CryoLetters 28(1), 13-22 (2007) CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK
CRYOPRESERVATION OF PEACH PALM ZYGOTIC EMBRYOS Douglas A. Steinmacher1,2, Cleber W. Saldanha3, Charles R. Clement4 and Miguel P. Guerra1* 1
UFSC, Universidade Federal de Santa Catarina, Dept Fitotecnia/LFDGV, 88040-900 Florianópolis, SC, Brazil 2 Present address: Department of Crop Science and Plant Ecology, Biocentre Klein Flottbek and Botanical Garden, Ohnhorststr. 18, 22609 Hamburg, Germany. 3 UFSM, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil. 4 INPA, Instituto Nacional de Pesquisas da Amazônia, Cx Postal 478, 69011-970 Manaus, AM, Brazil * Author for contact
[email protected] Abstract Cryopreservation is a safe and cost-effective option for long-term germplasm conservation of non-orthodox seed species, such as peach palm (Bactris gasipaes). The objective of the present study was to establish a cryopreservation protocol for peach palm zygotic embryos based on the encapsulation-dehydration technique. After excision, zygotic embryos were encapsulated with 3% sodium alginate plus 2 M glycerol and 0.4 M sucrose, and pre-treated or not with 1 M sucrose during 24 h, followed by air-drying. Fresh weight water contents of beads decreased from 83% and 87% to 18% and 20% for pre-treated or nonpretreated beads, respectively, after 4 h of dehydration. Sucrose pre-treatment at 1 M caused lower zygotic embryo germination and plantlet height in contrast to non-treated beads. All the variables were statistically influenced by dehydration time. Optimal conditions for recovery of cryopreserved zygotic embryos include encapsulation and dehydration for 4 h in a forced air cabinet to 20% water content, followed by rapid freezing in liquid nitrogen (-196°C) and rapid thawing at 45°C. In these conditions 29% of the zygotic embryos germinated in vitro. However, plantlets obtained from dehydrated zygotic embryos had stunted haustoria and lower heights. Histological analysis showed that haustorium cells were large, vacuolated, with few protein bodies. In contrast, small cells with high nucleus:cytoplasm ratio formed the shoot apical meristem of the embryos, which were the cell types with favorable characteristics for survival after exposure to liquid nitrogen. Plantlets were successfully acclimatized and showed 41±9% and 88±4% survival levels after 12 weeks of acclimatization from cryopreserved and non-cryopreserved treatments, respectively. Keywords: Bactris gasipaes, germplasm conservation, in vitro culture, acclimatization. INTRODUCTION Peach palm (Bactris gasipaes Kunth) was domesticated for its fruit and is widely distributed in the Neotropics (20). Today it is important for its fruit, popular throughout its traditional distribution, and for its heart-of-palm, a gourmet vegetable extracted from the 13
apical meristem. Various Latin American institutions have breeding programs for one or both uses, for which they have developed ex situ collections of its genetic resources. To date, ex situ conservation is accomplished only in field collections, which present several advantages for the plant breeder, but which are extremely vulnerable for various reasons (6). Unfortunately, peach palm seeds are recalcitrant (4), losing their viability after short periods of storage, so long-term ex situ conservation seed collections are not feasible. In this situation, cryopreservation is an alternative technique for long-term conservation. This technique maintains cell viability with extremely reduced metabolism at ultra-low temperatures, usually with liquid nitrogen (9). Different methods to ensure cell viability after exposure to ultra-low temperatures have been described (9). The encapsulation-dehydration technique (7) uses synthetic seed technology and includes two cryoprotection treatments: dehydration by evaporation and by osmotic potential. This technique was adjusted by Sakai et al. (26) with the addition of sucrose and glycerol into the alginate matrix. In addition, to improve the cells’ tolerance to dehydration, the use of a pre-treatment with sucrose at concentrations ranging from 0.3 to 1.5 M has been used (7,8,11,15,17,26). This improvement increases cell membrane stability (9), as well as accumulation of polysaccharides (3), and results in the fragmentation of large vacuoles into smaller ones (14). In palms, the development of cryopreservation protocols has been described for apical meristems of Phoenix dactylifera (2), plumular tissue of Cocos nucifera (16) and somatic embryos of Elaeis guineensis (8,10). Additionally, the use of zygotic embryos is also considered a good strategy and protocols have been developed for Cocos nucifera (1), Elaeis guineensis (12), Areca catechu (25), Veitchia merrillii and Howea fosteriana (5) using this explant source. The present study aimed to develop a protocol for the cryopreservation and plantlet regeneration from zygotic embryos of peach palm using the encapsulation-dehydration technique, in order to establish a long-term conservation technique. Histochemical analyses were used to better examine the morphogenetic responses. MATERIALS AND METHODS Plant material Open-pollinated seeds were obtained from plants at RECA (Reflorestamento Econômico Consorciado Adensado), Nova Califórnia, Rondonia, Brazil. The germplasm is a mixture of the Putumayo and Pampa Hermosa landraces. The kernels (i.e., zygotic embryo and attached endosperm) were surface-sterilized for 1 min in 70% ethanol, followed by 40 min immersion in sodium hypochlorite solution [60% commercial bleach (2.0-2.5% active chlorine) and 40% water, plus one drop of Tween 20® to each 100 ml]. The kernels were rinsed three times in sterile distilled water and the zygotic embryos were aseptically removed from the kernels under a stereoscopic microscope. The zygotic embryos were maintained on MS (22) culture medium, supplemented with Morel vitamins (21) and 0.3 M sucrose. The pH was adjusted to 5.8 prior to the addition of 7.0 g L-1 agar (Merse®) and autoclaved at 121°C (1 kgf cm-2) for 15 min, after which 30 mL culture medium was transferred to Petri dishes. In each Petri dish 25 zygotic embryos were inoculated and the cultures were kept in a dark chamber at 25 ± 2°C overnight. This procedure allowed selection of intact sterile zygotic embryos. Procedures for encapsulation and cryopreservation Intact zygotic embryos were selected and encapsulated in an alginate matrix composed of 3% sodium alginate, 2 M glycerol and 0.4 M sucrose (26). The beads were formed by 14
polymerization in a 100 mM CaCl2 solution for 30 min. The beads (4-5 mm) were then rinsed with distilled water and excess water was removed with a filter paper. Usually the zygotic embryos were localized at one extremity of the bead, but always inside of them. All the solutions used were autoclaved at 121°C (1 kgf cm-2) for 15 min. To evaluate the pre-treatment effect, MS liquid culture medium with 1 M sucrose was employed, and 100 mL of the culture medium was transferred to Erlenmeyer flasks (250 mL) and autoclaved for 15 min at 121°C. Twenty-four beads were placed in each flask and were kept in darkness at 25 °C on a horizontal agitator at 80 rpm for 24 h. To dehydrate the beads, they were placed in open Petri dishes and exposed to the airflow from the sterile chamber for variable periods (0, 2, 4 or 8 h). The curve of water loss was calculated by measuring the initial and final fresh weights of the samples during the dehydration. Drying 15 beads containing zygotic embryos at 105 °C for 48 h allowed the estimation of the dry weight. Water contents are presented on a %FW basis. In order to evaluate the cryopreservation effect, half of the beads were transferred to liquid nitrogen, while the remaining half was used to evaluate the germination potential of the non-cryopreserved zygotic embryos. For cryopreservation, the dehydrated beads were transferred to cryotubes (1.8 mL) and rapidly immersed in liquid nitrogen, where they remained for at least 24 h. Subsequently, the tubes were thawed in a water bath at 45 °C for 2 min. After the treatments all beads were submitted to a rehydration treatment (17) carried out in the cryotubes with MS culture medium plus 1 M sucrose. After 30 min, 0.5 mL of the culture medium was replaced by MS culture medium with 0.1 M sucrose for 10 min. This procedure was repeated three times. The zygotic embryos were then removed from the rehydrated beads and transferred to germination conditions. In vitro germination procedures The zygotic embryos were transferred to MS culture medium supplemented with Morel vitamins and 1.5 g L-1 activated charcoal. The pH was adjusted to 5.8 before the addition of 7.0 g L-1 agar (Merse®), and 10 mL culture media was transferred to test tubes and autoclaved at 121 °C (1 kgf cm-2) for 15 min. Two zygotic embryos were inoculated in each test tube and the cultures were maintained in a growth room at 26±1 °C with a 16 h photoperiod of 50-60 µmol m-2 s-1 provided by cool-white fluorescent lamps (Sylvania). The number of embryos turning brown in vitro was used to calculate percentage embryo oxidation. Histochemical analyses Samples were collected at different steps of the protocol and fixed in 0.1 M phosphate buffer (pH 7.3) containing 2.5% paraformaldehyde. Thereafter, samples were dehydrated in a graded alcohol series (30-100%), embedded in Leica Historesin® and 5 µM sections were obtained with a manual rotatory microtome (Slee Technik), placed on a glass slide and fixed by heat. The samples were stained with Coomassie brilliant blue R250 (Sigma®). All samples were collected from only one block and prepared together to avoid technique artifacts. Plantlet acclimatization Regenerated plantlets taller than 4 cm were selected for acclimatization. The haustorium, when present, was removed and the root system was reduced to 2 cm in order to facilitate management and to stimulate the development of new roots. The plantlets were rinsed in tap water to remove the culture medium and were transferred to commercial substrate (PlantMax® - electrical conductivity 1.5-2.0 dS m-1) with carbonized rice straw (1:1) in expanded polystyrene trays, containing 5x5 cm cells. The trays were placed inside a plastic box covered with glass to allow the entry of light and reduce water exchange. These plantlets were kept 15
under 16 h light periods with 100-130 µmol m-2 s-1 light intensity provided by cool-white fluorescent (Sylvania) and high pressure sodium vapor lamps (Empalux – VST). Gradual opening of the glass cover started after four weeks and the glass cover was completely removed 12 weeks after transplanting. Statistical procedure The present study was arranged in a tri-factorial 2x2x4 consisting of cryopreservation or not x pre-treatment with 1 M sucrose or not x four dehydration times. The experiment was conducted in a complete block design, with five blocks, where each block was represented by one seed tree (genotype). Each treatment contained six repetitions per block. Three months after inoculation the variables oxidation, germination and plantlet height were evaluated. The data were transformed to ( x + 5) and submitted to variance analysis; Duncan’s test at 5% significance was used to compare means. For the plantlet acclimatization the survival level was evaluated after 12 weeks and presented as mean ± standard error. RESULTS The cryopreservation treatment and the pre-treatment significantly reduced (p
