the year are as follows: Genetic Diagnosis cum Counseling Units The genetic unit established at CMC, Vellore on Molecular Genetics of Thalassemia, Hereditary, Bleeding Disorders and Leukemia”, collected samples from 225 patients and screened β globin gene mutations and these were characterized by DNA sequencing. Further 4 rare mutations that have not been reported earlier in the Indian population were identified. Common α globin gene deletions were screened by multiplex PCR in the heterozygous. Alpha globin triplications and quadruplications were screened by a multiplex PCR approach, In the area of leukemia diagnosis, the group has standardized the quantitative real time PCR for several transcripts. Excellent infrastructure has been established to undertake advanced molecular genetic research in blood disorders and providing services for bone marrow transplantation for thalassemia patients.

In the project implemented at JIPMER, Pondicherry on “Genetic susceptibility to essential hypertension in a South Indian Population”, standardization of genotyping procedures for ATI polymorphism, eNOS polymorphism and ACE polymorphism were done using PCR-RFLP and multiplex PCR methods. Allele specific PCR method was done using primers. DNA analysis of eNOS (Glu298Asp) and ATI (A1166C) polymorphism were carried out by PCR-RFLP method using Mbo1 and Dde1 restriction enzyme respectively. It was observed that individuals in the Tamilian population, with I/I genotype had an increased risk for hypertension when compared to D/D genotype, but the difference was not statistically significant. The A/A genotype occurred more frequently in cases when compared to control.

Under the project “Characterisation of mutations underlying Hemophilia A and B” at AIIMS, New Delhi, 143 samples of Hemophilia A&B have been collected

Others

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Under the multi-centric project on “Deafness in India : A network mission towards understanding the genes and mutations and their clinical outcomes”, clinical interview and examination was carried out to collect information on demographic profile of the families and molecular genetic analysis of the 14 genetic loci implicated in causation of deafness. The group also provided free patient services for audio logical and clinical assessment as a part. Seminars were conducted to educate the families regarding genetic causes of deafness and possible utility of the results obtained for the Cx26 mutations. The results obtained have implications for early detection and intervention of the disorder. With the results of six genes that have been analyzed in at least 300 affected families, the group expects to detect 45-50 pathogenic mutations in Indian populations.

The project implemented at NIMHR, Mumbai in decipherning role of homeobox protein HOXA 10 and decidualization, the research group studied expression of HOXA10 in a zone specific manner in the adult primate endometrium. The zone specific distribution was significantly modulated in vivo by progesterone. The changes in the expression of HOXA 10 associated by altered expression of its downstream effectors IGFBP-1 in vivo. The group at IITB, Mumbai under the project on identification and functional characterization of P. falciparum, characterized unknown function and showed the data very promising. This analysis will be useful in downstream analysis of the candidates. The group also attempted transient transfection of a-sexual blood stage parasites with some success. Transfections were carried out using two different electroporation conditions and new ncRNAs were identified by bioinformatics and cloning.

Under the project on molecular role of transcription factors in retinal diseases at NBRC, Manesar, the research group has identified retinal proteins that interact with NRL, a key rod photoreceptor specific transcription factor, expressed NRL as a GST fusion protein by sub-cloning the cDNA encoding the entire ORF of NRL without any extra sequences at 5' untranslated sequence. The protein was purified to homogeneity and used for protein-interaction studies. Two transcription factors, TBP and CREB-1 that interact with NRL were identified. The truncated carboxyl terminal half of NRL were identified produced strong interaction with both the proteins than the full length NRL protein.

Under the project on status of HFE gene frequency & other modifier genes of iron homeostasis in Indian population at SGPGIMS, Lucknow, DNA extraction has been done in the samples collected. The group identified ferroportin gene (A77D) and H63D gene mutation in thalassaemia group of patients and on screening observed high serum ferritin levels in subjects positive for H63D mutation and A77D mutation. Genomics, Clinical Proteomics and RNAi

Under the project on study of angiotensin converting enzyme insertion/deletion gene polymorphism of renal damage at AIIMS, New Delhi, about 120 samples were been studied. The most significant observation in the study was the influence of ACE I/D gene polymorphism in adversely influencing normal renal function. The group conducted genetic studies as well as assessment of molecular markers in the patients in the Paediatric Urology clinic of AIIMS. These patients are counseled on the possible influence of ACE I/D gene polymorphism on renal prognosis as well as the significance of molecular markers in further therapy.

DBT Annual Report 2006-07

Programmes have been supported on RNAi; clinical proteomics; whole genome sequencing of Mycobacterium w; microbial, functional, and comparative genomics. In the area of RNAi, the study is on screening interfering RNA sequences (siRNAs) made against the genes (protein kinases, protein tyrosine phosphatases, etc.) in the skeletal muscle cells. By siRNA silencing, a novel role for FAK has been demonstrated as a positive regulator of insulin signaling pathway leading to glucose transport and insulin sensitization. In another study, expression and

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expression profiling. This study is designed to test the hypothesis that these differentially expressed proteins (14-3-3 zeta and ALCAM) play an important role in the development and progression of oral cancer, either directly or by interacting with other cellular proteins, involved in cytoskeletal reorganization and loco regional spread of oral carcinomas.

profiling of the outer membrane proteins of Acinetobacter baumannii in native strain and its βlactam resistant strains using SDS PAGE has been carried out by a group at AIIMS, New Delhi. The resistant groups were characterized according to their outer membrane profiles and designation of profiles as A (ATCC type), A2 (ATCC type with prevalence of high molecular weight proteins) and U (Unusual profiling). Proteins of native and resistant strains have been identified and structural and functional characterization of OmpAb protein of native strain has been carried out.

In “The Mycobacterium w genome program: complete genome sequencing and comparative genomics”, 10X sequence has been completed with a sequencing efficiency of 87.84% and total sequence coverage of over 6 Mb. An extensive study of scaffolds and their linking pattern with 562 contigs from 10X assembly has been carried out which shows that the assembly has almost reached a plateau in terms of its lateral expansion in size and the total genome size of around 6 Mb has been expected. There are a total of 82 contigs oriented in the 24 scaffolds covering around 5.75 Mb (95.5%) of the genome sequenced with the largest scaffold of about 1.3 Mb in size. There are 58 sequencing gaps and 24 physical gaps in the genome data at the 10X stage of assembly that need to be filled to obtain a finished quality sequence and to carry out annotation work. The preliminary analysis of the sequencing gaps show that 22 out of these 58 have linking sub clones of a 5 kb library and the remaining 36 are linked by 2 kb sub clones. The detailed analysis and targeted gap filling for the same is currently under process. So far, information about sequencing gaps (58) and physical gaps (14) have been generated. Phylogenetic analyses based on high-resolution genome fingerprinting by FAFLP analysis, ERIC typing, and sequence analyses with the help of candidate gene sequencing like 16S rRNA, hsp65 and rpoB point to its relatedness to the M. avium complex. The study of genome assemblage trend of Mw indicates a size ~6.0 Mb which points to the possibility of Mw being the predecessor of M. avium complex species. This is an interesting observation considering the opportunistic nature of members of M. avium complex in contrast to a non-pathogenic attribute of Mw.

A combined approach of genomics and proteomics was used to understand the role of geneenvironment interactions in Parkinson's disease (PD). Several genes and proteins were identified that participate in pesticide induced PD. The study highlighted the contribution of several genes and proteins in understanding the basic mechanism of PD. Proteome profiles of striatal tissues were compared and differentially expressed proteins were identified. Three proteins significantly down regulated in treated groups were identified as enolase, glia maturation factor β (GMF-β) and complexin-I. The differential expressions of these proteins were also validated. Microarray profiling was done in the striatal tissues of animals treated with maneb+paraquat for 3, 6 and 9 weeks and upregulation/down-regulation of several genes including those involved in neuronal/apoptotic pathways were identified. Some sets of treated animals were further treated with L-DOPA and a significant alteration in protein and gene expression profiles were observed. Oral cancer is a major global health problem with no marked improvement in five year survival rates over the past decade. Understanding the molecular mechanisms involved in invasion and loco regional spread of oral tumors will pave the way for identification of key proteins implicated in these processes. A research group at AIIMS has recently identified some novel proteins in oral carcinomas that may be involved in interacellular adhesion and communication using high throughput gene

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The genetic basis of Type2 diabetes is heterogeneous and shows ethnic variations among different populations. Indians are one such ethnic group who are more insulin resistant and are considered to be a vulnerable high-risk population. A project was implemented for an extensive genetic and protein profiling using state-of-the-art technologies in the Chennai Urban Rural Epidemiology Study (CURES), on a large population at Chennai. Genes such as PPAR, PGC-1 , Adiponectin, UCPs, Resistin, INSR, IRS, Calpain10, PTPN1, GLUT, RAGE and VEGF are being studied on a large number of well characterized subjects and are correlated with clinical and biochemical investigations. Identification of the genes predisposing to Type2 Diabetes will contribute t o t h e u n d e r s ta n d i n g o f t h e m o l e c u l a r pathophysiology of the disease, which is crucial for development of effective treatment modalities and community based strategies to prevent the epidemic growth in the incidence of Type2 diabetes. The studies showed that: a) Pro12Ala polymorphism was highly frequent in South Asians but does not modulate the excessive risk for type 2 diabetes in this ethnic group; b) Thr394Thr polymorphism of PPARGC1A was associated with type 2 diabetes and body fat in Asian Indians; c) Among the four polymorphisms(866G/A, Ala55Val, 45bp I/D and 55C/T) of the uncoupling protein 2-3 (UCP2-3) examined at the UCP2-3 locus, the Ala55Val and 55C/T polymorphisms were associated with a significantly reduced risk of developing Type 2 diabetes in Asian Indians; d) In Asian Indians in the presence of obesity, the GD genotype of IRS-2 gene appears to be protective against, while the DD genotype increases susceptibility to, type 2 diabetes; and e) There was no significant difference in the frequency of the four polymorphisms, SNP-19, -43, 44 and -63 in the Calpain10 gene between NGT and diabetic subjects.

been identified under which many research projects have been sponsored. Efforts were also made to generate collaborative, multi-institutional network projects. Since, environmental pollutions are of different nature because of diversified industrial activities in the country, biotechnological interventions can provide the solutions for the environmental pollution problems through development of technologies, techniques, tools and processes. During the year, four brain storming sessions have been organized for generation of focussed, multi-institutional R&D projects in the area of Environmental Genomics, Environmental Biotechnology and Biodiversity Conservation. In addition two Task Force meetings were convened. During the year, progress of ongoing R&D projects and 18 completed projects were reviewed, 15 new R&D projects have been supported by Task Force. Sub-Committee of the Working Group for formulation of XI th Five Year Plan was constituted and its meeting was held in June, 2006 for finalization of priority areas for consideration in XI th Five Year Plan. Salient achievements in some of the R&D projects are : Biodegradation of Pesticides, Nitro-compounds, Xenobiotics, oil etc. Saurashtra Univesity has identified and characterized wood rotting strain 5A Trametes versicolor a white rot basidiomycete, which produces typical ligninolytic enzymes on natural lignocelluloses and in liquid cultures. The plant pigment extracts out along with ligninolytic enzymes from wheat straw infested by the white rot fungus, and hinders in the downstream processes as well as analysis of enzyme activities especially Lignin Peroxidase. A new protocol has been designed for the removal of plant pigments from the ligninolytic enzyme mixture. Efforts were made to develop a low cost, easy to handle and sustainable form of inoculum of white rot basidiomycetes for its application at field level in the remediation of dye

Environmental Biotechnology Department has made concerted efforts to identify priority areas for focussed R&D in the area of Environmental Biotechnology. 10 Major areas have

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infested soil. Culture was grown on grains to develop spawn. It colonizes grains within 3-5 days and spawn retains its property of inoculum till 2 months when stored at 4-8oC. Spawn grows rapidly and infests soil in 7 days in moist soil in a tube reactor. Thus bioremediation of soil could be demonstrated successfully by the use of spawn. Protocol for extraction of dyes from soil has been standardized.

presence of 1% (w/v) glucose and 0.5% (w/v) peptone. All isomers of picoline (α,β and γ) were degraded from pyridine raffinate along with pyridine by isolated bacterial strain and Aspergillus niger separately upto 95.99% and 96.98% respectively. There was more than 80% toxicity reduction in pyridine raffinate after bacterial and fungal degradation. The study concluded that biological detoxification of pyridine raffinate is possible only after chemical pretreatment.

IIT, Kanpur has isolated and characterized pure bacterial strains for the degradation of few xenobiotic compounds and toxic organic compounds. Nitroaromatic compounds were degraded by bacterial strains NP-1 and NP-2, which were identified to be Pseudomonas aeruginosa and Serratia marsecens. A bacterial strain PNS-1 was isolated for 4- Aminobenzene degradation during the study and was characterized to be belonging to the genus, Agrobacterium. Dimethylformamide (DMF) was degraded by 16S rRNA gene, which belongs to Paracoccus strain. Pathways for their degradation have been identified. Although plasmids have been detected in two strains, their actual role in degradation of specific growth substrates is yet to be delineated.

IIT, Bombay has isolated microorganisms degrading hydrocarbons in petroleum oil and in oily wastes in Rotating Biological Contacter (RBC). Specific means for enhancing adherence of oil degrading microorganisms to the rotating discs in the RBC was explored in a bench-scale RBC model and in a floor model. Two aliphatic hydrocarbon degrading cultures i d e n t i f i e d a s B u r k h o l d e r i a c e pa c i a a n d Exiguobacterium aurantiacum demonstrate significant enhancement when diesel was emulsified with Triton X 100 at twice its critical micelle concentration. Unidentified bacterial cultures enriched and isolated with naphthalene (designated as NG1, HNA1) were found to grow on emulsified diesel oil by degrading the aromatic fraction. A marine algal/ cyanobacterial culture obtained from Kurusadai Island, Tamil Nadu and B. cepacia was spiked in a bench scale RBC model fed with 0.5% n-

University of Delhi used biodegradation of endosulfan in soil microcosm as a model system with the aim to eventually develop a strategy for biodegradation of endosulfan contaminated sites. Conditions for growth of bacterium Bordetella sp. B9 was optimized prior to its use in microcosm studies. The study showed that soil bacteria are good degraders of endosulfan and therefore could be applied as a technology to decontaminate the endosulfan at contaminated sites. ITRC, Lucknow has developed microbial techniques for degradation of pyridine and picoline raffinate for safe disposal. The aerobic bacterial strain ITRC EM1 and ITRC EM2 were isolated and identified as Bacillus cereus (DQ435020) and Alcaligens faecalis (DQ435021). The bacterial consortia are capable to degrade pyridine from 20% pyridine raffinate up to 95.99 % within 7 days of incubation at temperature O range 35-37 C after chemical pretreatment in

Floor model RBC Reactor - Full set-up showing rotary shaker and overhead stirrer for preparation and temporary storage of emulsion before the emulsion is fed to the reactor

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Treatment Technologies: Anaerobic technology was applied as the primary treatment, which involves single-phase and biphasic systems. For biomethanation Anaerobic Fixed Film Reactors (AFFR) have been employed in the anaerobic system. It was observed that in single-phasic reactors, coconut coir was the best packing material with 55 % COD reduction at 6 days HRT, 31 kg COD 3 3 3 m /day with gas production of 3m /m day having methane content of about 60-65%. The biphasic reactor was packed with charcoal with 68 % COD 3 3 reduction with gas production of 4m /m /d having high methane content. Both the packaging materials are economically beneficial. Biodegradation and decolourization of anaerobically treated distillery spent wash was achieved by exploiting lignolytic ability of white rot fungus Trametes versicolor. Maximum decolorization 71 ± 2 % was observed at o 30 C under shaking condition in acidic pH range of 55.5 at an inoculum size of 10% (v/v) with 70 % COD reduction.

Floor model RBC Reactor - Close-up view of the reactor

hexadecane was also explored. Another algal cyanobacterial culture was obtained from Powai Lake, IIT Bombay that could be acclimatized to diesel oil. Exposure to oil caused a loss in species diversity and thin filamentous algae was found to predominate. Such oil tolerant algae can facilitate formation of biofilm in growth reactors.

Bacterial consortium DMC, comprising Pseudomonas aeruginosa PAO1, Stenotrophomonas maltophila and Proteus mirabilis appeared a good choice in terms of low additional nutrient requirement for biodegradation and decolourization of post biomethanated distillery effluent. Production of high value compounds like enzymes (xylanase, cellulase) from treated effluent by solid state fermentation is being investigated.

Distillery waste treatment (including deodorization) At NEERI, Nagpur, potential bacterial cultures were isolated for deodorization of sulphurous compounds. The isolated cultures were characterized for colony morphology, biochemical and physiological characteristics and are under the process of identification. The isolated cultures have been used f o r t r e a t m e n t o f w a s t e g a s c o n ta i n i n g dimethylsulphide in a biofilter system packed with wood chips and compost. The bench scale unit is under evaluation for optimization of process parameters viz Effective Bed Retention Time (EBRT), loading, moisture, pH of the medium, recovery after shock load etc.

Dye, paper and pulp mill waste treatment (including deodorization) Sardar Patel University had selected Common Effluent Treatment Plant (CETP) of Vatva, which treats the wastewater of 525 small-scale industries. The existing CETP of Vatva consists of equalization and aeration tank. They have developed a composite plan for treatment comprising of physiochemical, biological anaerobic and aerobic processes in sequence. This has improved treatment efficiency from 40-45% to 94% COD removal and 30-40% to 89% decolorisation.

Sardar Patel University focussed on improvisation of existing anaerobic treatment technologies by introducing potential anaerobes, developing efficient bioreactors and exploring additional microbial treatments to remove color from molasses effluent.

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IIT, Mumbai has used bimetallic Magnesium Pallidium catalyst to effectively decolorize and degrade reactive black 5, sunset yellow FCF and tartrazine dyes. Colour removal was more than 95% +4 o o within 24hrs of reaction using Mg/Pd or Mg /Pd alumina. Heavy metal removal At IIT Chennai, a new bacterial strain Arthrobacter rombhi RE has been isolated. A continuos aerobic system was developed which was able to achieve 95% of Cr (VI) reduction (initial concentration: 25mg/L) and around 90% COD reduction (initial concentration: 2000mg/L and molasses as the carbon source) at a Hydraulic Retention Time (HRT) of 12hrs. Nolina recurvata (Palam flower), Ganoderma lucidum (wood rotting fungus) and few aquatic weeds have been extensively studied for biosorption potential. Various instrumental techniques have also been developed for biosorption of Cr (VI) and Cr (III). Metal resistant P. putida strain KNP9 was studied in chickpea at G.B Pant University of Agriculture and Technology, Pantnagar. The KNP9 strain is resistant to 1.8mm of lead acetate and 0.546mm CdCl2 respectively. Probability of metal accumulation was evident by increase in size of bacterium in presence of cadmium and lead as seen by Scanning Electron Microscopy (SEM). It has been found that the metal resistant 'P' solubilizing bioinoculant KNP9 is reducing metal toxicity in chickpea in the presence of cadmium and lead. The ability of microorganisms to survive and reproduce in metal habitat may depend on genetic as well as physiological adaptations. Thus, cadmium and lead resistant strains can be exploited for bioremediation/ detoxification at sites containing respective metals. TERI, New Delhi has characterized the arbuscular mycorrhizal (AM) fungal germplasm for two major activities. 1) Functional characterization of AM fungal isolates of CMCC (Centre for Mycorrhizal Culture Collection)

2) Optimization of protocol for molecular characterization. Based on sequence data from 5.8S and 18S ribosomal RNA genes, primers were designed to specifically amplify DNA fragment including the ITS (Internal Transcribed Spacer). The resulting PCR product can be used to identify the fungal symbionts at the genus level by DNA sequencing. Optimisation of protocol for partial sequence analysis of CMCC germplasm was done by using Glomous specific primer. Biodegradable plastics Scientists at Agharkar Research Institute, Pune have isolated alkalophilic and salt tolerant bacteria from Lunar Lake, which are able to synthesize Polyhydroxyalkanoates (PHA). The process has been optimized for different parameters like temperature, pH, inoculum density and incubation period. FTIR (Fourier Transform Infrared Spectroscopy) and H NMR (Nuclear Magnetic Resonance) analysis confirmed the presence of copolymer hydroxybutyrate and hydroxyvalerate in 96:4 proportion. The organism could utilize agro waste like wheat bran for production of the polymer. Bacterial strains, taken from available gene pool including different agro climatic zones (Leh, Chamoli, Pithoragarh and artificially developed soil bed in Pantnagar) were screened for their ability to utilize polymers as 'C' source. Eight strains have shown better degradation potential against these polymers and have been identified based on 16S rDNA sequence and was submitted to NCBI database. TGDTG-DTA (Thermal Gravimetric-Differential Thermal Gravimetric and Differential Thermal Analysis) profiles of consortium treated versus control samples were compared depicting different patterns of exotherm and endotherm peaks in thermograms which confirmed biodegradation of the compound. In a project, at TERI, New Delhi, 262 bacterial strains were screened for PHA production. P. genus was the most frequently isolated PHA producer from the contaminated sites. Fifteen strains were selected and

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different seasons under two types of irrigation practices at field capacity viz. surface and subsurface. Plant growth was quantified in terms of

analyzed for the selection of best substrate for growth. Different carbon source like propionate, octanoic and decanoic acid were used as sole source of carbon in the nutrient deficient media to check for PHA production. A combination of glucose and propionate was found to be the best substrate, in which most of the selected isolates showed good growth. Bioconversion of solid waste and Biomethanation Fisheries College and Research Institute, T h o o t h i k k u d i i n Ta m i l N a d u p e r f o r m e d Vermicomposting of seafood processing waste. The seafood processing wastes, bulking agents and inoculum cow dung slurry or microbes Trichoderma/Azotobactor were mixed homogeneously in different ratios. Composting methods were employed for 21-25 days. Compost was then subjected to vermicomposting by using exotic earthworm Eudrilus eugeniae for 25 days which led to odourless, nitrogen rich biocompost. A biocompost yard is under construction.

Schematic Flow diagram of high rage Bioemthanation of poultry litter based on smart

Self mixing Anaerobic Reactor Technology (SMART) has been developed and patented by IICT, Hyderabad and ANGRAU, Hyderabad for High Rate Biomethanation from poultry litter. The process consists of feed preparation system, two number of Self Mixing Anaerobic Reactor (SMAR) in series with solid liquid separation system. The study revealed that approximately 60% of volatile solids in the litter 3 could be removed yielding 0.33m CH4/(kg VS reduced) of methane at 20 days of residence time. This design is efficient as it has low residence time and methane produced could be used for brooding within the farm saving LPG and dried bio-manure could be sold as fertilizer.

Hight rate Biomethanation of Poultry litter : bench scale studies conducted at IICT

Bioremediation

It has been observed that Phargmites is the most tolerant species followed by Arundo and Typha , whereas Scirpus species were the least tolerant.

biomass, chlorophyll and proline contents. Important findings are: Marshy species :

University of Rajasthan, Jaipur has identified the most tolerant plant species for the development of Ecotechnology for distillary waste treatment. 9 terrestrial (7 glycophytes and 4 halophytes) and 5 marshy species were studied in spent wash in

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During the study period, identification of economically important species such as henna (Lawsonia in cosmetics), Acacia (for fodder and

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energy) and Atriplex (shoots for fodder and seed for oil) for phytoremediation of distillery spent wash was completed. Phragmites and Arundo having very high organic matter production may provide organic substrate (filler) for bio-composting of spent wash a technology presently considered to be the most ecofriendly. The plantation of proposed herbaceous species (surface feeder for moisture and nutrients) with tree (deep feeder) will not only improve landscape and its general environment, but it will also prevent ground water pollution.

concentration of trace elements in fruits and vegetables from contaminated soil were found to be higher. The degradation was monitored by gravimetric loss of the crude oil with time. The results of the study suggest that the degradation was prominent at pH 7.5. Centre for Environmental Management of Degraded Ecosystems, University of Delhi collected data on functional diversity among rhizosphere bacteria which was analyzed to understand their ecological significance in nutrient enrichment and colonization potential of the host grass species. Bacterial isolates which produce extremely efficient siderophores for chelation of iron and trace elements were identified. Rhizospheres of these grasses harbour a high proportion of multi-functional bacteria, which need detail investigation. Lysogeny seems to be one of the important strategies among rhizosphere bacteria for their persistence, survival and multiplication in nutrient-stressed and degraded habitats and in assisting the host plant for better colonization and biomass production. Phages form an important component of rhizosphere bacterial community and understanding of their role in evolution in structure and function of rhizobaterial community will lead to improvement of efficacy of microbial biotechnologies useful for restoration of stressed and degraded habitats. Plant growth promoting ability of selected rhizobacterial isolates using host grass species and other model plant species is being initiated.

Mercury resistant E.coli strains have been isolated from different mercury polluted sites of India viz Yamuna river sites I, II, and III, Delhi; Hindon river, Ghaziabad; Kalu river; Bombay; GTB Hospital, Delhi; Floodwater, Delhi; Kalindi Kunj, Delhi; whereas the sample collected from the Dal Lake, Srinagar, Kashmir, was taken as control. Amplicons of putative merA genes were purified and cloned into plant expression vector pB1121. These recombinant vectors had been screened out by PCR and restriction digestion for the presence of the putative merA gene was performed. Construct of mercuric reductase gene (merA) in plant binary vector was made and it was mobilized in to Nicotiana tabaccum callus. It is under selection and screening process. After transgenics screening, molecular analysis of transgenics and its field trial studies will be initiated to utilize the outcomes of this research study. Present investigation revealed that the seven selected strains used in the study showed significant level of tolerance against HgCl2 of the different E.coli isolates. Dal Lake sample showed maximum tolerance to HgCl2, i.e., 55µg/ml, and the sample collected from Kalu river tolerated the lowest concentration of HgCl2 (25µg/ml). The isolated E.coli showed the following order of incidence of mercury resistance : Dal lake > Kalu River> Flood Water > Yamuna River > GTB Hospital

Cleaner Technologies (including Hydrogen production from waste, desulphurization, chemicals /value added products from waste etc.) Enterobacter cloacae IIT-BT 08, a facultative anaerobic bacterium has been reported to be a high yielding strain for biological hydrogen production. Spent media from dark fermentation by Enterobacter cloacae strain DM11 is found to have the ability to photo produce hydrogen by Rhodobacter spheroides strain O.U 001 in a hybrid reactor system with maximum conversion efficiency of acetate into gaseous hydrogen about 81%. In a batch system, the

IASST, Guwahati assessed physiochemical characteristics including heavy metals of the soil near the five Group Gathering Stations (GGSs) operated by the ONGC Limited for their eventual remediation and reclamation. It was found that

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yield of hydrogen in the first stage was about 3.31 -1 mol H2 (mol glucose) and that of the second stage -1 was about 1.5-1.72 mol H2 (mol acetic acid) . The overall yield of hydrogen in two-stage hybrid process considering glucose as preliminary substrate was found to be higher compared to a single stage process. Further, light conversion efficiency of hydrogen was found to be inversely proportional to the incident light intensity.

Biosensors/Biomarkers Industrial Toxicology Research Centre, Lucknow aims to detect the functional genes and study their expression levels in natural environments in a high throughput format such as a DNA microarray. They have developed 50-mer based oligonucleotide microarray based on most of 2,402 known genes involved in biodegradation and metal resistance. The oligos arrayed were specific to genes involved in biodegration of synthetic compounds like polyaromatic hydrocarbons (PAHs), monocylic aromatic compounds like BTEX, textile dyes, metal and nitro aromatic compounds and pesticides. This specific gene-chip is being validated with few organisms having genes for well-characterized degradative pathways.

Feasibility of E.cloacae IIT-BT 08 for biohydrogen production from sludge has been studied. In the process two potential hydrogen producers, Bacillus coagulans IIT-BT S1 and Citrobacter freundii IIT-BT L139 were also isolated. Heat-treated sludge was found to be better substrate for the enhancement of the production of hydrogen. 1% (w/v) supplementation of dextrose for 15% (v/v) heat treated sludge was recorded to give maximum hydrogen yield of 121.38 mol H2/g COD after 32 hrs. of fermentation. The process has been scaled-up successfully to 20 L with significant improvement in H2 yield.

Biodiversity conservation Wildlife Institute of India, Dehradun and CCMB, Hyderabad have attempted to standardize the protocol for isolation of DNA from tissue samples and toe tips. Four new species of anurans belonging to the genus Rhacophorus, Polypedates, Philautus and Bufo are diagnosed by morphological and molecular characters and also compared with all related congenerics and their distinctiveness has been demonstrated. The study has proved that allopatric speciation and convergence has played an important role as well as usefulness of biotechnological tools in the evolution of endemism of amphibians and reptiles in Western Ghats.

TERI, New Delhi has collected samples from Indian oil reservoir for microbial diversity of hyper thermophilic sulphate reducing bacteria. The DNA of the purified strains was sequenced by Microseq sequencing kit. Total culture independent studies based on 16S rDNA and DGGE (Denaturing Gradient Gel Electrophoresis) analysis indicted diversity of the Sulphate Reducing Bacteria (SRB) population in the various oil wells of Oil India Ltd, Assam. Out of 31 biocides tested, two (Navdeecide AM 5015, and Navdeecide 91) were found to be effective against sulphate reducing bacteria. These o O biocides were tested at 37 C and 55 C. This study : resulted in two important findings

Molecular tools such as genome-wide profiling Single Primer Amplification Reaction (SPAR) methods, namely, Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat PCR (ISSR PCR) are used to resolve Equisetum taxonomy at NBRI, Lucknow. Two species of Equisetum which are E. ramosissimum and E. arvense have been analyzed and then were classified systematically. Study has clearly shown the utility and resolution power of the SPAR profiles for carrying out or inferring inter- species relationships as well as diversity amongst accessions of the same species of Equisetum.

(i) Sulphate reducing bacteria are indegenous to the oil reservoirs, and (ii) Fermentative bacteria also lead to the production of sulphide, in addition to the sulphate reducing bacteria.

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ATREE, Banglore is working on two critically endangered plant species of Western Ghats Dysoxylum malabaricum and Garcinia indica. Protocols have been standardized for raising number of seedling.

Material of Jatropha” has been launched with the emphasis on collection and characterization of

Mission Mode Programme on Bioenergy and Biofuels Biofuels The mission programme initiated by the Department during 10th Plan concentrated on perfecting technologies for establishing bioenergy plantations for different agro-climatic zones with the involvement of local people; developing a cost effective economically viable technology for production of ethanol using alternate feed stock especially lignocellulosic wastes and efficient high yielding strains and recombinant strains of microorganisms; increased biodiesel production through improved planting material and improved transesterification process and production of hydrogen from algae and bacteria. Duriong the year activities continued inall area. Some salient achievements are:

Jatropha in demanstration plot

Bioenergy plantation Under the demonstration plots established in different agroclimatic zones for a number of energy crops, a total of 22 ha has been covered for sustainable utilization of the Bioenergy crops. Necessary linkages at grass root level are being fostered. Technologies for utilization of raw material for energy production are being developed and demonstrated. Main species are Jatropha curcas, Exocecaria agallocha, Avicennia marina, Salicornia brachiata, Cassia siamea, Albizzia lebbeck, Hardwickia binata, Dalbergia sissoo, D. sericia, Ficus glomerata, Leucaena leucocephala and Acacia nilotica. Agrotechnology packages have been developed for these species and are being documented. Technologies for nursery raising and harvesting are being transformed to the grass root.

On farm trial Jatropha plantation; at farmers field

superior material from across the country based on yield and oil content. 14 centres were supported covering 12 states. Nearly 1000 accessions of Jatropha have been collected and characterized for oil quality and quantity. 8.02 lakhs plants of superior material (>30% oil content and approximate 2t / ha yield) have been produced, covering 300 ha. Besides, experimental plots have been laid for standardization of agrotechnology package, seed collection time and seed storage conditions.

Biodiesel Micromission on “Production of Quality Planting en

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Operational guidelines for collection, characterization and production of quality planting material have been brought out. All accessions collected are being finger printed and superior accessions are being conserved at NBPGR, New Delhi. This is the first scientific documentation of superior germplasm. Work has also been initiated on multi-locational trials of the superior accessions collected in terms of increased yield and oil quality. R&D programmes are being initiated for improvement of Jatropha; studies include marker assisted selection, prospecting of genes associated with oil production pathway, metabolic pathway engineering etc.

and higher than C33 at low proportion. Cultivation of B.braunii strain was scaled up to 100 L level in both circular and race way ponds and further to 1000L and 3000L in raceway ponds under out door conditions, which is a major achievement. B.braunii growth and hydrocarbon production in outdoor ponds was evaluated in different seasons and maximum biomass (2 gL-1) was obtained during Oct-Dec and Feb-Apr. Hydrocarbon production correlated with biomass yields and maximum hydrocarbon content 28% (w/w) was observed during winter season (NovDec) using three-phase partitioning system. Conditions for lipase production from Pseudomonas cepasia strain have been optimized. Reusability of P. cepasia lipase immobilized on celite in the laboratory has also been studied at IIT, New Delhi. It was found that same could be used four times without loss in activity. P. cepasia was found to give high transesterification activity. 97-99% conversion of Jatropha oil by celite immobilized P. cepasia lipase towards biodiesel production.

Programmes have also been supported for testing the potential of other tree borne oil seeds standardizing plant production technologies and transesterification. Seeds of Heynea trijuga, Sapium sebiferum, Aleurites moluccana, Mesua ferrea and Pongamia pinnata were collected, processed to separate the kernels and oil extracted and estimated from seeds / kernels. Physico-chemical properties of the oils were determined. The methyl esters prepared from the oils were analysed by GLC to determine the fatty acids composition. Heynea trijuga oil has been evaluated at IIP for biofuel properties and has shown encouraging results.

Since Biofuels is one of the identified priority areas, it becomes imperative to have improved Jatropha plantation which provides raw material for biodiesel production. A brain storming session was held in the department to identify priorities in biodiesel and bioethanol. Under biodiesel, the focus is on : An integrated breeding programme for developing mapping populations including polyploidy and induced mutation for genetic improvement, marker assisted selection, development of micro-satellite markers for fingerprinting of elite genotypes and markers for yield and oil content and quality. Gene based association mapping study will also be taken up with an emphasis on oil content and quality. Besides, prospecting of genes associated with oil production pathway; metabolic pathway engineering for improved oil production will also be looked into.

Trans-esterification of curcas oil on a pilot scale (10 kg batch) has been carried out at IIP, Dehradun. Use of Pongamia, Salvadora and Madhuca as a raw material for biodiesel has been studied at NBRI, Lucknow. Agrotechnology package for cultivation has been worked out. In a study supported at CFTRI, Mysore, indigenous Botryococcus braunii strains were isolated from fresh water samples collected from Kodaikanal, Chennai, Mamallapuram and purified and identified them as A race based on their hydrocarbon profiles. All the strains were established in autotrophic medium and growth profiles were studied. All the indigenous strains produced long chain hydrocarbons of C22 to C33 chain length as major ones

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Bioethanol Various projects have been supported to produce ethanol using alternate feed stocks as a raw material. Sweet sorghum, lignocellulosic wastes, fruit &

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being optimized, before economic evaluation is done.

vegetable waste were found to be suitable as feed stock for bioethanol production. Cost economics of bioethanol production is being worked out at pilot scale.Thermotolerant yeast strain (Saccharomyces cerevisiae) identified, characterized and studies towards complete utilization of starch for ethanol production were carried out. In a study supported at University of Delhi South Campus, New Delhi, methodology has been developed for chemical hydrolysis of lignocellulosic wastes like Lantana camara and Prosopis juliflora into fermentable sugars and subsequent conversion into ethanol will be refined. The lignin degrading fungal isolate are being characterized. Recombinant microbial strains have been identified, which show enhanced ethanol recovery.

Based on discussion with experts future priorities under bioethnaol programme have been identified. These include development of a low cost cellulose / hemi cellulose for enzymatic hydrolysis which could be achieved either through natural selection of microorganism or development of a recombinant microorganism(s) or microbial consortia. Studies would also be initiated to develop an efficient process for production/scale up of the enzyme either through solid state or submerged fermentation. Simultaneous fermentation of both pentose and hexose for ethanol production either through development of recombinant microorganisms or coculture would also be taken up. Impetus will be on development of an efficient biological pre-treatment system for delignification.

In an ongoing project on bioethanol production at Osmania University, Hyderabad, efficient cellulose producing microorganisms have been identified. Pentose utilizing fusant has been constructed by protoplast fusion between thermotolerant yeast and Candida utilis. Fusant strain has been sent to IMTECH, Chandigarh for molecular characterization. Cloning, expression and confirmation studies are in progress. Ethanol fermentation upto 20 litre level was carried out. Further optimization work is going on. Cyanobacterial strain, Phormidum sp. BDU-5 has also been used for degradation of different cellulosic wastes like P. julflora, L. camara and coir pith. By products released during degradation are being studied. Besides, phenolic compounds like 3, 4dimethoxy cinnamic have been isolated and furher purification is being carried out by solvent extraction and column chromatography.

Hydrogen from Biomass Various efforts have been made to explore production of biological hydrogen from available biomass sources. In a study supported at Bharathidasan University, Trichirappalli, marine cyanobacteria have been screened for hydrogen photoproduction both in argon and nitrogen atmospheres under light regimes. All nonheterocystous filamentous strains tested produced hydrogen in nitrogen atmosphere with slight reduction. Production of hydrogen in nitrogen atmosphere finding totally disproves the earlier findings that hydrogen could not be produced in nitrogen atmosphere. By methane supplementation (5% - 20%) in nitrogen atmosphere the reduction of hydrogen rate could be revoked. Besides, a survey was carried out along the coast from Mimesal to Mandapam and Rameswaram and Kurusadai islands for marine photosynthetic bacterial collection. The survey yielded more than 50 different isolates of different groups of photosynthetic bacteria. This would be helpful in designing the integrated bioreactors for hydrogen photoproduction and the work in this direction is going on.

The substrates were delignified and hydrolysed to obtain fermentable sugars using Candida shehatae, Pichia stipitis and co-culture of both. In a12 L batch fermentor, Candida shehatae maximum ethanol yield was 18.78 g/L (0.65 g/g), followed by 16.72 g/L (0.62 g/g) by P. stiplis and 12.48 g/L (0.48 g/g) in coculture after 36 hrs. The optimization of enzymatic saccharification has been carried out and is further

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Chapter- 6

Biotechnology for Societal Development Programmes for SC and ST Population

of large size fish seed. Technology on floating cages was demonstrated through installation in Halali Reservoir in Bhopal with effective utilization of feed to benefit cooperative societies/self help groups, graduate and postgraduate entrepreneurs. Techniques of crab and lobster fattening were disseminated in coastal villages of Tamil Nadu as viable alternative livelihood. The activity was extended for post tsunami rehabilitation programmes. About 1000 farmers have been benefited through the implementation of the projects. Fattening techniques transferred to fisher folk helped to sustain income generation by coastal villages. DRDA has taken up the extension of the activity through establishment of crab fattening unit for dissemination of technological know-how. Model crab fattening units were replicated in Pudukottai District and 60 women were trained. Around 261 fish farmers benefited through implementation of the programme.

Biotechnology-based Programme for SC/ST population has benefited around 65,000 people through 50 ongoing projects and 12 new ones during the year. The projects supported cover cultivation of medicinal and aromatic plants, production of biofertilizers and biopesticides, organic farming, aquaculture, mushroom cultivation as well as human healthcare interventions etc. Universities, Krishi Vigyan Kendras and voluntary/non-governmental organizations have been involved in the implementation of the projects. The target group received the benefit of hands-on training and field demonstrations to bring greater awareness. Salient achievements of the programme are as follows: Integrated Aquaculture Activities on integrated aquaculture were undertaken through awareness programme conducted for the farmers in West Bengal. Farmers were trained in maintaining the ducks, poultry birds, pigs in integration with fish culture in addition to their domestic farm based activities. Around 3500 families were benefited through integrated fish farming. Integrated scampi-fish culture was undertaken in Allahabad district through small scale production of prawn seeds at the hatchery. Villagers were trained on technicalities of seed production and freshwater prawn culture. Honey production was also taken up to help target population in additional income generation. Farmers were trained on technologies through monoculture and polyculture at Palayamkottai in Tamil Nadu. Different species of murrels, Channa striatus, C. punctatus and C. micropeltus, were collected and successfully breed. Breeding and feeding technologies developed were standardized and disseminated to the beneficiaries. Cage culture activity was taken up for rearing of fingerlings in the reservoir with faster growth, high survival and reduces transportation losses and cost

Techno-economically feasible treatment of parboil rice mill waste followed by recycling through aquaculture was taken up in Gondia District in Maharashtra with a view to generate additional

Harvesting of fish and prawn of a polyculture pond in Mysore

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source of income to surrounding local people. Based on the standard design practice and laboratory studies, an effluent treatment plant and a fish pond were constructed. Experimental protocol was standardized on fish culture and a low-cost extruder based fish feed production unit fabricated. Fortified fish feed pellets with essential minerals and vitamins have been tested on fish and performance of standardized fish feed formulation was studied. Training programmes on feed preparation was organized. The demonstration of technology was undertaken for selected youths belonging to SC/ST weaker sections to train them in wastewater aquaculture and value addition of by-products from a typical parboil rice mill industry.

know-how for on farm fertility management, vermiculture and it's enrichment was undertaken in Rajastan through training programs conducted using agriculture waste, crop residues & cow dung. Around 6000 farmers were trained on various bio-dynamic preparations and composting. Linkage established with the exporters to enable direct market of cotton, banana, turmeric and pulses. Farmers fetches around 20-25% additional revenue for their organic certified produce. In Tirupati district of Andhra Pradesh, SHGs formed are creating awareness on productive use of agricultural waste for vermicomposting and mushroom cultivation and its advantages in income generation, employment, enhancing food nutritive value and pollution abatement. Training programmes on compost production were undertaken at Kerala benefiting around 400 farmers. A vermi-mela was organized for technology dissemination to the farmers at Aligarh for two days, which was attended by farmers and district agriculture extension officials.

Animal Husbandry Project was undertaken to train farmers on various aspects of brooding, hatching, production and reproduction using locally available materials in Mizoram. Quail farming has been popularized among the tribal community and school children of Mizoram, as it is easy for them to rear and earn. Trained beneficiaries started rearing Quails through adoption of technology packages through erection of bamboo housing and maintaining sex ratio. Pig rearing is also very common in Mizoram and some farmers are rearing both quails and pig together to earn extra income.

Biocontrol Agents Popularization programme on use of Trichoderma spp. was undertaken at National Botanical Research Institute, Lucknow to educate and train farmers using cheap agricultural wastes. Mass multiplication of Trichoderma was undertaken using locally available raw material, protecting high value crops against several soil-borne diseases. Packets of mother culture of potential strain of Trichoderma were distributed to the farmers along with the extension material.

Organic Farming Aspects on organic farming through dissemination of

Mushroom Cultivation The activity was promoted as an alternate income generation source in Tamil Nadu utilizing locally available agriculture residues, coir pith, mat, banana dried leaves, handloom waste and paddy straw available at the villages. Various aspects on mushroom cultivation such as spawn preparation, selection of raw materials, bed preparation, harvesting, processing and sale of mushroom were demonstrated. Around 500 beneficiaries were benefited through cultivation of mushrooms, product preparation, commercialization and marketing etc. The training programmes on oyster mushroom cultivation were undertaken for SC/ST and Weaker

Quail farming by tribal folk in Mizoram

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section in the Western Dun Valley to benefit around 234 people from 18 villages of Sahaspur Block of DehraDun. A commercial spawn production unit was established and marketing linkages were established. Doon Valley Mushroom Cooperative is helping the beneficiaries in marketing the produce in the local market. The beneficiaries are earning a good income through sale of their produce in addition to consumption in their daily meals to improve their nutritional status. Medicinal Plants and Plantation Crops Herbal plantation production through nursery

Aspects on biodiversity conservation were pursued to improve the livelihood of communities dependent on forests. High yielding varieties viz. large cardamom, black pepper, ginger were introducted for farmers and cultivation was promoted in Trichy district. Self-help groups were established for successfully producing and selling the compost. Farmers were trained on scientific cultivation of medicinal plants, organic farming, water management techniques & marketing strategies. Farmers realized lucrative income through cultivation of medicinal plants as compared to conventional cash crops. Sensitization programme was undertaken for SHGs to introduce non-timber forest produce collectors and herbal healers at Tirupati District, Andhra Pradesh. Model for development of herbal medicines and its cultivation in kitchen gardens were demonstrated to identified SHGs. Training programmes were conducted on herbal medicine preparation, nutritive foods, sustainable harvesting techniques and cultivation of MAPs. Model manufacturing units are being established for manufacturing licensed medicines through tribal involvement. Integrated horti-forest herb cultivation was undertaken in Nagalnd and Manipur through transfer of technology on cultivation of citronella, lemongrass, patchouli, bamboo and mushroom to benefit around 250 people. Demonstration units on essential oil, bamboo and mushroom cultivation were established. Identification of superior germ plasm and selection of plus trees after extensive survey in different parts of Madhya Pradesh was undertaken. Trees of Aonla, Achar, Harra, Baheda and Mahua are being used far clonal propagation. Seedlings of these species have been produced and budding were successfully carried out. Flowering and fruiting in Mahua was

achieved in one year old clonal plants through clonal propagation technique. Successful epicotyls grafting in Mohua was achieved. Training programmes on activation of bud material and collection of activated bud were imparted to the villagers. The knowledge of aftercare of clonal plants was also given to them through field trainings. Around 6000 clonal plants were distributed to 218 tribal beneficiaries. Programmes for Women During the year, 186 new proposals received (AprilNovember). Out of these, 20 were supported after their evaluation through three tier system. The Task Force reviewed 62 completed as well as ongoing projects. Over 16,000 women benefited though these projects under three major categories viz. (i) economic empowerment using softer biotechnologies, (ii) awareness on nutrition and health including importance of traditional food/healthcare and (iii) technical empowerment including capacity building, developing training modules etc. Over 8,000 women were benefited directly through biotech packages for floriculture, horticulture, cultivation of mushrooms, medicinal and aromatic plants, biofertilisers, organic farming, vermicomposting, sericulture, bee keeping, aquaculture, animal husbandry, poultry farming and value added food items. Approximately similar numbers benefited indirectly through programmes for human health and awareness generation. Highlights of the projects are given below : Medicinal and Aromatic Plants Approximately 800 women from J&K, Uttaranchal,

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Chandigarh, Gujarat and Karnataka states were trained in cultivation practices of medicinal plants of commercial importance as well as conservation of local species. They were also trained in preparation of semi-processed products. Major achievement of some these projects are : (i) Over 150 women from Lolab valley & Tangmarg were trained in cultivation, semi-processing & value addition of four medicinal plants. These women (are selling the semi-processed items in local market) while growing their own plants in their nurseries and are earning about Rs. 200/- per month/person. (ii) In another project, supported through S&T Council, Chandigarh, over 230 women were trained in cultivation of Amla along with lemon grass as an inter-cropping covering an area of about 25 ha. Interested women were also trained in preparation of Amla products for which marketing has been established through two agencies namely Unnati Biofresh and Sunstar Overseas Ltd. (At Supi, Uttaranchal, women were trained in vegetative propagation of high yielding variety of potato (Kufri jyoti and Kufri chipsona) and French bean) (iii) Through TERI, unit at Supi Uttaranchal women were trained in Orgainc cultivation of digitalis, swertia, oregano and lavender as the commercial crops for medicinal and aromatic importance. (iv) A training cum demonstration programme was arranged through Zandan Foundation for tribal people of South Gujarat. Interested 41 women were trained in cultivation of Kauncha, Ashwagandha, Senna and Kalmegh. (v) Through NGO, Rishi Herbal, Bangalore, 208 women have cultivated Coleus, Vinca, Patchouli, Solanum and Artmisia at their own land, covering an area of 45 acres. (vi) In an attempt to conserve and propagate local plants of medicinal and aromatic importance, a project was supported to H.P.K.V., Palampur. Through this project, 23 training camps were organized in Lahaul Spiti distt. and over 330 beneficiaries were trained in cultivation practices for Aconitum heterophyllum, Picrorhiza kukrrooa, colchicum luteeum and Valeriana jatamansi species.

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Women participating in medicinal plants cultivation

Women were also encouraged for entrepreneurship through cultivation of aromatic plants and their processing. Over 700 women from Uttaranchal, Himachal Pradesh and Madhya Pradesh were trained in cultivation of Patchouli, Lavender and

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aromatic grasses. These women are getting good return through sale of semi-processed essential oils procured at oil extraction units set up at their own land or community land. The high lights are as follows

Horticulture In an attempt to provide quality hybrid rice seed to the farmers, a project was supported to TNAU, Coimbatore, where the technology had already been perfected. Through this project, about 200 progressive farm women were trained in cultivation practices at their field covering an area of 4 ha; about 5000 kg of CORH-3 hybrid rice seed was produced which has provided additional profit of more than Rs. 10,000/ ha. In another project supported to TERI, New Delhi, women from Supi, Uttaranchal were trained in vegetative propagation of high yielding variety of potato (Kufri jyoti and Kufri chipsona) and French bean.

(i) At G.B. Pant University of Agriculture & Technology, Uttaranchal, tissue culture techniques has been perfected from leaf & nodal explants of Patchouli. Over 300 women were trained in complete technology including cultivation, harvesting and oil extraction. An extraction unit has been installed for the beneficiaries and market linkage established for their product. (ii) Under a roject supported at HRG, Shimla, over 100 women from 23 families of two blocks of Mandi distt. were selected for training in organic cultivation of lavender. They were provided with 50,000 plants raised in nursery covering an area of 5 ha. The beneficiaries were also trained in rearing those plants and collecting the spikes for Lavender processing. Individual beneficiary has been able to earn around Rs. 3000-4000/- per month through half ha; of land.

Organic Farming / Vermicompost Organic farming has been taken in a big ways This sector not only deals with application of various bioagents but production of biopesticides, biofertilizers based on local strains and vermicomposting. Thousand of women are benefited with several projects supported throughout the country. A examples are as follows : (i) A formulation of bio-control agent (Pseudomonas fluorescens + Trichoderma harzianum and P. fluorescens + Paecilomyces lilacinus) developed at Indian Institute of Horticultural Research, Hessarghata was tested at farmers field. About 300 rural farmers were trained to produce the formulation for the treatment of seeds, nursery substrate to be used for raising the seedlings of the horticultural crops in the polyhouses, nursery soil mixture and nursery beds in the open field conditions.

(iii) In another project supported to an NGO, at Bhopal, over 300 women belonging to SC/ST category were selected from 6 villages. They were trained in cultivation of aromatic grasses and oil extraction. A low cost extraction unit was also set up. Women are earning approx Rs. 200/- per month through sales of essential oils of mint and lemon grass. Floriculture

(ii) In another project supported to Sri Padmavati Mahila Vishwavidyalaya, Tirupati, 320 members from 8 villages of 3 mandals of Chittur Distt. were trained in preparation and marketing of phosphate solubilising biofertilizer enriched vermicompost. The individual beneficiary is earning around Rs.570/-.

Over 300 women from Shillong, Itanagar and Chandigarh were trained in complete packages of hardening of micropropagated plants of commercially important varieties of orchid viz. Dendrobium fimbriatum var. oculatum, D. longicornu, D.lituiflorum, C. pendulum, C. aloifolium and C. giganteum in their backyard. The beneficiaries were also trained in vegetative propagation of local species and post-harvest technologies. Also 100 women from Papum Pare Distt., Itanagar were trained in establishing hardening facilities using local resources such as bamboo.

(iii) Through JSS Krishi Vigyan Kendra, Suttur, Karnataka, farmers were trained in large scale production of vermicompost enriched with vesicular arbuscular mycorrhiza (VAM). The farmers were educated on the benefit of organic cultivation of various vegetable and horticulture crops. 30

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vermicompost units were set up at beneficiarie's land. The trained beneficiaries are selling their surplus vermicompost @ Rs. 3/kg and are able to earn additional income.

Mushroom Cultivation About 150 women from Tarra and Dondekhurd villages around Raipur were trained in cultivation and processing of oyster and milky mushroom. Out of these, 13 women were trained in spawn production technology. These women have established spawn production unit at Panchayat's land and are selling their product under the name of Swa Shakti Samiti. The unit has earned Rs. 68,000/- in six months.

(iv) A low cost biofertiliser production unit has been set up at Kuttathavaranpathi for the benefit of farmers from 8 villages under Rediarchatram block through a project supported at MSSRF, Chennai. (v) At UAS, Dharwad, 350 women from 12 villages were trained in vermicomposting. 70 units were set up at beneficiaries site. A hatchery for rearing the quality worms Eudrilus euginae, was set up at demonstration unit. The beneficiaries were encouraged to use their vermicompost at their farm. The surplus vermicompost was purchased by the University @ Rs. 2.5/kg and were demonstrated in various horticultural crops like pomegranate. On an average each family is earning Rs. 500-2000/- per month through the sale of additional vermicompost.

Through Manipur Science & Technology Council, a spawn production unit for Pleurotus and demonstration units for Oyster mushroom including few local species were set up. Selected 55 women were trained in mushroom cultivation including identification of edible variety, mushroom diseases and pests maintenance of spawn, harvesting and marketing. On an average each family is earning approximately Rs. 600/- per month by selling fresh mushroom @ Rs. 50 per kg. At TBGRI, Thiruvananthapuram, 25 strains of Pleurotus were collected from different places throughout Kerala state. They were screened for better yield, their availability throughout the year using RAPED and ITSRFLP methods. Seven training-cum-awareness fair were organized, over 10,000 people participated in these 3- day fair, out of which 2000 women were trained in Pleurotus cultivation at three satellite units. They were provided with quality spawn and assisted in setting up their units. 75 women are selling fresh mushroom @ Rs. 100/kg in local market.

(vi) Under a project at College Damoh, MP, over 1000 women were educated on the benefit of vermicompost, recycling of agrowaste and organic cultivation of vegetables and horticultural crops. Out of these, 200 beneficiaries from 8 villages were trained in production and application of Vermicompost, They were provided earthworms and assisted in setting up Vernicompost units at their land. The individual beneficiary is now earning Rs. 200-400 per month through the sale of compost. (vii) In another project supported at Bhubaneshwar through an NGO, 500 women were trained in converting coir pith to vermicompost, 5 demonstration unit set up at beneficiaries land and market linkages established after making 25 SHGs of trained women.

Bee Keeping A project was supported to an NGO, HESCO, Dehradun to promote entrepreneurship through bee keeping. Under this project about 400 women from two different altitude were selected and low cost wodden hives were introduced for lower altitude and upgraded wall hives in higher altitude. The lantana hives became very popular among the beneficiaries for its being cheaper as well as availability of the lantana in the region. They were also trained in valueaddition like extraction, purification, packaging for honey. The interested farmers were also trained in lantana box preparation. The trained women have already started their units and are selling their products in local market @ 150/- .

(viii) At Sri Sathya Sai Institute of Higher Learning, Anantpur, 40 women were trained in seed collection, drying, pulverizing and collecting neem oil by cold compression method. A neem processing unit has been installed at sub-centres. The beneficiaries have been trained to operate these units. On an average each beneficiary is earning around Rs. 6,500/- per month through the sale of neem oil.

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Poultry and Livestock Farming

aquarium, backyard breeding tanks for commercial and local of ornamental fish. The trained women are earning good amount through these projects.

In an attempt to strengthen livestock industry through biotechnology, projects were supported for improved production and productivity for cattle a project was supported to Veterinary College & Research Institute, Namakkal. A total of 200 women from 10 villages were trained in detection of mastitis and their control, low cost feed for better milk yield in cattle. Interested women were also trained in preparation of anti helminthic mineral blocks for sheep and goats. These women are earning good amount through the sale of these mineral blocks @ Rs. 35/- each. Also training was provided to selected women in entrepreneurship through poultry farming including low cost hatchery, feed and disease aspect. In order to strengthen the animal feed sector through protein and vitamin rich azolla, a project was supported to an NGO at NARDEP, Vivekananda Kendra, Kanyakumari. Under this project 1000 women were trained in low cost technology for azolla production and its application as supplement animal feed. The project had shown significant improvement in production & productivity in poultry & cattle. Over 1000 women from Mutharasanallur, Pirattiyur, Nachikurichi, Maruthanddakurichi and Melavaaladi villages of Tiruchirapally were trained on desi chicken rearing and incubation using custimised hatchery unit. Out of these 100 women have set up a small poultry farm and are earning Rs. 35,000/- per annum.

Sericulture Under project for economic empowerment to women in through Sericulture projects are supported at Karnataka, Andhra Pradesh and West Bengal. A total of 130 women were trained in organic cultivation of Mulberry, rearing of silkworm including disease management. They were also trained in preparation of various decorative items using the cocoon waste for additional income through the by-product of silk industry. Healthcare Through a project supported to Mahavir Hospital, Hyderabad, attempts were made to create awareness on lead toxicity to women and children working in paints or batteries industries. The blood samples from these population were screened for chemical, biochemical and clinical data. The affected population were also provided counseling and treatment. Kowing the fact that Vitamin D deficiency affects more to growing children and women a project was supported SGPGIMS & KGMC, Lucknow, Through the project, 200 girls from 6 villages were selected on random bases. Their detailed health status and serum alkaline phosphatase (SAP) level recorded. The affected population was advised to take 600 unit of calcium in the form of Calcium Carbonate and at least 15 minutes exposure to sun. As fluorosis (dental or skeletol) is endemic in large parts of UP, Karnataka, Tamilnadu, Andhra Pradesh and Rajasthan a project was supported to Saraswati Dental College, Lucknow. Through this project, attempts were made to find out the genetic linkages among the children with skeletol or dental fluorosis. A population of 5024 from 7 villages around Lucknow were screened and it was noted that 28% of adolescent girls and 43% of pregnant women have biochemical osteomalacia. Through a project supported at Kidwai Memorial Institute of Oncology, Bangalore, a total of 3,000 women were screened for Carcinoma Cervix using Pap Smear test; 28 positive cases were referred to hospital for detailed diagnosis and treatment.

Aquaculture Through College of Fisheries, Mangalore, a dedicated demonstration facility has been installed at Bengre village having 16 racks with the capacity of 20 kg fish each and dry fish storage cabinets. Approximately 140 women were trained in various activities for maintaining a fish village by these fisherwomen. About 40 women were trained in hygienic fish drying and packaging, 37 women in aquarium fish breeding and feed pellet making, 20 women in Laboratory analysis of dry fish, 20 women in laboratory analysis of water & ice and 26 women in handling computers. An attempt to train women in entrepreneurship through ornamental fish three projects were supported at T.N., Orissa and West Bengal. Over 700 women were trained in the preparation of pelleted fish feed, glass tanks, mother

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department supported 6 projects for rehabilitation of Tsunami affected people and the area. These projects were able to provide employment opportunities to affected people at Tamilnadu, Pondicherry and Andaman & Nicobar Islands through softer biotechnology packages like cultivation of horticulture crops, vegetable, seaweed, poultry & goat rearing etc. Over 600 people were trained in various technologies and setting up their own units for sustainable employment. The project supported for prevention of outbreak of Malaria after Tsunami attack had made a significant impact to the people at Car Nicobar. The project supported for ecorestoration of devastated coastal land at Andaman Islands is an attempt to provide bioshield for the coastal line. The beneficiaries trained for softer biotechnologies for economic empowerment through poultry rearing, seaweed cultivation and organic farming have already started earning a good amount through the sale of their produce.

Fruiting in Dhingri bags

Network Programme for Prasad Kit Under the Network Project for Prasad Kit, emphasis was given on involvement of community in preparation of offerings using local bioresources and negotiations with authorities of local shrines for marketing. Over 150 local people got employment through five ongoing projects around major shrines like Male Mahadeshwara Temple & Biligiri Rangan Temple at Bangalore in Karnataka, Sri Bade Haniman Ji shrines & Hanuman Temple at Allahabad, U.P., Kaliyar Sharif Dargah, Udhampur and churches of Thrissur. Through project supported at Ashoka Trust for Reseacrh in Energy & Environment (ATREE), Bangalore, women from 56 SHGs from Male Mahadeshwara Hills (MM Hills) and 17 SHGs from Biligiri Rangaswamy Temple (BRT) were trained in ladoo preparation, raising nurseries of sacred plants including sixteen native species. In another project supported to Parivartan Vikas Sansthan, Udamsingh Nagar, Uttaranchal, 60 women from 4 villages around Kaliyar Sharif Dargha were trained in preparation of sweet bread, the traditional tabarruk, ladoos from maize and rice. The beneficiaries were also trained in incense making as well as candle making. Waqf Board of Dargah assured the marketing of all the products prepared by these women. It is expected that 5 new projects will be supported during this year.

Programmes for Rural Areas Biotechnology-based Programme for rural areas has benefited around 35,000 people through 30 ongoing projects and 8 new ones during the year. The projects supported cover cultivation of medicinal and aromatic plants, production of biofertilizers and biopesticides, organic farming, aquaculture and ornamental fish breeding, mushroom cultivation, genetic counseling, bee keeping and product development etc. Universities, Krishi Vigyan Kendras and voluntary/non-governmental organizations have been involved in the implementation of the projects. The target group received the benefit of hands-on training and field demonstrations to bring greater awareness. Ornamental Fish Breeding An integrated aquafarming project was undertaken at two districts namely Puri and Khurda of Orissa by undertaking activity in water spread area of about 6.9 ha. Fish-poultry activity was undertaken with mushroom, vegetable cultivation and plantation of crop like banana and drumstick. Ornamental fish breeding units were set up through introduction of live bearers such as Guppy, Balloon molly, Black molly, Sword tail and egg layers like Gold fish, Rosy barb and Gourami. Training programmes were

Programme for Rehabilitation of Tsunami Affected People After the Tsunami disaster in December 2004, the

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conducted on mushroom cultivation, brooding and management of ducklings and chicks, vaccination, aquarium setting, marketing of ornamental fish, land preparation and management for nursery plants etc.

reliable sources and building certified nurseries and availed subsidy besides the benefits that will accrue from the sale of these saplings. Black pepper and ginger saplings were distributed amongst the farmers for cultivation. Square Metre Vegetable Garden was introduced as a green house technology to enabled farmers to cultivate a variety of vegetables, mainly: coriander, spinach, fenugreek, beetroot, carrot, cauliflower, tomato, capsicum, broccoli, lettuce and round chilli from a small piece of land for higher economic return. Vermicomposting approached was introduced and five units were established involving five self-help groups for producing and selling the compost.

Bee Keeping Training and demonstration was conducted on bee keeping and honey processing in two blocks of Jagdeeshpur and Musafirkhana in Sultanpur District of UP. Ninety farmers were trained on bee keeping, honey production, value addition, product making and marketing. Trained beneficiaries were introduced to banking schemes for financial support. Kisan Credit Cards were provided through banks and National Kisan Clubs were formed by the trainers for further development. Trainers' training was organized.

Product Development A protocol was standardized for oil extraction from trash fish and its purification at Kanyakumari District, Tamil Nadu. Chemical analysis such as acid value, free fatty acids, -carotene and HUFA content were analyzed. Vitamins A, D, E and K compared with the values obtained for commercial cod liver oil. Dried fish were powdered and different ingredients and vitamin mineral premix were used to prepare poultry feed by altering the ratio of fish waste meal. Capsule formation and shelf life of fish oil capsule are being evaluated and efficacy of the prepared poultry feeds is being tested. Training programmes were conducted to disseminate the technology to the target community.

Medicinal Plants Organic cultivation of medicinal and aromatic plants was undertaken in Uttarkashi District of Uttaranchal for income generation and sustainable development of the rural community. Nursery was established and the planting material was distributed to the farmers in three villages, Ranadi, Hitandu and Matholi to motivate them to cultivate medicinal plants. The species under cultivation were Coleus forskholii, Rauvolfia serpentina and Pelargonium graveolens. Farmers' visits were arranged to the nursery and learn about the prospects of cultivation of medicinal plants. About 25 farmers showed interest and agreed to undertake the cultivation in their respective fields. To conserve biodiversity and improve the livelihoods of communities dependent on forests, a programme was undertaken in Darjeeling, West Bengal. High yielding varieties were introduced and depending upon the suitability of land and climatic condition, different feasible interventions were designed with the involvement of target population. Around 6250 seedlings of large cardamom were raised by 30 households and are being sold as well as transplanted in the beneficiaries' field. Progressive farmers are raising multiple saplings procured from

Genetic Disorders The programme was taken up in Balanagar Mandal of Andhra Pradesh to create awareness on genetic disorders in rural folk and training the primary health centre workers about identification and prevention of genetic disorders. Demonstration and training programmes were conducted to benefit Anganwadi workers and multipurpose health assistants. Posters and handouts on genetic disorders in English and local language were distributed. The data obtained from the survey on prevalence of genetic disorders has been documented.

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Chapter- 7

Bioprocess and Product Development Biotechnological Approaches for Food and Nutritional Security

analyze the expression of Immunoglobulin receptors (IgG & IgA) on colonocysts isolated from stool before and after intervention by flow cytometry. Network R&D programme on identification, screening and mass production of probiotic strains and placebos for clinical trials was initiated. A cell culture-based assay has been developed to evaluate attenuation of the inflammatory response by test probiotic bacteria. Two human epithelial cell lines and a human macrophage cell line were grown in monolayers and the effects of pathogenic bacteria as well as individual bacterial components including lipopolysaccharide and muramyl dipeptide on chemokine production was tested. Interleukin-8 was identified as the most prominent inflammatory chemokine. Probiotic bacteria from various sources were tested to identify those which suppressed IL-8 production in response to stimuli. Eight different bacteria (4 Lactobacilli and 4 Bifidobacteria) were identified for inclusion in a probiotic cocktail. One billion of each of these bacteria were administered daily to Swiss albino mice before induction of experimental colitis. Compared to controls that did not receive the probiotic cocktail, treated mice showed significant reduction in ulceration and increase in healing suggesting that the probiotic modulated inflammation and repair in colitis. Analysis of the cytokine and chemokine mRNA response in these mice using reverse transcriptase qPCR is underway. Pathway-targeted human inflammatory chemokine array, analysis revealed that enteropathogen upregulated neutrophil chemoattractants, while downregulating monocyte and macrophage chemoatractant proteins. The probiotic were observed to reduced neutrophil chemoattractant response without altering monocyte and macrophage chemoattractant response.

The main emphasis during the year was on development and use of nutraceuticals and probiotics for holistic health. Brain storming sessions were convened to generate inter-institutional projects for studying the relationship of nutrition with reference to chronic diseases and on cellular & molecular nutrition. Probiotics for holistic health : a) Molecular characterization of Probiotics: Human faecal samples, human milk and raw milk samples from different sources were used for the isolation of Lactobacilli. Molecular techniques for identification and typing of indigenous probiotic cultures have been standardized to conserve probiotic strains ex situ and create a well catalogued probiotic culture bank for their commercial and research uses. b) Immuno-modulatory effect of Probiotics: Colonization and immuno modulatory effect of probiotic strains in Indian children are being studied. The levels of cytokines (IL-1,IL-6, IL-10 and TNF-alpha) in stool samples of subjects, before and after intervention were monitored to

c) Probiotics for reproductive health: Phase-I clinical trials on safety and capability of

Induction of chemokines by V. cholerae

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probiotic selected strains of Lactobacilli to colonize in vagina for reproductive health of women have been initiated, to determine whether Lactobacilli can colonize in vagina of women depleted of such organisms. The safety, lack of side effects and efficacy of replenishment for preventing episodes of vaginosis are being determined.

4.91 ± 0.40, 21.68 ± 2.69 and 3.91 4.91 ± 0.26 ppb, respectively. Further work on the validation of ELISA method with microbiological assay employing Lactobacillus leichmannii as an assay organism is in progress. f) Food mixtures with Probiotics: Standardization and quality evaluation of banana based probiotic fermented food mixtures are being accomplished. Initial standardization of the 14 different combinations of food mixtures based on raw banana flour, defatted soya flour, green gram flour, tomato, papaya and mango have been carried out. Banana flour was the main ingredient in the food mixture which ranged from 50 to 70% in all combinations. A total of 56 food mixtures with the above raw ingredients were developed and subjected to organoleptic evaluation and out of this, 14 combinations were selected based on their sensory qualities, for further studies.

d) Bacteriocins from Probiotics: Natural isolates of lactic acid bacteria isolated from the rhizosphere were screened for bacteriocin production and , strain LR/14 has been found to be potential bacteriocin producer. This strain was identified as Lactobacillus plantarum LR/14 by biochemical tests and 16SrDNA sequencing. The crude bacteriocin of strain LR/14 was heat and pH stable with shelf-life upto 2 years was checked. The mode of action was observed to be bactericidal with broad host range against related as well as some food-borne pathogens like Listeria monocytogenes, Staphylococcus and Salmonella.

Nutraceuticals a) Reduced calorie fats: Various novel reduced calorie fats have been developed. The conditions for the transesterification were optimized at laboratory scale and further upscaled. In a typical reaction, sunflower oil was transesterified with ethyl behenate in presence of specific enzyme Lipozyme. Reaction products from five similar reactions were pooled and distilled. Various structural lipids have been prepared and the products have been analyzed for RP-HPLC and physico-chemical properties. b) Tea Polyphenols: Theaflavins, (TF) is a high valued neutraceutical found in Black Tea in limited amounts, through the bioconversion of catechins from tea by the immobilized polyphenol oxidase (PPO) enzyme system native to tea. Identification of suitable matrix for the enzyme immobilization, isolation of tea PPO, kinetic studies of the immobilized enzyme system, number of turnovers of immobilized enzyme system to Theaflavins, stability of the immobilized enzyme matrix, isolation of tea PPO in soluble form, alogwith characterization of Theaflavins product through UV, IR and mass spectrometery, were completed. 100% conversion of tea catechins to Theaflavins were achieved with the system with repeated turnover of

E-Test (VA & CIP) - probiotic [C]

e) Probiotics as Potential Source of Vitamin B12: Various Propionibacteria are being explored as potential source of Vitamin B12 and functional probiotic ingredient in a dairy based nutraceutical formulation. Twenty two cultures were identified as dairy Propionibacteria. Molecular Characterization with the help of genus-specific primers from the 16S23S rRNA intergenic spacer regions for dairy propionibacteria, isolated organism are under process. The estimation of Vitamin B12 in milk has been standardized using immunosorbent method. Using ELISA kit, the values of vitamin B12 in cow milk, buffalo milk and goat milk were observed to be

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recorded 50 times batch run without any loss of efficiency of conversion rate.

d) Beta-carotene from Alga: A total of 272 salt pans seawater samples were collected along the coasts of Andhra Pradesh and Tamil Nadu. Most of the samples contained the quadriflagellate alga like Tetraselmis. Only a few samples known to contain a mixture of naked green algal species Dunaliella, thus indicating the rare occurrence of the species. The algal colonies appeared after 20 days were isolated and maintained. Dunaliella strains were investigated for their growth study and total carotenoids and potential strains have been identified for enhanced beta carotene production. In another study, carotenoids and fatty acid composition of some selected Indian brown and red seaweeds were analysed and phenolic content and antioxidant activity of extracts from seaweeds were also analysed. The brown seaweeds analysed included Sargassum marginatum, Padina tetrastromatic and Turbinaria connoides; while, the red seaweeds included Acanthophora spicifera, Euchema kappaphycus and Gracilaria folifera. The lipid content in various seaweeds varied between 1.0 and 3.0% (dwb) with P. tetrasromatica showing the highest content. Glycolipids were found to be a major lipid class, followed by neutral and phospholipids, in brown as well as red seaweeds. Brown seaweeds were found to contain fucoxanthin as the major pigment. Fucoxanthin content of fraction obtained by column chromatography was found to be >90%. Among the various solvent fractions obtained, butanolic fraction of E. kappaphycus was found to contain the highest. The red seaweeds showed relatively higher antioxidant activity than brown seaweeds as indicated by radical scavenging activity.

In another study, potential of black tea and its constituents in reversal of multidrug resistance and as bio-enhancer are being studied. The non-toxic concentrations of tea polyphenols were found to effectively reverse the drug resistance as evident by p-glycoprotein expression (through western blotting) and increased drug uptake of doxorubicin (through flow cytometry). Potential of tea polyphenols as bioenhancer in cancer chemoprevention studies, experiments on hepato-carcinogenesis and skin carcinogenesis were undertaken revealed that polyphenolic constituents of both varieties of tea, green and black are providing significant protection against cancer induction in vivo models. c) Enhanced Omega-3 fatty acid content in foods: Alpha linolenic acid (ALA) is an essential omega-3 fatty acid required in the diet that gets converted in the body to Eicosapentaenoic acid and Docosahexaenoic acid that form precursors respectively for eicosanoids and membrane components especially, brain and retina. Many microorganisms can desaturate the abundant plant linoleic acid (LA) to ALA that is poorly present in plant oils. Work to develop recombinant yeast strains capable of converting LA to ALA extracellularly was carried out. More than 100 yeast isolates were collected, mostly from the premises of plant oil mills from different parts of the country, and grouped into forty independent types based on colony and cellular characteristics. Genomic DNA was isolated from the forty different strains and used for screening of omega-3 desaturase gene by PCR. Primers were designed based on sequence homology of a published yeast omega-3 desaturase with those present in algae, fungi and plants. When compared across different phylogenetic groups there is over all poor sequence similarity (23%-29%), with the exception of cyanobacetria and Arabidopsis which showed 45%-50% similarity. Three aminoacid clusters, 16-22 aa long with 45%-50% similarity among all the known desatuarse, was used for designing primers. Seven out of forty samples yielded ~600 bp amplicon. These were further confirmed by PCR using a different set of primers and 2 DNA hybridization studies. The PCR positive strains were able convert LA to ALA.

e) Microbial Production of Nicotinamide: Nicotinamide is one of the important vitamins B3, which is mainly used in pellagra and niacin deficiency. It also has an antioxidant and cytoprotective effect. The other form of vitamin B3 is nicotinic acid, which is equally important as nicotinamide. In this project a large number of microbes have been screened for both nicotinamide and nicotinic acid production. The nitrile hydratase activity of these strains was determined by assaying the enzyme activity. The HPLC method was developed to determine 3-cyanopyridine, nicotinamide and nicotinic acid. Good nitrile

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hydratase activity was shown by eight strains among the different strains screened for this purpose. Reactions conditions are being optimized with the isolated strains.

plants was analyzed.Protocol for solid phase extraction and purification by gel-filtration chromatography of microbial siderophores was devised. The protocol for production and purification of phytosiderophores was standardized and trials are in progress. Three (microbial siderophores, phytosiderophores & synthetic)-Fe chelates were made and its comparative account on absorption in wheat plants have been assessed. Field trials were conducted to test efficacy of siderophorgenic pseudomonads wherein yield, quality and iron content in wheat grains was observed to be improved by almost 18-23%.

f) Folate Supplementation: The study investigated the optimal level of folate supplementation in the presence and absence of Vit. B12 using rat model. In the first protocol, there were three treatment groups (12% protein with folate supplementation at 2, 4 & 8 mg/kg diet) and one control group (18% protein). There was no difference observed in brain weights of pups from different groups at birth. In contrast, males pups from both 4 and 8 mg folic acid supplemented groups had significantly lower (p