Cryopreservation of somatic embryos An overview

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1 Indian Journal of Biotechnology Vol 4, January 2005, pp Cryopreservation of somatic embryos An overview Sonali Dixit Sharma Avestha Gengraine Technologies Pvt. Ltd., Discoverer 9th Floor, Unit 3, International Tech Park, Bangalore , India Received 8 September 2003; revised 9 December 2003; accepted 22 December 2003 Development of methods for long-term conservation of somatic embryos is important because they are potential explants for manipulations and conservation of in vitro maintained germplasm. This review discusses about the cryopreservation of somatic embryos with special reference to techniques involved and genetic stability of cryopreserved germplasm. Keywords: cryopreservation, genetic stability, somatic embryos IPC Code: Int. Cl. 7 A 23 B 7/024, 7/055, 9/10 Introduction Somatic embryogenesis is a prerequisite for economic multiplication of plants either used for commercial propagation or breeding programmes; raising fully transformed plants after mutagenesis or gene transfer. It refers to the initiation of embryos from previously differentiated somatic cells, which can be usually induced by the auxin analogue 2,4-D 1. Unlike cells of other eucaryotes, almost all plant cells have the capacity to become embryogenic under defined conditions. This characteristic relies on reprogramming of gene expression and triggers many structural changes, which are similar to those found in zygotic embryos 1. Somatic embryos are also important for management and conservation of in vitro produced materials for they give an appreciably higher rate of multiplication compared to any other clonal propagation system. Further, somatic embryos are of significance for germplasm conservation of those species where shoot tip is either recalcitrant (Ipomoea batatas) or in vitro culture is not possible because of larger size (Hevea brasiliensis, Phoenix dactylifera, Elaeis guineensis, Coffea canephora) 2. Furthermore, the embryos originate from a single somatic cell and, thus, can maintain genetic fidelity of the plants from which they are obtained. Additionally, they make a potential candidate for transformation as regeneration from them is comparatively less cumbersome, which in many cases becomes a limiting factor in producing Author for correspondence: Tel: / ; Fax: sonali-dixit@lycos.com transgenics. Somatic embryos have been recognized as an attractive tool for synthetic seed production and mass propagation of elite genotypes 3. Further, application of biotechnologies based on protoplast isolation and fusion relies on embryogenic cell lines, which are also suitable for transformations 4. Somatic embryo production is being steadily increased, as essential factors become better understood 5. However, prolonged maintenance of tissues in culture reduces its embryogenic potential and increases chances of genetic instability 6. Cryopreservation, i.e. storage of plant propagules at ultra-low temperature (-196 C) usually that of liquid nitrogen (LN), is the only available option for safe, cost-effective and long-term conservation of those tissues that would be retrieved in ready form for further manipulation or use. Cryopreservation protocols have been developed for more than 80 different plant species cultivated under various forms, including cell suspensions, calluses, apices, somatic and zygotic embryos. Significant cryopreservation clone banks have been established for somatic embryogenesis clonal propagation programs in Canada, France, New Zealand, Sweden and the USA. The most recent information indicates the application of this technology to 7 species each of Picea and Pinus, 4 species of Larix, and 1 species each of Abies and Pseudotsuga, as well as several hybrids of Larix and Picea. An estimated eight to ten thousand genotypes have been stored worldwide 7. Cryopreservation being cost-effective offers advantages over other conservation strategies. The cost incurred in standardization of protocols substantially contributes to the total cost for the cost

2 48 INDIAN J BIOTECHNOL, JANUARY 2005 of components involved in cryopreservation have been distributed over a period of 50 years as accessions once kept are believed to remain viable for a indefinite period of time. The setting up of a cryopreservation facility requires just a cryo-tank in addition to the main tissue culture facility. However, LN and chemicals contribute to a variable cost. Thus, cryopreservation is much cost-effective, safe and reliable in comparison to other storage techniques, such as in vitro conservation, slow growth, ex situ conservation, filed gene banks, etc 8, and provides an efficient tool and logical choice for long-term conservation of plant genetic resources. Storage of Embryogenic Tissues at Different Temperature Regimes Several reports are available pertaining to storage of embryogenic tissues at ambient and below ambient temperatures. Caplin 9 reported that mineral oil overlays of 6-45 mm depth on carrot and grape tissues, cultured at 26 C for up to days, greatly inhibited tissue growth. Although, the tissues remained green and resumed normal growth when transferred to fresh medium. Later, using mineral oil overlays, tissue cultures of various medicinal plants were stored for 4-6 months without subculture 10. A low residual water content of dried callus of carrot, cultured on 0.58 M sucrose for 16 days, allowed freezing till -80 C and storage under low humidity conditions at ambient temperature. At 15 C and 25% relative humidity, the callus could be stored for more than 2 years without loss of viability and plantlets successfully regenerated from them within four weeks of reculture 11. Moriguchi et al 12 reported that the embryogenic calluses of grape could be stored under layers of liquid silicone cover at C for 360 days. Nadel et al 13 stored the embryogenic callus, proembryogenic clumps and somatic embryos of celery at 4 C for 24 weeks. The various reports on storage of somatic embryos at ambient and below ambient temperatures have shown the maximum storage period for two years. However, using cryopreservation, i.e. storage at 196 C, the embryogenic tissues/somatic embryos could be stored for more than 5 years in case of silver birch without loss in recovery rates 14. If we extrapolate the data, it can go virtually for indefinite period of time. Keeping in view the advantages of cryopreservation, efforts have been be made to extend it to a large number of genotypes and species. Techniques Used for Cryopreservation of Somatic Embryos Several reports on cryopreservation of somatic embryos have been published describing a range of different techniques, viz. vitrification, encapsulationdehydration, pre-growth, pre-growth desiccation and desiccation. All these techniques are based on minimizing the available freezable water with minimum injury. The following section discusses about each of these techniques to provide an overview. Whereas, Table 1 summarizes the advantages and disadvantages associated with some of these techniques. Vitrification In cryopreservation protocols, it is necessary to avoid inter-cellular ice-crystal formation. Techniques developed recently seek to avoid the problems of ice crystal formation by inducing the process of vitrification, in which water undergoes to a less damaging non-crystalline glassy state 15. One approach to this problem has been the development of vitrification cocktail technique, which exposes the cells to the mixture of cryoprotectant compounds, such as glycerol and poly ethylene glycol (PEG) 16. Successful vitrification requires the use of a highly concentrated yet effectively non-toxic solution of cryoprotectants. The key to success of cryopreservation by vitrification is to carefully standardize the procedures for dehydration and cryoprotectant permeation and to prevent injury by chemical toxicity and/or excessive osmotic stress during the treatment. In case of vitrification, the explants are treated with loading solution (MS M glycerol M sucrose) for 20 min at room temperature, followed by treatment with plant vitrification solution [PVS2; MS + 30% glycerol + 15% ethylene glycol + 15% DMSO (dimethyl sulfoxide) M sucrose] at room temperature or at 0 C. The explants are then frozen rapidly in LN, followed by rapid thawing and treatment with unloading solution (MS M sucrose) for 20 min (Fig. 1). Loading was necessary for vitrification of nonacclimated rye protoplasts 17. Equilibration of Brassica compestris cells within 1:5 PEG minimized dehydration injury during transfer to concentrated vitirification of the cells while cooling in liquid nitrogen 18. Incubation time of explants has to be species specific in PVS2. It is 3 min at 25 C for cultured cells

3 SHARMA: CRYOPRESERVATION OF SOMATIC EMBRYOS 49 Fig. 1 Flow diagram of process involved in cryopreservation of somatic embryos using vitrification technique of navel orange 19, 7.5 min at 0 C for rice embryogenic cells 16 and 15 min at 0 C for meristems of white clove 20. The PVS2 has also been found to have characteristic temperature dependent exposure time 17. Encapsulation-dehydration Fabre and Dereuddre 21 were the first to develop encapsulation-dehydration technique (Fig. 2). The procedure involves the encapsulation of somatic embryos in calcium alginate beads, dehydration using osmoticum (sucrose) and air drying treatments, followed by direct immersion in LN or controlled cooling to an intermediate temperature (using a programmable freezer) before transfer to LN. After storage, the beads are re-warmed at room temperature and explants returned to standard culture conditions. It is possible to regenerate whole plants as the developing shoot emerges from the beads encapsulating embryogenic tissues 22. However, it is necessary for some species to excise the somatic embryos from the bead to permit re-growth 23. In encapsulation-dehydration technique 21, extraction of water results in progressive osmotic dehydration in place of use of toxic cryoprotectants to induce dehydration tolerance. Further, additional loss of water is obtained by evaporation and subsequent increase in sucrose concentration in the beads. Thus, the sucrose molarity in the beads increases markedly during the drying process and reaches or exceeds the saturation point of the sucrose solution, resulting in glass transition during cooling to -196 C 24. Fig. 2 Flow diagram of process involved in cryopreservation of somatic embryos using encapsulation-dehydration technique Thermal analysis of beads and encapsulated embryos suggested that after dehydration the beads and tissues undergo glass transition at sub-zero temperatures 24. It would, thus, appear that the encapsulation/dehydration might offer a novel approach to cryoprotection via vitrification as this technique would allow storage of plant organs at higher temperatures than that of liquid nitrogen, thus avoiding risk of ice recrystallization. Also, as PVS can be unstable and rewarming is critical, glassy state obtained using encapsulation-dehydration may be more stable and encapsulated tissues can be rewarmed slowly at ambient temperature without loss of survival. The concentration of sucrose used and duration of dehydration are important factors to be standardized for developing a protocol for encapsulationdehydration of a particular species. Sucrose has been generally used as a cryoprotectant and successfully applied to a number of species. As the explant tissues are delicate, very high concentrations (i.e., >0.5 M) of sucrose proved to be toxic in case of sweet potato (personal communication). Cryopreservation of Dioscorea bulbifera somatic embryos revealed the significant effect of sucrose on post-thaw recovery rates. Preculture of encapsulated embryos in 0.5 M sucrose for 7 days followed by 4 hrs of dehydration gave optimum recovery rates 25. Hatanka et al 26 reported successful cryopreservation of somatic embryos of coffee, progressively precultured for 3-4 days on medium supplemented with increasing concentration of sucrose, dehydrated to about 13%

4 50 INDIAN J BIOTECHNOL, JANUARY 2005 water content and plunged in LN. About 63% embryos survived cryopreservation, of which 50% grew directly without formation of secondary embryos and non-embryogenic callus. Recently, an efficient cryopreservation protocol was established for several genotypes of citrus using the encapsulation-dehydration technique. High survival rates (75-100%) were consistently obtained after 1 day pre-growth in 0.75 M sucrose, desiccation down to 20-25% moisture content in the beads and direct immersion in LN. The histological study showed that embryos, subjected to the encapsulation-dehydration, accumulated high sucrose levels which appear to ensure the recovery of the whole embryo after cryopreservation 27. Pregrowth and Pregrowth Desiccation The pregrowth technique consists of culturing samples in the presence of cryoprotectants, then freezing in LN (Fig. 3). The pregrowth technique has been developed for a number of Musa species and Ipomoea batatas somatic embryos 28. However, the pregrowth desiccation refers to the preculture of the explant on medium to induce desiccation tolerance so that it can be desiccated and cryopreserved with minimum cryoinjury (Fig. 3). Studies on pre-growth desiccation suggest that pre-culture of the explant on high sucrose or ABA or proline induces desiccation tolerance, which enables the explants to be desiccated/dried and directly frozen in LN. Nitzsche 11 demonstrated that carrot callus precultured on Gamborg s medium, supplemented with 10 mg/l ABA and 50 g/l sucrose, tolerated desiccation and regenerated into plants after storage at -80 C for a few days. The plant regeneration is also reported from dried somatic embryos of alfalfa 32. The nodal segments of Asparagus, pretreated on 0.7 M sucrose and dehydrated to lower water content in silica gel, has been successfully cryopreserved 33. Somatic embryos of melon were cryopreserved with 65% survival after preculture with 10 mg/l ABA and controlled desiccation (relative humidity of 56-65%) 34. Changrun et al 35 demonstrated that desiccation tolerance of oilseed rape somatic embryos increased significantly after preculture for 7 days in the presence of 10 mg/l ABA and 81% survival was achieved using stepwise slow drying in which relative humidity was progressively lowered from 97%- 75%. In a recent report, drying of the somatic embryos before the application of the cryoprotectants resulted in a significantly higher viability after cryostorage as compared to when the material was first exposed to the cryoprotectants and then dried 36. Fig. 3 Flow diagram of process involved in cryopreservation of somatic embryos using pre-growth, pre-growth desiccation, desiccation techniques. Desiccation Desiccation is a very simple procedure requiring only dehydration of the plant material before rapid freezing by direct immersion in LN (Fig. 3). It is mainly employed with zygotic embryos, as other materials are relatively more delicate and sensitive to dehydration. Usually, desiccation is performed by placing the embryos in the air current of a laminar airflow cabinet 37. However, more precise and reproducible desiccation conditions are achieved by placing the embryos in a stream of compressed air or in an airtight container with silica gel 37. Optimal survival rates are obtained when the water content of the embryos is around 10-20% 25. Freezing is usually rapid but positive results have been obtained using slow cooling with several tropical forest tree species 37. In a recent study, effects of drying and cryopreservation on survival of spruce (Picea glauca (Moench) Voss and Picea glauca engelmannii complex) somatic embryos (SEs) were investigated with the aim of developing simple and robust protocols for embryo storage 38. Somatic embryos dried over salt solutions of known water potential (Ψ) survived the removal of virtually all free water to a relative water content (RWC) of approximately 0.13, a value similar to that observed for spruce zygotic

5 SHARMA: CRYOPRESERVATION OF SOMATIC EMBRYOS 51 embryos from dry seed. Highest survival (> 80%) after freezing in LN was observed in embryos predried to Ψ of 15 to 20 MPa, which yielded RWC close to predicted bound (apoplastic) water values. In a recent study, three techniques, viz.: (i) encapsulation/dehydration; (ii)pregrowth/dehydration; and (iii) vitrification, were compared for cryopreserving embryogenic masses (EMs) sampled from embryogenic cultures of mango (Mangifera indica L.) 39. In all experiments, EMs were sampled from embryogenic cultures during their exponential growth phase and pretreated for 24 hrs on solid medium containing 0.5 M sucrose before freezing. Using the vitrification technique, recovery ranged from 94.3 % after treatment of EMs with the PVS3 vitrification solution for 20 min. With the pregrowth/dehydration technique, limited recovery (8.3 %) was achieved after desiccation of EMs for 1 hr to 58.5% moisture content. However, no recovery was achieved after using the encapsulation/dehydration technique despite reduction in moisture content (fresh wt basis) of EMs, which ranged from 78.3 % (without dehydration) to 40.8 % (after 6 hrs dehydration). It is worthwhile to note that the best technique to be employed for cryopreservation has been species specific. For example, if the species is cold hardy/or tolerant to low temperatures (like plants growing in temperate or subtropical region), pregrowth and/or pregrowth desiccation can be followed. On the other hand, if the species is sensitive to low temperatures (like the plants growing in tropical region), encapsulation-dehydration or vitrification could be better choice for it needs more effective cryoprotection because of their cell physiology. However, each technique has its own advantages and disadvantages as mentioned in Table 1. Important Practical Issues before Routine Use of Somatic Embryos for Cryopreservation Certain issues need to be addressed before going in for routine use of somatic embryos. One of them has been the genetic stability of plants produced using somatic embryos for long-term conservation. It is important to analyze the genetic stability of the longterm preserved tissues. Studies on genetic fidelity of somatic embryo generated populations have yet to be carried out. However, published reports on genomic stability of tissue culture regenerants from embryogenic cell suspensions suggest that the plants are genetically, morphologically, cytologically and behaviorally uniform 40,41. Population of Picea plants, derived from embryogenic tissue, has been analyzed for isozyme systems but no qualitative variation was found at any of the loci examined in comparison to control plants 42. RAPD analysis of somatic embryo regenerants has also highlighted their potential for cryopreservation 43,44. Similarly, no variation in RFLP profile was detected in mitochondrial plastid and nuclear genomes in a representative sample of regenerants of Pennisetum 45. In few instances, although variation was observed but it was shown to be a manifestation of a pre-existing variation within the explants used for induction of somatic embryos 46. Even, the DNA content of nuclei isolated from embryogenic culture and from plantlets regenerated from somatic embryos has been found to be the Table 1 Advantages and disadvantages of commonly used cryopreservation techniques Technique Advantages Disadvantages Slow freezing Stability from relatively non toxic cryoprotectants Vitrification Encapsulation-dehydration No special equipment needed, fast procedure, fast recovery No special equipment needed, non toxic cryoprotectants, simple thawing procedures Requires expensive equipment, slow recovery, low applicability to tropical species Vitrification solutions are toxic to many plants, cracking is possible, requires careful timing of solution changes Requires handling each bead several times, some plants do not tolerate high sucrose concentrations Desiccation Easy, useful for many temperate trees species Requires expensive freezing equipments, larger storage space, recovery requires grafting or budding, works best in cold temperate regions Pregrowth Pregrowth desiccation No special equipment needed, simple, useful for many temperate trees species No special equipment needed, simple, useful for many temperate trees species Only appicable to winter hardy buds, seeds/zygotic embryos of a few species, works best in cold temperate regions Requires expensive freezing equipments, larger storage space

6 52 INDIAN J BIOTECHNOL, JANUARY 2005 same 47. Recently, a total of 2247 bands were obtained in plants regenerated from somatic embryos of Pinus, which exhibited no aberration in RAPD banding pattern among the tested plants 48. The extent of variation among the clones of somatic embryos derived population is dependent on induction and maturity of somatic embryo 25. If at any stage, i.e. induction, maintenance or maturity of embryogenic tissues, intervening non-embryogenic callus is present, there is a possibility of variation 49. However, in a recent study on genetic stability of somatic embryos generated populations of sweet potato, using RAPD markers and morphological markers and morphological descriptors, revealed no significant variation among the clones 25. Another important issue has been the genetic and agronomic stability of plants regenerated from frozen somatic embryos; however, little documentation is available on the subject. At the cytological and molecular level, RFLP and RAPD patterns and electrophoretic profiles of plants regenerated from cryopreserved cell suspensions proved to be identical to those of non frozen control plants Interestingly, a recent study of RAPD assays of cryopreserved embryogenic callus cultures of open pollinated Abies cephalonica revealed considerable variation in DMSO treated nonfrozen samples; 16.8% of the produced RAPD profiles showed interclonal variation, while background variation was seen in 1.7% of the control amplification. These results indicate the significance of monitoring genetic fidelity of the cryopreserved material as cryoprotectants may cause a risk to fidelity of plant material 54. Oil palm trees developed from cryopreserved embryos showed vegetative and floral development comparable to that of control plants 55. A study conducted on agronomical characteristics of plants regenerated from cryopreserved embryogenic tissues of banana showed no difference to the normal plants at the agronomic level 56. Varietal stability was recorded in plantlets developed from cryopreserved meristems of sugarcane pertaining to botanical characteristics and yield components evaluated 57. A recent study compared the field performance of sugarcane plants originating from three different sources, i.e. control, non-cryopreserved embryogenic calluses, cryopreserved embryogenic calluses and macropropagated material of the same commercial hybrid. Significant differences were observed between treatments only during the first six months of field growth of sugarcane stools. However, these differences disappeared by12 month of field growth 58. More recently, the RAPD, morphological and biochemical analyses of plants regenerated form cryopreserved embryogenic tissue revealed no significant variation in all the 60 plants tested 59. When we intend to use the embryogenic systems for long-term conservation of genotypes, another point to be kept in mind is the possibility of successful induction of embryogenesis in large number of genotypes. In case of sweet potato, somatic embryogenesis had been induced in more than 25 accessions 23,25 and genotypic variations in response to embryogenesis have been observed. Some accessions respond to the tune of 60 % frequency and in some induction was not possible at all 23. Conclusion In vitro somatic embryos offer a wide potential in plant biotechnology. Therefore, preservation of these elite cell lines is imperative. However, somatic embryogenesis is still very much an empirical science; each species, and sometimes genotype requires different conditions. Plants where cryopreservation of somatic embryos have been worked out are shown in Table However, before going in for routine use of cryopreservation of somatic embryos, it is important to investigate whether somatic embryogenesis is possible to large number of genotypes and somatic embryo generated population can be genetically stable, which has been a major limitation. Additionally, in vitro maintenance of these elite cultures in a particular stage suitable for cryopreservation is tricky and needs specific manipulations with respect to culture conditions. Further, extensive studies are required, using more sophisticated molecular techniques, to assess genetic stability of the cryoconserved germplasm for their more confident use. Even, the effect of longer duration storage on the conserved germplasm needs to be studied. Never the less, application of cryopreservation technique to somatic embryos offers availability of these cell lines today and in distant future when confronting situations would demand reconstruction of genomes for sustainable production. Cryopreservation is highly cost-effective in comparison to other in vitro conservation strategies as it does not require frequent sub-culture, continuous check of cultures or maintenance in sophisticated growth chambers. All that is required for storage is liquid nitrogen, which is very cheap. In recent years,

7 SHARMA: CRYOPRESERVATION OF SOMATIC EMBRYOS 53 Table 2 Species where cryopreservation of somatic embryos has been worked out Species Technique Aesculus hippocastinium Desiccation method 60 Asparagus officinalis Vitrification 61 Brassica napus With/without encapsulation 35 Camellia japonica Desiccation 62 Citrus sinensis Osb. var. brasiliensis Tanaka Vitrification/DMSO 51 Citrus DMSO 63 Cnidium officinale Vitrification 64 Coffea arabica Partial drying 29 Coffea canephora Encapsulation-dehydration 65 Daucas carota Desiccation 66,67 Elaeis guinensis Desiccation 31 Gossypium Vitrification 68 Ipomoea batatas With/without encapsulation 23,69 Juglans regia Desiccation 70 Manihot esculenta Partial drying 36 Macropidia fuliginosa Vitrification 71 Oryza sativa Vitrification 16,72 Olea europea Encapsulation-dehydration, Encapsulation-vitrification 73 Phoenix dactylifera Partial drying 29 Picea DMSO treatment 74, Desiccation 75 Pisum sativum Partial drying 29 Pinus DMSO/sucrose 50,76 Saccharum officinale DMSO/sucrose 77 Melon Desiccation 34 Mangifera indica Encapsulation-dehydration, Vitrification, Pregrowth desiccation 39 major advances have been witnessed in cryopreservation with the development of new vitrification-based freezing techniques that are operationally simple, efficient and have a broad applicability. Moreover, continuing research in cryopreservation by various teams worldwide is progressively improving our understanding of mechanisms involved, which would allow the reduction in empirical developments. It can, thus, be expected that the utilization of somatic embryo cryopreservation in genetic resource conservation and biotechnology programmes will steadily increase in the coming years. References 1 Philips G C, Hubstenberger J F & Hansen E E, Plant regeneration from callus and cell suspension cultures by somatic embryogenesis, in Plant cell tissue and organ culture, edited by O L Gamboorg & G C Philips (Narosa Publishing House, New Delhi) 1998, Blackesley D, Mazrooei A & Henshaw S, Cryopreservation of embryogenic tissue of sweet potato (Ipomoea batatas): Use of sucrose and dehydration for cryoprotection, Plant Cell Rep, 14 (1995) Redenbaugh K, Fujii J A & Slade D, Synthetic seed technology, in Cell culture and somatic cell genetics of plants, vol 8, edited by I K Vasil (Academic Press, New York) 1991, Abdullah R, Cocking E C & Thompson J A, Efficient plant regeneration from rice protoplasts through somatic embryogenesis, Bio-Technology, 4 (1986) Williams E G & Maheswaran G, Somatic embryogenesis: factors influencing coordinated behaviour of cells as an embryogenic group, Ann Bot, 57 (1986) Hao Y I & Deng X X, Occurrence of chromosomal variations and plant regeneration from long-term cultured Citrus callus, In Vitro Cell Dev Biol Plant, 38 (2002) Cry D, Role in clonal propagation and germplasm conservation of conifers, in Cryopreservation of tropical plant germplasm: Current research progress and applications, edited by F Engelmann & H Takagi (JIRCAS/ IPGRI) 2000, Withers L A & Engelmann F, In vitro conservation of plant genetic resources, in Biotechnology in agriculture, edited by A Altman (Marcel Dekker Inc. New York) 1997, Caplin S M, Mineral oil overlay for conservation of plant tissue cultures, Am J Bot, 46 (1959) Augereau J M, Courtois D & Petiard V, Long-term storage of callus cultures at low temperatures or under mineral oil layer, Plant Cell Rep, 5 (1986) Nitzsche W, One year storage of dried carrot callus, Z Pflanzenphysiol, 100 (1980) Moriguchi T, Kozaki I, Natsuta N & Yamaki S, Plant regeneration of grape callus stored under a combination of low temperature and silicone treatment, Plant Cell Tissue Organ Cult, 15 (1988) Nadel B L, Sakai A, Amano Y & Matsuzawa T, Cold storage and efficient conservation of somatic embryogenesis of celery into transplantable plants, Sci Horict, 44 (1990) Ryynanen L, Effect of early spring birch bud type on postthaw regrowth after prolonged cryostorage, Can J For Res, 29 (1999) Sakai A, Kabayashi S & Oiyama I, Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasieleinsis Tanaka) by vitrification, Plant Cell Rep, 9 (1990) Huang C N, Wang J H, Yan Q F, Plant regeneration from rice (Oryza sativa L.) embryogenic cells cryopreserved by vitrification, Plant Cell Rep, 14 (1995) Langis R & Steponkus P L, Vitrification of isolated rye protoplasts: protection against dehydration injury by ethylene glycol, Cryo Lett, 12 (1991)

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