Plant Cell Rep (2002) 21:118–124 DOI 10.1007/s00299-002-0490-8
CELL BIOLOGY AND MORPHOGENESIS
D.H. Touchell · V.L. Chiang · C.-J. Tsai
Cryopreservation of embryogenic cultures of Picea mariana (black spruce) using vitrification
Received: 3 December 2001 / Revised: 14 May 2002 / Accepted: 15 May 2002 / Published online: 27 June 2002 © Springer-Verlag 2002
Abstract This study reports on the first use of a vitrification procedure for the successful cryopreservation of embryogenic cultures of a coniferous species. Using Picea mariana embryogenic cultures, we obtained the highest survival by first preculturing embryogenic masses on semi-solid medium containing 0.8 M sorbitol for 48 h followed by incubation in PVS2 cryoprotective vitrification solution at 0°C for 30 min and direct immersion in liquid nitrogen. The replacement of sorbitol with 1.6 M glycerol also resulted in high survival. When sorbitol and glycerol were used in preculture treatments, 9 of 11 embryogenic lines survived liquid nitrogen treatments. We also demonstrated that 100% post-liquid nitrogen survival of mature somatic embryos could be obtained without pretreatments. A brief desiccation pretreatment, however, increased uniformity of somatic embryo germination. Keywords Cryopreservation · Vitrification · Picea mariana · Somatic embryogenesis Abbreviations ABA: Abscisic acid · BA: 6-Benzyladenine · 2,4-D: 2,4-Dichlorophenoxyacetic acid · LM: Litvay medium · LN Liquid nitrogen · NAA: α-Naphthaleneacetic acid · PVS: Plant vitrification solution
Introduction Picea mariana (black spruce) is a coniferous species native to North America. The tree is grown as an important forestry species and has commercial value to the paper Communicated by S.A. Merkle D.H. Touchell · V.L. Chiang · C.-J. Tsai (✉) Plant Biotechnology Research Center, School of Forestry and Wood Products, Michigan Technological University, 1400 Townsend Drive, Houghton, MI 49931, USA e-mail:
[email protected] Tel.: +1-906-4872914, Fax: +1-906-4872915
and pulp industries (Tautorus et al. 1990; Klimaszewska 1995; Tremblay and Tremblay 1995). Interest in the species has led to the development of somatic embryogenesis, enabling rapid mass propagation to provide raw material for future wood demands (Adams et al. 1994; Tremblay and Tremblay 1995; Klimaszewska 1995; Charest 1996). However, embryogenic cultures of P. mariana are characteristically fast growing and require frequent maintenance, which over the long term are labor-intensive and space-consuming. Serial cultures also increase the risk of tissue loss through adverse culture conditions, somaclonal variation or the loss of embryogenic potential. To alleviate problems associated with culture maintenance, cryopreservation of embryogenic cultures has been developed for the ex situ conservation of valuable genetic lines of coniferous species (Cyr 2000; Häggman et al. 2000). Cryopreservation allows tissues to potentially be stored indefinitely with minimal maintenance and risks. Successful cryopreservation procedures aim to reduce water content to levels at which ice crystal formation is non-lethal. For embryogenic tissues and cell lines of coniferous species, this is routinely achieved through slow cooling regimes (for review, see Häggman et al. 2000). Although the specific conditions vary among species, the slow cooling regimes involve pretreating tissues with a cryoprotectant or osmoticum, then slowly cooling the tissues (0.2–10°C/min) to a terminal temperature (–30°C to –40°C) to further promote desiccation, after which the tissues are directly immersed in LN. Tissues are warmed rapidly and recovered on appropriate media. Using slow cooling procedures, successful cryopreservation, with high recovery and regeneration frequency, has been demonstrated for P. mariana embryogenic cell lines (Klimaszewska et al. 1992; Adams et al. 1994). Similar procedures have been developed for embryogenic tissue of many coniferous species (for review, see Cyr 2000; Häggman et al. 2000). Indeed, Cyr (2000) reports that there may be between 8,000 and 10,000 conifer embryogenic lines cryostored worldwide. However, slow cooling procedures require expensive equipment and a care-
119
ful manipulation of tissues, cryoprotective treatments and cooling rates (Benson 1999). Alternatively, a less expensive version of this approach using Nalgene freezing containers and a –80°C freezer for gradual cooling of samples has been reported (Percy et al. 2000). In comparison, vitrification is a simplified cryostorage procedure that does not require the use of expensive programmable cooling devices (Pennycooke and Towill 2000) or a –80°C freezer. Vitrification procedures avoid ice formation by using highly concentrated cryoprotective solutions to desiccate and penetrate cells in order to form a highly viscous intracellular solution that forms a metastable glass at low temperatures. It has become the preferred method for cryopreservation over the last decade, with over 140 species and cultivars being successfully cryostored (Sakai 2000). However, there are no reports of it being used for embryogenic cultures of conifers (Häggman et al. 2000). Vitrification protocols have primarily been used to cryostore shoot apices (Touchell and Dixon 1999; Sakai 2000); however, recently they have been more widely applied to include embryogenic tissues (Turner et al. 2000). Based on work done with shoot apices, the key steps in the procedure are: (1) culture condition (i.e. age of culture), (2) preculturing on medium containing sugars or sugar alcohols, (3) cryoprotection through a PVS (e.g. PVS2, Sakai et al. 1990) and (4) recovery after LN exposure. In light of the apparent simplicity of the vitrification protocol and the successful application of the procedures to diverse species and tissue types, the aim of the investigation reported here was to explore the use of the protocol for embryogenic tissue of P. mariana. This was achieved through investigating two key steps of the vitrification protocol: (1) preculturing conditions and (2) exposure to the PVS. In addition, the ability of mature somatic embryos to survive LN treatment was investigated through manipulation of water content by desiccation.
Materials and methods Plant material Open-pollinated Picea mariana seeds (obtained from Le Groupe Conseil en AmJnagement Forestier, Quebec) stored at 4°C were surface-sterilized for 7 min with 1% sodium hypochlorite containing 2 drops of Tween 20 and then washed three times, 5 min each, with sterile distilled water. Seeds were imbibed in sterile water for approximately 4 h, and zygotic embryos were aseptically removed from the seeds and placed onto somatic embryogenesis induction medium. To estimate viability, we placed three replicates of ten zygotic embryos onto media without growth regulators and monitored germination. Culture media and conditions Embryogenesis induction medium consisted of half-strength (1/2) LM (Litvay et al. 1981) supplemented with 1 g/l casein hydrolysate, 500 mg/l L-glutamine, 9.1 µM 2,4-D, 4.5 µM BA and 30 mM sucrose and solidified with 3 g/l Phytagel (Sigma, St. Louis, Mo.). The pH of the media was adjusted to 5.8 before autoclaving. L-Glutamine was filter-sterilized and added to cool sterile media
before the latter was poured into petri-dish plates (95×15 mm, approx. 20 ml each). Zygotic embryos (ten per plate) were maintained in the dark at 25°C and subcultured onto fresh medium every 4 weeks. Embryogenic masses were maintained on the same medium with a 2- to 3-week subculture interval. Somatic embryo maturation was achieved within 4–8 weeks by placing embryogenic masses on 1/2-LM supplemented with 1 g/l casein hydrolysate, 500 mg/l L-glutamine, 20 µM ABA and 180 mM sucrose and solidified with 6 g/l Phytagel. Tissue was kept in the dark at 25°C and subcultured to fresh media every 2–3 weeks. Mature somatic embryos were then used for cryopreservation treatments or germinated in the dark on 1/2-LM supplemented with 1 g/l casein hydrolysate, 500 mg/l L-glutamine, 60 mM sucrose and solidified with 3 g/l Phytagel. Germinated somatic seedlings were maintained on the same medium in Magenta GA-7 vessels (Sigma) for seedling development at 23±2°C in a growth chamber under a 16/8-h (day/night) photoperiod (light intensity: 160 µE/m2 per second). Four- to eight-week old somatic seedlings were transferred to a soil mix (perlite:peatmoss:vermiculite:top soil = 1:1:1:1), acclimated in a mist chamber for 2 weeks and then grown in a greenhouse. Cryostorage Embryogenic masses (line BS 001) 3 mm in diameter were cultured on solidified embryogenesis induction media supplemented with sorbitol (0.0, 0.2, 0.4, 0.6, 0.8 or 1.0 M) for 48 h. Ten masses were then placed in a 1.2-ml cryovial (Nunc) containing 1 ml of PVS2 (see below; approximate ratio of fresh embryogenic masses to PVS2 was 1:10, v/v) and incubated at 0°C for different intervals (0, 10, 20, 30 or 40 min). To evaluate the cryostorage potential, we then directly immersed the treated embryogenic masses in the cryovials in LN and stored them for 30 min, followed by warming in a 40°C water bath and three washes of 5 min each with liquid 1/2-LM media containing 1.0 M sucrose. Embryogenic masses were recovered on embryogenesis induction medium in the dark. Survival was monitored every 2–3 days. To test the effects of the preculture treatment, we treated embryogenic masses in the same manner but only incubated them in PVS2 for 0, 20 or 40 min without LN exposure. Five replicates of ten embryogenic masses were used for each trial. Survival was scored when new cell proliferation was observed. The plant vitrification solution (PVS2) used for all studies was modified from Sakai et al. (1990). The medium consisted of 30% (w/v) glycerol, 15% (w/v) DMSO and 15% (w/v) ethylene glycol in liquid 1/2-LM supplemented with 0.4 M sucrose. Influence of length of preculture Embryogenic masses (line BS 001) were incubated on semi-solid embryogenesis induction media containing 0.8 M sorbitol for 1, 2, 3, 4 or 7 days before being treated with PVS2 for 30 min at 0°C, exposed to LN and recovered in the same manner as described previously. Three replicates of ten embryogenic masses were used for each treatment. Influence of sugars To test the influence of sugar/sugar alcohol type in the preculture media, sorbitol was replaced with mannitol (0.8 M), sucrose (0.4 M and 0.8 M), glucose (0.8 M) or glycerol (0.8 M and 1.6 M). Three replicates of ten embryogenic masses were used for each treatment. Applicability to other clones The success of the vitrification procedure was evaluated for an additional ten (making a total of 11) embryogenic lines. Embryogenic masses were precultured on media containing 0.8 M sorbitol
120 or 1.6 M glycerol for 48 h before being treated with PVS2 for 30 min at 0°C, exposed to LN and recovered in the same manner as described previously. Three replicates of ten embryogenic masses were used for each treatment. Cryopreservation of mature somatic embryos The suitability of mature somatic embryos (after 8–12 weeks of maturation) for cryostorage was also investigated for four selected clones. Mature somatic embryos were desiccated for 0, 30, 60, 90 or 120 min in a laminar flow hood at ambient temperature. Ten desiccated embryos were placed in a 1.2-ml cryovial and immersed in LN for 30 min. Embryos were warmed in a 40°C water bath and recovered on embryogenesis induction media without plant growth regulators. Control somatic embryos were desiccated but not exposed to LN. For each clone, three replicates of ten somatic embryos were used for each trial. Statistical analysis To better understand the effect of various cryostorage treatments on the survival of embryogenic masses, we fitted the data with several statistical models using the generalized linear model of the binomial family and logit link (Agresti 1990). Such models are appropriate for survival data that are assumed to be binomially distributed. The fitting accuracy was assessed using Akaike’s information criterion statistic. The relationship of sorbitol and PVS2 pretreatments on survival was best described using a quadratic model (Fig. 1B). Similarly, the influence of sorbitol preculture time on survival was also best described using a quadratic model (Fig. 2). For experiments concerning the influence of sugars on survival, a saturated model (i.e. one parameter for each sugar treatment) was best (Fig. 3). Parameter estimates for each model were calculated by the software package SPSS version 10.0 (SPSS 2000).
Fig. 1 Survival of embryogenic tissue treated with different levels of sorbitol for 48 h and exposed to PVS2 at 0°C for 0–40 min: A without LN treatment, B after LN treatment. Data points in B are predicted probabilities of survival based on a general linear model with a quadratic term for sorbitol concentration and PVS2 exposure time
Results Somatic embryogenesis Approximately 90% of mature zygotic embryos germinated within 3 days when placed on 1/2-LM media with no growth regulators. When cultured on somatic embryogenesis induction medium, embryogenic callus was first visible 3–4 weeks after culture initiation but was predominantly observable 6–8 weeks after initiation. Embryogenic tissue was separated from non-embryogenic tissue over several subculture cycles. Eleven embryogenic lines derived from different zygotic embryos were maintained for cryostorage and future studies. For all of the embryogenic lines, growth rates ranged between four- and fivefold over 14 days. When placed on maturation media, approximately 200–500 somatic embryos per gram fresh mass were obtained within 8–12 weeks. Following germination, somatic seedlings were regenerated, transferred to soil mix, acclimated and grown in a greenhouse. Cryopreservation The survival of embryogenic masses (from embryogenic line BS 001) incubated for 48 h on media containing sorbitol (0.0–1.0 M) remained at 100% (Fig. 1A, 0 min).
There was no apparent differences in growth between all sorbitol treatments and the no-sorbitol control. Treatment of embryogenic masses with PVS2 at 0°C for 20 min had no adverse influence on the survival of embryogenic cultures. However, incubation for 40 min resulted in a slight reduction in survival (Fig. 1A). Similar responses to PVS2 treatment were observed for all of the sorbitol concentrations tested. All embryogenic tissues (with or without sorbitol and PVS2 treatments) showed signs of recovery and growth after 3 days. Following LN exposure, the highest survival was obtained when embryogenic masses were pre-treated for 48 h on 0.8 M sorbitol media and incubated for 30 min at 0°C in a PVS2 (Fig. 1B). Variations in this sorbitol concentration and PVS2 incubation time resulted in a significant decline in embryogenic tissue survival. No survival was obtained when tissues were precultured on media supplemented with no or 0.2 M sorbitol. Survival was first observed 10–14 days after warming. After the initial lag phase, embryogenic tissue growth was the same as that of the untreated controls. Similarly, when embryogenic tissues were transferred to maturation media 12 weeks after LN treatment, somatic embryo production and subsequent germination and somatic seedling conversion were consistent with that of the untreated controls (data not shown).
121 Table 1 Survival percentage of embryonic masses from 11 embryogenic lines of Picea mariana pre-cultured on media containing either 0.8 M sorbitol or 1.6 M glycerol, treated with PVS2 at 0°C for 30 min, exposed to liquid nitrogen, warmed and recovered. Values represent means of three replicates of ten embryogenic masses ± standard error (ND not determined)
Fig. 2 Survival of embryogenic tissue treated with 0.8 M sorbitol for different lengths of time and exposed to PVS2 at 0°C for 30 min followed by LN treatment. Data points are predicted probabilities of survival based on a general linear model with a quadratic term for length of sorbitol pretreatment. The vertical bars are standard errors for each prediction
Fig. 3 Survival of embryogenic tissue treated with different levels of sugars or sugar alcohols for 48 h and exposed to PVS2 at 0°C for 30 min followed by LN treatment. Data points are predicted probabilities of survival based on a saturated model for sugar pretreatment
The length of the preculture was critical to the survival of embryogenic tissues exposed to LN. The survival of embryogenic masses precultured for 2 days or 3 days on media containing 0.8 M sorbitol was not significantly different (Fig. 2). However, a significantly lower survival rate was observed when embryogenic masses were precultured for 1 day or 4 days, while no survival was obtained when tissues was precultured for 0 days or 7 days (Fig. 2). Preculture on medium supplemented with different sugars or sugar alcohols was investigated as a means to improve survival following LN treatments. The use of 0.8 M sorbitol remained the best treatment (Fig. 3), although good survival was also obtained when the tissue was incubated on media supplemented with glycerol adjusted to contain a similar number of hydroxyl groups as
Line
0.8 M Sorbitol
1.6 M Glycerol
BS 001 BS 002 BS 003 BS 006 BS 010 BS 011 BS 015 BS 016 BS 020 BS 021 BS 025
50.4 ± 2.9 30 ± 5.8 0 3.3 ± 3.3 46.8 ± 2 66.7 ± 6.8 20 ± 0 13.3 ± 8.9 0 30 ± 21.2 0
35.0 ± 2.8 36.6 ± 3.4 0 3.3 ± 3.3 23.3 ± 3.4 26.7 ± 3.4 33.3 ± 8.9 26.7 ± 6.8 63.3 ± 14.0 0
0.8 M sorbitol (28.896×1023). Lower (>two-fold) survival was obtained with mannitol, a structural isomer of sorbitol. No survival was obtained using sucrose, glucose or 0.8 M glycerol in the preculture medium (Fig. 3). Based on these results, we evaluated a vitrification protocol on 11 embryogenic lines that involved preculture on semi-solid medium supplemented with 0.8 M sorbitol or 1.6 M glycerol for 48 h followed by a 30-minlong incubation at 0°C in PVS2 prior to exposure to LN (Table 1). Survival varied among the lines and between treatments. Lines BS 003 and BS 025 did not survive liquid treatment, and line BS 006 only had low survival. BS 001 and BS 010 had higher survival percentages when a 0.8 M sorbitol pre-culture was used, while lines BS 002, BS 015, BS 016, BS 020 and BS 021 showed a higher survival using 1.6 M glycerol in the pre-culture medium (Table 1). Notably, lines BS 011 and BS 021 displayed high recovery rates, with survival (as indicated by cell proliferation) being observed within 7 days after LN treatment compared to the 10–14 days required by all the other lines. Independent of line, new growth was occasionally detected up to 8 weeks after LN treatment. For lines BS 002, BS 006, BS 010, BS 011 and BS 016, recovered embryogenic masses were transferred to maturation medium. Somatic embryo production and germination were consistent with that shown by the controls (data not shown). Cryopreservation of somatic embryos In a further study, the ability of mature somatic embryos to tolerate desiccation and LN treatments was investigated. Somatic embryos matured on media supplemented with 180 mM sucrose and 20 µM ABA for 8–12 weeks were dried in a laminar flow hood prior to LN exposure and recovered on media without growth regulators. Somatic embryos from all embryogenic lines survived cryostorage without desiccation or with only a brief (up to 60 min) desiccation period (Table 2). A brief desicca-
122 Table 2 Survival percentage of mature somatic embryos, representing four embryogenic lines of P. mariana, desiccated for different lengths of time and exposed to liquid nitrogen (LN). Drying (min) 0 30 60 90 120
BS 002
BS 003
Values represent means of three replicates of ten embryos ± standard error (ND not determined) BS 010
BS 030
Control
LN
Control
LN
Control
LN
Control
LN
100 100 40±10 0 0
100 100 40±6 0 0
100 100 100 100 90±0
100 100 100 100 80±4
100 100 100 ND 100
100 100 100 ND 100
100 80±17 100 ND 30±0
100 100 90±0 ND 40±6
tion increased the uniformity of germination (data not shown). Line BS 002 appeared to be susceptible to desiccation with the embryos unable to tolerate drying for longer than 60 min (Table 2). However, it was noted that these embryos appeared to be hyperhydric prior to desiccation. For all lines, embryos desiccated for 90 min and 120 min showed abnormal and stunted growth after LN exposure. Desiccation for up to 60 min did not adversely influence post-LN somatic embryo germination and somatic seedling development. Furthermore, when cryostored somatic embryos (with a 30- or 60-min desiccation) were recovered on embryogenesis induction medium, 60–80% produced embryogenic masses within 8 weeks.
Discussion Cryostorage is an important tool for securely maintaining living tissues in a genetically stable state for prolonged periods of time. For coniferous species, cryostorage is routinely achieved through the use of slow cooling procedures (Cyr 2000; Häggman 2000), which requires expensive equipment to carefully manipulate cooling rates (Benson 1999; Percy et al. 2000). In the investigation reported here we demonstrate for the first time the successful cryostorage of conifer embryogenic tissues using a simple vitrification protocol. Survival following cryopreservation in which vitrification protocols have been used is dependent on minimizing desiccation and freeze injury through the treatment of cells with protective agents (Charoensub et al. 1990). In the present investigation, embryogenic masses of P. mariana showed the highest survival (50–67%) following LN exposure when treated with 0.8 M sorbitol for 48 h, followed by a 30-min-long incubation at 0°C in a modified cryoprotective agent, PVS2. All of the other sorbitol concentrations and exposure times to PVS2 that were tested resulted in lower survival. Several studies on shoot apices of woody tree species, such as apple and mulberry (Niino and Sakai 1992) and Eucalyptus granitcola (Touchell et al. 2002) have also demonstrated the necessity for specific cryoprotective treatments and the careful application (e.g. time and temperature) of concentrated PVS2. Although in the literature the precise exposure times varied among species, it is generally
agreed that exposure to vitrification solutions, such as PVS2, is necessary to adequately desiccate tissues in order to minimize injury through intracellular ice formation (Niino and Sakai 1992; Thinh et al. 1999). On the other hand, prolonged exposure to vitrification solutions results in injury from chemical toxicity or excessive desiccation. To achieve optimal desiccation through PVS2 treatment, adequate preculturing regimes are required (Pennycooke and Towill 2000). Preculturing regimes on media containing sugars or sugar alcohols seem to be a critical step in the successful cryostorage of tissues using the vitrification procedure. During preculture with sugars or sugar alcohols, tissues are subjected to a mild osmotic stress, which acts to induce responses that lead to increased desiccation tolerance, such as increased levels of ABA and late embryogenesis-abundant proteins (Charoensub et al. 1999; Reinhoud et al. 2000). Furthermore, the sugars and sugar alcohols used in the preculture media may replace water and form hydrogen bonds with membrane phospholipids, thus stabilizing membranes during dehydration and cooling (Crowe and Crowe 1986; Turner et al. 2001a). In the present study, culturing embryogenic tissue clumps on medium supplemented with 0.8 M sorbitol for 2 days or 3 days prior to cryoprotective and LN treatments was effective in inducing dehydration and freezing tolerance. Preculturing for 1 day resulted in lower survival, which can be attributed to an insufficient acquisition of desiccation tolerance or cryoprotection (Turner et al. 2001b). Preculturing for longer than 3 days also resulted in lower survival, most likely due to tissue growth and changes in the physiological condition. Among the several different types of sugars and sugar alcohols tested, sorbitol appeared to be the most effective cryoprotectant for P. mariana embryogenic tissues, followed by glycerol (at 1.6 M) and mannitol, whereas sucrose and glucose were ineffective. Sorbitol and glycerol have also been successfully used in the cryopreservation of several Australian herbaceous and woody species (Turner et al. 2001b; Touchell et al. 2002). Turner et al. (2001a) suggested that the success of sorbitol and glycerol as cryoprotectants could be attributed to their stereochemical arrangements. The stereo-orientation of the hydroxyl groups of these sugar alcohols may allow for more efficient hydrogen bonding and packing around the membrane bilayer, thus providing increased desicca-
123
tion and freeze tolerance (Turner et al. 2001a; Crowe and Crowe 1986). However, it is interesting to note that, for P. mariana, glycerol was only effective when added at a concentration of 1.6 M. This concentration provides for 28.896×1023 hydroxyl groups, the same number of hydroxyl groups as 0.8 M sorbitol. This result thus supports the observation of Turner et al. (2001a) who showed that the number of hydroxyl groups present in the preculture media for shoot apices of Anigozanthos viridis had a greater impact on survival than the molar concentration of the sugar alcohols. Together, this suggests that the action of sugar alcohols in the preculture media is not solely one of osmoconditioning but also one of cryoprotection. In terms of developing a simplified vitrification protocol for the cryopreservation of a large collection of P. mariana embryogenic lines, glycerol may prove to be a superior alternative to sorbitol as a cryoprotectant due to the variable responses of different embryogenic lines to the vitrification protocol shown in this study. While the sorbitol pretreatment was effective for 8 out of 11 lines with respect to survival after LN exposure and yielded the highest survival rate (i.e. 67% for line BS 011), pretreatment with glycerol resulted in a better survival for five of the eight lines and provided cryoprotection to an additional embryogenic line (BS 020) that did not survive after sorbitol treatment. Variability in the cryostorage response of coniferous embryogenic cultures has also been observed using slow cooling procedures. Adams et al. (1994) showed that only 10 of 15 embryogenic lines of Picea mariana survived LN treatment, while Häggman et al. (1998) reported that only seven of nine embryogenic lines of Pinus sylvestris were tolerant to LN. The variation in response to LN treatment has been attributed to the physiological condition of the cultures. Although no link between physiological condition and the ability of any given tissue to survive LN treatments could be made in the present study, it was noted that cryopreservation treatments were lethal to suspensor cells (data not shown). Thus, the ratio of suspensor cells to embryogenic cells may influence tissue survival and recovery. An alternative to embryogenic tissue is the cryopreservation of mature somatic embryos, which has been suggested by Cyr (2000) as a potential strategy for conserving conifer germplasm. Mycock et al. (1995) demonstrated that somatic embryos of Coffea arabica, Manihot esculenta, Phoenix dactylifera and Pisium sativum survived LN exposure when desiccated in a laminar flow hood for 60 min. In previous reports, spruce and western white pine somatic embryos have survived LN treatments if dried to remove freezable water (see Cyr 2000; Percy et al. 2000). However, for Picea mariana, we have demonstrated that mature somatic embryos can survive LN exposure without desiccation treatments. The simplicity of our system and the high recovery rate of cryostored mature somatic embryos, as compared to the embryogenic tissues, could be especially valuable for conifer genetic transformation. Somatic embryos of conifer
species can be transformed via particle bombardment and readily induced (>90%) to form embryogenic tissue through secondary embryogenesis (Charest et al. 1996). In conclusion, this study has for the first time described simplified vitrification protocols for the cryopreservation of coniferous embryogenic cultures without the requirement of slow cooling. Clearly, further studies are necessary, with an emphasis on optimizing physiological conditions, to ensure that the procedure is applicable to large germplasm collections. In addition, the genetic fidelity of cryostored germlines will need to be assessed due to the high concentration of cryoprotectants used in vitrification protocols. Our data also suggest that mature somatic embryos may provide an alternative source of germplasm for maintaining elite lines in cryostorage. Acknowledgements We would like to thank Dr. John Vucetich for his advice and assistance with statistical analyses and two anonymous reviewers for their helpful comments.
References Adams GW, Doiron MG, Park YS, Bonga JM, Charest PJ (1994) Commercialization potential of somatic embryogenesis in black spruce tree improvement. For Chron 70:593–598 Agresti A (1990) Categorical data analysis. Wiley, New York Benson EE (1999) Cryopreservation. In: Benson EE (ed) Plant conservation biotechnology. Taylor and Francis, London, pp 83–95 Charest PJ (1996) Biotechnology in forestry: examples from Canadian Forest Service. For Chron 72:37–42 Charest PJ, Devanter Y, Lachance D (1996) Stable genetic transformation of Picea mariana (black spruce) via particle bombardment. In Vitro Cell Dev Biol Plant 32:91–99 Charoensub R, Phansiri S, Sakai A, Yongmenitchai W (1999) Cryopreservation of Cassava in vitro-grown shoot tips cooled to – 196°C by vitrification. Cryoletters 20:89–94 Crowe LM, Crowe JH (1986) Stabilisation of membranes in anhydrobiotic organisms. In: Leopold AC (ed) Membranes, metabolism, and dry organisms. Comstock, Cornell University, New York, pp 210–230 Cyr D (2000) Cryopreservation: roles on clonal propagation and germplasm conservation in conifers. In: Engelmann F, Takagi H (eds) Cryopreservation of tropical plant germplasm. Current research progress and application. Japan International Research Centre for Agricultural Sciences, Tsukuba/ International Plant Genetic Resources Institute, Rome, pp 261–268 Häggman HM, Ryynanen LA, Aronen TS, Krajnakova J (1998) Cryopreservation of embryogenic cultures of Scots pine. Plant Cell Tissue Organ Cult 54:45–53 Häggman HM, Aronen TS, Ryynanen LA (2000) Cryopreservation of embryogenic cultures of conifers. In: Jain SM, Gupta PK, Newton RJ (eds) Somatic embryogenesis in woody plants, vol 6. Kluwer, Dordrecht, pp 707–728 Klimaszewska K (1995) Somatic embryogenesis in Picea mariana (Mill.). In: Jain SM, Gupta PK, Newton RJ (eds) Somatic embryogenesis in woody plants, vol 3. Kluwer, Dordrecht, pp 67–79 Klimaszewska K, Ward C, Cheliak, WM (1992) Cryopreservation and plant regeneration from embryogenic cultures of larch (Larix × eurolepis) and black spruce (Picea mariana). J Exp Bot 43:73–79 Litvay JD, Johnson MA, Verma D, Einspahr D, Wergrand K (1981) Conifer suspension culture medium development using analytical data from developing seeds. Technical paper series, vol 115. Just Paper Chemistry, Appleton, Wis. pp 1–17
124 Mycock DJ, Wesley-Smith J, Berjak P (1995) Cryopreservation of somatic embryos of four species with and without cryoprotectant pre-treatment. Ann Bot 75:331–336 Niino T, Sakai A (1992) Cryopreservation of alginate-coated in vitro grown shoot-tips of apple, pear and mulberry. Plant Sci 87:199–206 Pennycooke JC, Towill LE (2000) Cryopreservation of shoot tips from in vitro plants of sweet potato [Ipomoea batatas (L.) Lam.] by vitrification. Plant Cell Rep 19:733–737 Percy RE, Klimaszewska K, Cyr DR (2000) Evaluation of somatic embryogenesis for clonal propagation of western white pine. Can J For Res 30:1867–1876 Reinhoud PJ, Van Iren F, Kijne JW (2000) Cryopreservation of differentiated plant cells. In: Engelmann F, Takagi H (eds) Cryopreservation of tropical plant germplasm. Current research progress and application. Japan International Research Centre for Agricultural Sciences, Tsukuba/International Plant Genetic Resources Institute, Rome, pp 91–102 Sakai A (2000) Development of cryopreservation techniques. In: Engelmann F, Takagi H (eds) Cryopreservation of tropical plant germplasm. Cryopreservation of tropical plant germplasm. Current research progress and application. Japan International Research Centre for Agricultural Sciences, Tsukuba/ International Plant Genetic Resources Institute, Rome, pp 1–7 Sakai A, Kobayashi S, Oiyama I (1990) Cryopreservation of nucellar cells of navel orange (Citrus sinesis Osb. var Brasiliensis Tanaka) by vitrification. Plant Cell Rep 9:30–33 SPSS (2000) SPSS version 10.0 user’s guide. SPSS, Chicago
Tautorus TE, Attree SM, Fowke LC, Dunstan DI (1990) Somatic embryogenesis from immature and mature zygotic embryos, and embryo regeneration from protoplasts in Black spruce (Picea mariana Mill.). Plant Sci 67:115–124 Thinh NT, Takagi H, Yashima S (1999) Cryopreservation of in vitro grown shoot tips of banana (Musa sp) by vitrification method. Cryoletters 20:163–174 Touchell DH, Dixon KW (1999) In vitro preservation. In: Bowes BS (ed) A colour atlas of plant propagation and conservation. Manson, London, pp 108–118 Touchell DH, Turner SR, Bunn E, Dixon KW (2002) Cryostorage of somatic tissues of endangered Australian species. In: Towill LE (ed) Cryopreservation of plant germplasm II, vol 50 Springer, Berlin pp 373–390 Tremblay L, Tremblay FM (1995) Maturation of black spruce somatic embryos: sucrose hydrolysis and resulting osmotic pressure of the medium. Plant Cell Tissue Organ Cult 42:39–46 Turner SR, Tan B, Senaratna T, Bunn E, Dixon KW, Touchell DH (2000) Cryopreservation of the Australian species Macropidia fuliginosa (Haemodoraceae) by vitrification. Cryoletters 21:379– 388 Turner SR, Senaratna T, Touchell DH, Bunn E, Dixon KW, Tan B (2001a) Stereochemical arrangement of hydroxyl groups in sugar and polyalcohol molecules as an important factor in effective cryopresrvation. Plant Sci 48:489–497 Turner SR, Senaratna T, Bunn E, Tan B, Dixon KW, Touchell DH (2001b) Cryopreservation of shoot tips from six endangered Australian species using a modified vitrification protocol. Ann Bot 87:371–378
