A CRYOPRESERVATION PROTOCOL FOR EMBRYOS OF THE ENDANGERED SPECIES ZIZANIA TEXANAChristina Walters1*, Darren H. Touchell1, Paula Power2, James Wesley-Smith3 and Michael F. Antolin4 1USDA-ARS National Center for Genetic Res ources, 1111 S. Mason Street, Fort Collins, Colorado, USA2US Fish and Wildlife Service, San Marcos National Fish Hatchery and Technology Center, San Marcos, Texas, USA3 Electron Microscope Unit, Univ of Natal, Durban, South Africa4Department of Biology, Colorado State University, Fort Collins, Colorado, USA * for correspondence (email: firstname.lastname@example.org) Summary Seeds of the endangered species Zizania texana are recalcitrant, making it difficult to preserve the remaining genetic diversity of this sp ecies in genebanks. Excised embryos can be cryopreserved using solution-based cryoprotection protocols. Survival following cryoexposure increased from less than 5% to about 75% by preculturing embryos in high concentrations of sugars, bathing them in cryoprotectant solutions, and partially drying them to water contents of about 0.6 g H2O/g dry mass. Keywords: cryopreservation, desiccation tolerance, em bryo, recalcitrant, vitrification, wildrice, Zizania INTRODUCTION Zizania species are aquatic grasses that produce recalcitrant seeds (13,16,20). Z. texana grows in a 4 mile stretch of the San Marcos Ri ver in Texas and is a Federally-listed endangered species (2). Preserving Z. texana seeds in ex situ genebanks will forestall the continued erosion of genetic diversity of this species and provi de a wider genetic base for improving its close relative, Z. palustris , which produces gourmet wildrice. As with all recalcitrant species, Z. texana cannot be stored in seed genebanks using conventional protocols, which require drying to about 5% water content and storing at 18C (5). Recalcitrant seeds of many species have been successfully cryopreserved by optimizing water contents (reviewed by 3, also 10,18,19,20) and cooli ng rates (23) to cryogenic temperatures so that both freezing and desiccation damage are lim ited. For unknown reasons, this approach has not been effective with Z. texana , giving less than 20% survival for seeds dried to 0.2 to 0.4 g/g and cooled at about 500C/sec (Wesley-Smith et al., unpublished). In this paper, we report successful cryopreservation of embryos of Z. texana using solution-based cryoprotection protocols (frequently called â€œvitrificationâ€ because of the glassforming properties of the cryoprotectants glycerol and DMSO) more typically used for shoot apices (e.g., 1,3). These protocols involve inc ubating tissues in a protective sugar solution, exposing them to cryoprotectants, and then drying them to appropriate water contents before CRYOLETTERS 23 (5): 291-298 SEP-OCT 2002
rapidly plunging them into LN (1,3). The optimum combination of sugar, incubation time, cryoprotectant concentration, and final water c ontent vary among species , cultivars, cell types and provenance. Solution-based cryoprotection is sometimes used with systems that are relatively tolerant of desiccation (e.g. 6,8,17), although this type of application is usually unnecessary since the cells survive drying to wate r contents where water freezing is not observed or is not damaging (11,19,20,23). MATERIALS AND METHODS Plant material and viability assay Zizania texana seeds rarely mature in the wild (12). Plants were grown in containers offsite at Southwest State University, San Marcos, Texas. Seeds were harvested weekly or biweekly from April through July, packaged in moist paper towels and sent immediately by courier mail to Fort Collins, Colorado, where they were stored in moist paper at 5 C until used (within 8-9 months). Seeds from all harvests were pooled before experiments were begun in September to ensure adequate supply of similar pl ant material for a complete set of experiments. Embryos used for cryopreservati on studies were excised from fully matured grains, which were usually green with a hard endosperm, according to maturity classifications developed previously for Zizania (13,20). Embryos germinated about 16 h after excision. To preclude the confounding effects of germination, experiments we re started within 3 h of excising embryos. To prevent imbibitional damage in dried embryos, embryos were slowly rehydrated on damp filter paper before viability assessments. Viability assays consisted of surface-sterilizing embryos with 1.0% commercial sodium hypochlorite solution for 5 min, washing 3 times in sterile distilled water, and placing embryos in culture on Linsmaier and Skoog (LS) basal salts, supplemented with 60 mM sucrose and solidified with 0.8% agar (7). Cultures were incubated at 25 C in the dark for 4 d before moving them under cool white fluorescent lights (100 Einsteins m-2s-1) according to Touchell and Walters (18). Embryo survival was scored when the coleoptile elongated to half the length of the embryo (appr ox. 4 d). Experiments were conducted within a 4 month period, with assessments of sugars a nd sugar alcohols conducted simultaneously within the first 2 months and preculture, PVS2 exposure time and dry time manipulated in the third month. Water relations studies and survival follo wing the established procedures was verified in the forth month, and these studies exhausted the s eed supply. Five replicates of 10 embryos were used for each trial. Drying and exposure to liquid nitrogen Following application of cryoprotective solutions, embryos were dried to different moisture contents using Â“flash drying Â“ pro cedures developed previously (23,19,20,21). Excised embryos were held in a stream of nitrogen gas flowing at approximately 10 liters/min, and moisture content was manipulated by drying time (0 to 300 min). Embryos, dried to different water contents , were mounted on ultramicrotomy specimen holders (Leica, Austria) using a thin film of gl ycerol as an adherent. Specimen holders with up to 5 embryos attached were mounted in a comp ressed-air device, and individually injected into LN subcooled to a slush (c. 210Â°C) within 10 s of axes contac ting the glycerol solution. Cooling rates achieved using this procedure were about 250 C/s as measured by the data recorder described by Wesley-Smith et al . (23). Embryos remained in LN for 30 min, after which they were warmed by submerging naked axes into water at 40 C with vigourous mixing to enhance warming. Cryoprotective solutions Developing the optimum cryoprotective treatment required determining the most effective sugar or sugar alcohol, and its optimum con centration (0 to 1.2 M). Optimum times of incubation (0 to 4 d), exposure to a modified plan t vitrification solution, PV S2 (14) (0 to 60 min),
050100150200250300DRYING TIME (min) 0 0.5 1 1.5 2WATER CONTENT (g HOH/g dry wt)after cryoprotection before cryoprotectionand drying (0 to 60 min) were also assessed. Optimization experiments were done sequentially, with a 48 hr preculture on a concentrated s ugar medium, a 30 min exposure to PVS2 at 0 C, and a 30 minute drying time used as the default treatment unless that variable was specifically studied. The efficacy of various sugars and sugar alcohols in protecting Z. texana embryos during LN exposure was measured by incubating freshly excised embryos for 48 h on standard culturing medium (LS salts solidified with 0.8% agar and containing 60 mM sucrose) further supplemented with 0 to 1.2 M solutions of sorbito l, mannitol, xylitol, ribitol, sucrose, glucose or raffinose. The water potential of supplemented medium was meas ured using a thermocouple psychrometer and varied from Â–0.45 to Â–3.85 MPa, depending on the sugar concentration. Embryos were then exposed to PVS2 (30 min) and partially dried ( 30 min) (as described in the previous paragraph) before LN exposure. Embryos used in experime nts to determine the optim um time of preculture, PVS2 exposure and drying were precultured on the standa rd LS medium further supplemented with 0.8 M sorbitol. RESULTS Freshly excised (e.g. no exogenous cryoprotectants) embryos of Z. texana contained about 2.0 Â± 0.1 g H2O/g dry mass (= 0.66 g H2O/ g fresh mass). Embryos were dried to water contents less than 0.3 g/g dm within 150 min of flash drying (Fig 1). Survival decreased abruptly for embryos dried to water contents less th an 0.3 g/g (Fig 2). Embryos showed 5% survival after cryogenic exposure, irrespective of partial drying treatments (Fig 2), necessitating the use of cryoprotective solutions . Figure 1. Drying time course for Z. texana embryos before and after a cryoprotective treatment of 48 h preculture in 0.8 M sorbitol followed by 30 min exposure to modified PVS2 solution. Lines are exponential decay curves fit to time course data and are used to aid the eye.Generalized procedures for solution-based cryoprotection (e.g., 1,3) were applied to excised Z. texana embryos. All of the steps (preculture with sugars, exposure to a vitrifying solution, partial drying) were necessary for survival. Preculture for 48 hours on LS medium supplemented with additional sugar alcohols (Fig 3) or sugars (Fig 4) gave increased survival, though the concentration giving maximum benefit varied among the different sugars and sugar alcohols. Raffinose gave the best protection at the lowest concen tration (46% survival at 0.2 M), but was relatively ineffective at higher concentrations. Maximum survival (> 70%) was achieved using 0.8 and 1.0 M sorbitol. Most sugars and sugar alcohols gave optimum concentrations, with survival decreasing if concentration exceed ed 0.4 M (raffinose), 0.6 M ( mannitol, xylitol, glucose) or 1.0 M (sucrose, sorbitol).
00.511.52WATER CONTENT (g water/gdw) 0 25 50 75 100SURVIVAL (%)LN exposed, + cryoprotection no cooling LN exposed, cryoprotectionSUGAR ALCOHOL CONCENTRATION (M)SURVIVAL (%) A -sorbitol B -ribitol D -xylitol C-mannitol 1 0.50 1 0.50 100 0 50 0 100 0 50 0 SUGAR ALCOHOL CONCENTRATION (M)SURVIVAL (%) A -sorbitol B -ribitol D -xylitol C-mannitol 1 0.50 1 0.50 100 0 50 0 100 0 50 0 Figure 2. Survival of Z. texana embryos dried to different water contents (circles) and then exposed to LN with (open squares) and without (closed squares) cryoprotection. Data for cryoprotected embryos are replotted from data in Fig 5C using the time course from Fig 1.Exposure times for preculturing, bathing in PV S2, and flash-drying were optimized using embryos precultured on LS medium additionally supplemented with 0.8 M sorbitol (Fig 5). Survival was highest if embryos were precu ltured for 1-2 d, with longer times giving poor survival (Fig 5A). Embryos precultured for 2 d in 0.8 M sorbitol gave highest survival when bathed in PVS2 for 30 min, with toxic effects becoming evident after longer exposure times (Fig 5B). Partial drying was required for embryo surv ival following LN exposure, with 30 min drying giving highest levels (Fig 5C). The cryoprotec tion procedure lowered the initial water content of embryos from about 2 to about 1.6 g/g and increased drying rates (Fig 1). Highest survival was achieved when embryo water contents were reduced to about 0.6 g/g, with further reductions in water content giving reduced survival (Fig 2). Figure 3. Survival of Z. texana embryos following 48 hours preculture in LS medium further supplemented with varying concentrations of sorbitol (A), ribitol (B), mannitol (C) and xylitol (D), followed by 30 min exposure to PVS2, 30 min flash drying (circles), and cryo-exposure (squares). The bars represent the standard error of 5 replicate treatments, each replicate consisting of 10 embryos.
0 25 50 75 100 0 25 50 75 100 00.250.50.751 0 25 50 75 100S U R V I V A L ( % )SUGAR CONCENTRATION (M)C glucose B raffinose A sucroseFigure 4. Survival of Z. texana embryos following 48 hours preculture in LS medium further supplemented with varying concentrations of sucrose (A), raffinose (B) and glucose (C), followed by 30 min exposure to PVS2, 30 min flash drying (circles), and cryo-exposure (squares). The bars represent the standard error of 5 replicate treatments, each replicate consisting of 10 embryos. DISCUSSION This paper describes a protocol that can be used to cryopreserve excised embryos of the endangered species Z. texana . Embryos of Z. texana do not survive drying to water contents less than 0.3 g/g (Fig 2) and are therefore termed Â“recalcitrantÂ” because they do not survive standard storage protocols used in genebanks fo r orthodox seeds (5). Unlike its congener Z. palustris , fully mature Z. texana embryos do not survive typical cryopreservation in the absence of cryoprotectants. Protectant solutions can usually be avoided if cellular water is made unavailable for freezing by either sufficient drying (reviewed by 3, also 5,18,19,20) or by restricting the time available for ice crystal growth at higher wa ter contents (23). Exogenous application of cryoprotectants to excised embryos of Z. texana increased survival following LN exposure from < 5% to about 70% (Fig 2). The methodology to cryoprotect Z. texana embryos using exogenous solutions may be improved further through a better understanding of how the variables of the treatment interact [i.e. sugar, concentration, preculture time, PVS2 exposure, and drying time] as well as how the condition and provenance of embryos affect the response to cryoprotecting treatments. For example, 0.8 M sorbitol was deemed optimum for a 2 day preculture treatment, though it is possible that a shorter exposure time would reveal that sugars, rather than sugar alcohols, gave superior performance. Additionally, survival ma y be further enhanced if embryos were treated immediately after harvest rather than after a few months storage. Our experiments did not exclude the possibility that variables interact, but we had a limited number of seeds and so were unable to carry out all combinations of treatments.
SURVIVAL (%) 0 50 100 TIME (d) TIME (min) 0 153075 45 0 50 100 C-flash-drying A-preculture 0 12 4 3 60 0 50 100 B-PVS2 exposureSURVIVAL (%) 0 50 100 TIME (d) TIME (min) 0 153075 45 0 50 100 C-flash-drying A-preculture 0 12 4 3 60 0 50 100 B-PVS2 exposureFigure 5. Effects of various exposure times during cryoprotective treatments on survival of Z. texana embryos cooled to liquid nitrogen. In A, embryos are precultured for 0-4 d on LS medium further supplemented with 0.8 M sorbitol and then bathed in PVS2 for 30 min followed by 30 min flash drying. In B, embryos previously precultured for 2 d in 0.8 M sorbitol are exposed to a modified PVS2 soution for 0-60 min. In C, embryos, precultured for 2 d in 0.8 M sorbitol and bathed for 30 min in PVS2, are flashed dried for 0-60 min. The bars represent the standard error of 5 replicate treatments, each replicate consisting of 10 embryos . Preculturing embryos in nutrient media additi onally supplemented with sugars or sugar alcohols was required for survival of Z. texana embryos exposed to LN (Figs 3-4). Different sugars gave variable effects on the overall survival achieved, the concentration that gave maximum survival, and possibly (though not tested) the exposure time that gave maximum benefit. The mechanism by which sugars protect embryos may provide insights into the specificity of different sugars, concentrations and kinetics. Sugars and sugar alcohols may play a direct role in protecting cells from damage (e.g. 15), and so preculture in high concentrations may be regarded as a period when protectants are loaded into cells. Alternatively, the high concentrations of sugars and sugar alcohols dur ing the preculture period may affect the metabolic status of embryos with sugars serving either as substrates or osmoti ca. In this case, the concentration and kinetic effects are critical si nce embryos begin to germinate at higher water potentials (> 0.8 MPa), suffer from Â“pathologicalÂ” metabo lism at lower water potentials (< -3 MPa), and produce protective proteins and carbohydrates at intermediate water potentials (9,21,22). The bimodal effect of water potential on embryo metabolism is consistent with the low survival of embryos precultured at both low and high sugar concentrations (w = 0.45 or 3.9 MPa in media with 0 or 1.2 M sugar added, respectively) (Figs 3-4) or for longer incubation times (Fig 5A). Endogenous production of prot ectants may account for the higher survival of embryos precultured in intermediate sugar con centrations [0.4 to 0.8 M sugar solutions gave 1.4 to 2.5 MPa, with only slight differences observed among sugars (data not shown)]. Whether sugars are directly protective or induce the pr oduction of protectants through an osmotic effect, the specificity among different sugars and concentra tions is probably related to the rate at which they penetrate into the cytoplasm and are metabolized within cells. Exposure to PVS2 and partial drying were also necessary for survival of embryos exposed to LN. Glycerol and DMSO are potent inhibitors of ice formation, as is desiccation which increases intracellular viscosity (22,23). However, PVS2 can be toxic (4) and prolonged
exposure results in diminished survival (Fig 5B). Also, a comparison of water content versus survival of embryos before cryoprotecting pro cedures and following pr otection and cryoexposure (Fig 2) shows lower survival at water contents < 0.5 g/g in the treated samples, suggesting that desiccation damage is exacerbated by exposure to low temperatures (Fig 2). Interestingly, cryoprotection did not appear to increase the desiccation tolerance of Z. texana embryos though it clearly increased the rate at which embryos dried (Fig 1). It is unknown why embryos of Z. texana required exogenous application of cryoprotectants to survive cryoexposure. The water-content-limits for drying without damage and for cooling without freezing transitions were coincident at about 0.30 g/g (Fig 2 and 20, respectively), giving the false impression that wa ter content and cooling rates could be balanced to minimize both freezing and desiccation damage, as has been achieved in many other species (reviewed by 3, also 5,18,19,20,23). The experiment s presented here provide no insights into whether unprotected Z. texana embryos were damaged by freezing or desiccation. If damage resulted from freezing injury, then we hypothesize that the relationship between water content and intracellular viscosity [which dictates required cooling rates for successful cryoexposure (23)] differs in Z. texana compared to other recalcitrant embryos that are more amenable to cryoexposure. Differences in in tracellular viscosity among tissues have been reported previously (11) and were predicted for Z. palustris embryos excised from brow n and green seeds based on calorimetric measurements of water transitions (20). In this case, exogenous cryoprotection presumably increased intracellular viscosity to levels which prevented freezing damage when embryos were cooled at 250Â°C/sec. A lternatively, if damage to cryoexposed Z. texana embryos resulted from desiccation damage, then we hypothesize that low temperature exposures exacerbate desiccating stresses in unprotected Z. texana embryos as was demonstrated for immature Z. palustris embryos (20). In this case, exogenous cryoprotectants either directly stabilized cell structures or induced metabolism which gave Z. texana embryos a similar cryophysiology as mature brown Z. palustris embryos. CONCLUSION Embryos of Z. texana survive exposure to liquid nitrogen following cryoprotective treatments which include 1-2 d preculture in high c oncentrations of sugar or sugar alcohols, brief exposure to a cryoprotectant solution and partial drying. Each step in the methodology must be optimized as over-exposure has detrimental effects. The mechanisms of protection during cryogenic exposure are unknown, but may be elucidated with additional experiments that explore the interaction among cryoprotective steps. Acknowledgments The authors acknowledge Dr. Kathyrn Kennedy (formerly of US Fish and Wildlife Service and now with Center for Plant Conservation) for inspiring research on this species. REFERENCES 1.Bajaj YPS (1995) Cryopreservation of Plant Germplas m I. Biotechnology in Agriculture and Forestry vol 32. Springer, Berlin. 2.Emery WHP & Guy MN (1979) Reproduction and embryo development in Texas wildrice ( Zizania texana Hitchc.) Bull of the Torrey Botanical Club 106, 29-31. 3.Engelmann F (1997) Importance of desiccati on for the cryopreservation of recalcitrant seed and vegetatively propagated species. Plant Genetic Resources Newsletter 112, 9-18. 4.Fahy GM (1986) The relevance of cryoprotectant toxicity to cryobiology. Cryobiology 23, 1-13. 5.FAO/IPGRI (1994) Genebank Standards . Food and Agricultural Organization of the United Nations/International Plant Genetic Resources Institute, Rome. 6.Gagliardi, RF, Pacheco GP, Valls JFM & Mansur E (2002) Cryopreservation of
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