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Influence of days of culture on cryoprotectant-supplemented medium and of terminal freezing temperature on the survival of cryopreserved pea shoot tips
CRYOBIOLOGY
28, 288-293 (1991)
Influence of Days of Culture on Cryoprotectant-Supplemented Medium and of Terminal Freezing Temperature on the Survival of Cryopreserved Pea Shoot Tips SEAN MCADAMS,* Departments
of *Biological
DORAISWAMY RATNASABAPATHI?, ROBERT A. SMITH*
Sciences and TMathematics Science, Philadelphia,
and Physics, Pennsylvania
Philadelphia 19104
AND
College
of Pharmacy
and
Germplasm preservation, via shoot tip cryopreservation, is one method used to ensure access in the future to a broad genetic base. A method commonly used to achieve this goal is slow cooling in the presence of a cryoprotectant down to - 30 or -40°C followed by immersion in liquid nitrogen. Two factors that influence survival are days of culture on cryoprotectant-supplemented medium and the terminal temperature prior to immersion in liquid nitrogen. Pea (Pisum sativum L. cv Early Alaska) shoot tips were cultured on solid B5 pea shoot medium containing 5% Me,SO. The time that the shoot tips remained on the cryoprotectant-supplemented medium prior to freezing was varied (0 to 4 days). Percentage survival was evaluated at - 10, -20, - 30, and -4O”C, with and without immersion in liquid nitrogen, using three-way factorial analyses of variance. For shoot tips not immersed in liquid nitrogen, number of days of culture did not influence survival, whereas terminal freezing temperature did have an effect. Optimum survival was at - 10 and -20°C. For shoot tips immersed in liquid nitrogen, terminal freezing temperature had no effect, whereas number of days of culture did influence survival. The largest number of survivors was obtained after 2 days of culture. In addition, the interaction between terminal freezing temperature and exposure to liquid nitrogen was significant, indicating additional damage as a result of this step alone. That is, there was decreased survival as a result of transfer from the terminal freezing temperature to liquid nitrogen, and this damage was greater at - 10 and - 20°C than at - 30 and - 40°C. These results suggest that the dehydration levels at - 10 and - 20°C might be very close to those at -30 or -40°C. 8 1~1 Academic press, I~C.
Controlled freezing and low-temperature storage of isolated meristems have the potential of providing suitable and reliable means of germplasm preservation (7). However, the viable freezing of plant samples to a low temperature is still in a highly empirical state (5). Slow cooling is one method employed in cryopreservation. Slow cooling is thought to prevent intracellular ice formation and thus prevent freezing injury (6). With this technique, samples are cooled to about - 30 or - 40°C. During this time it is thought that extracellular freezing occurs and this results in a protective dehydration (6). This slow cooling is then followed by direct immersion of the samples in liquid nitrogen. However, there
is a “window” between excessive and insufficient dehydration (lo), in which damage should be minimal. Some of the factors that influence survival during slow cooling are cooling rate (8, 15>, concentration of cryoprotectant (16), culture on cryoprotectant-supplemented medium prior to freezing (1, 4, 7), terminal freezing temperature (17)) and time at the terminal freezing temperature before immersion in liquid nitrogen (2, 18). In this study, the effects of number of days of culture prior to freezing on a medium containing cryoprotectant and the terminal freezing temperature on survival of cryopreserved pea (Pisum sativum L. cv Early Alaska) shoot tips were determined. Since protection or damage may occur either during the initial slow cooling phase or during the phase of rapid cooling from the
Received April 20, 1990; accepted June 23, 1990. 288 001 l-2240/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
CRYOPRESERVATION
terminal temperature to liquid nitrogen (9), these effects were determined with and without exposure to liquid nitrogen, using three-way factorial analyses of variance.
OF
PEA
SHOOT
TIPS
289
ture for each day, with and without immersion in liquid nitrogen (five shoot tips/tube, two tubes/replication, five replications). Shoot tips, without washing, were then transferred to 16 x lOO-mm glass culture MATERIALS AND METHODS tubes containing 2.5 ml of solid pea shoot Pea seeds (P. sativum L. cv Early medium without cryoprotectant. The tubes Alaska) were disinfected and aseptically were wrapped in Parafilm and incubated for germinated in the dark for 5 days at 28°C 6 weeks under the previously mentioned (7). Shoot tips approximately 1 to 2 mm in growth chamber conditions. Only shoot length containing several leaf primordia and tips with healthy apical meristems and leaf subapical stem material were dissected primordia were judged as surviving. aseptically from the germinated peas. Five Data were analyzed using three- and twoshoot tips were cultured together in each 16 way factorial analyses of variance with an x loo-mm glass tube containing 2.5 ml of arcsine transformation (3). Fisher’s least solid pea shoot medium. Pea shoot medium significant difference (LSD) test was used consisted of B5 medium containing 0.5 t.~iW to locate optimum conditions. benzyladenine, 2% sucrose, and 0.8% agar RESULTS (7). The medium was supplemented with 5% dimethyl sulfoxide (Me,SO) for culturThe effects of days of culture on cryoproing of shoot tips prior to freezing. tectant-supplemented medium and terminal The shoot tips were placed in a growth freezing temperature, with and without imchamber with a 20°C day and 15°C night mersion in liquid nitrogen, are presented in and a 16-hr photoperiod (120 PE rnp2 Table 1. The results for shoot tips not imset- ‘) (7). The number of days shoot tips mersed in liquid nitrogen were not predicwere cultured on cryoprotectanttive of results for shoot tips that were imsupplemented medium was varied from 0 to mersed in liquid nitrogen. For shoot tips 4 days. Following this, five shoot tips were not immersed in liquid nitrogen, the numtransferred to each 15-ml polystyrene cen- ber of days of culture on cryoprotectanttrifuge tube containing 0.5 ml of freezing supplemented medium did not influence solution (pea shoot medium without the survival (P = 0.85), whereas terminal agar but with 5% Me,SO). For tips im- freezing temperature was significant (P < mersed in liquid nitrogen, 2.0-ml polypro0.05). Optimum survival was at - 10 and pylene cryovials were used. All shoot tips -20°C (P < 0.05). Survival decreased at were left in freezing solution at room tem- 30°C and again at - 40°C. For shoot tips perature for 1 hr. immersed in liquid nitrogen, terminal freezThe tubes containing the shoot tips were ing temperature did not influence survival then transferred to a Virtis refrigerated bath (P = 0.45), whereas number of days of culchamber containing 95% ethanol at 0°C. ture on cryoprotectant-supplemented meSamples were cooled to - 10, -20, - 30, dium did (P = 0.02). The largest number of or -40°C at a rate of 1.3”Umin. Half the survivors is found at 2 days of culture. Fishsamples were thawed, if necessary, immeer’s LSD test revealed that the results for 2 diately upon removal from the alcohol bath. days of culture were significantly different The other half were immersed in liquid ni- (P < 0.05) from those for all other days trogen for 30 to 60 min before thawing. except Day 4. However, the results for 4 Samples were thawed in a 50°C water bath days of culture are not significantly differuntil the ice plug melted. A total of 50 shoot ent from those for any other days of culture tips were tested at each terminal temperaexcept Day 0. Thus, the results suggest that
290
MCADAMS,
RATNASABAPATHI,
TABLE
AND
SMITH
1
Inthrence of Days of Culture on Cryoprotectant-Supplemented Medium and Terminal Freezing Temperature, with and without Immersion in Liquid Nitrogen, on Survival of Pea Shoot Tips Percentage survival at each terminal freezing temperature
Days of culture
Shoot tips not immersed in liquid nitrogen - 10°C
- 20°C
0 1
100
2 3 4
100 100 98
98 98 92 92 94
100
Shoot tips immersed in liquid nitrogen
-30°C
-40°C
30 42 48 68 54
18 24 22 22 28
-10°C
- 20°C
- 30°C
0 16 18 0 0
2 12 32 0 4
4 2 14
-40°C 0 2 12
10
10
30
14
Note. See Materials and Methods for the conditions of the experiment. Percentage survival is the number of shoot tips out of 50 tested with healthy apical meristems and leaf primordia 6 weeks after treatment.
2 days of culture on cryoprotectantsupplemented medium is the best choice. There was no interaction (at the 0.05 level of significance) between days of culture and terminal temperature or between days of culture and liquid nitrogen. However, exposure to liquid nitrogen revealed a statistically significant (P < 0.01) decrease in survival, indicating additional damage as a result of this step alone. In addition, the interaction between terminal freezing temperature and exposure to liquid nitrogen (Fig. 1) was found to be highly significant (P < 0.01). The decrease in survival from exposure to liquid nitrogen is much more marked at - 10 and - 20°C than at - 30°C. The damage due to exposure to liquid nitrogen at -40°C is slightly less. For shoot tips not exposed to liquid nitrogen at - 10 and -20°C there was less significant variation in viability observed between experiments. However, at -30 and -40°C as well as for all temperatures for shoot tips exposed to liquid nitrogen, the variation was highly significant.
tips were examined. These effects were examined with and without exposure to liquid nitrogen, since damage or protection may be a result of the initial cooling to the terminal temperature or of the transfer to liquid nitrogen and thawing (9). Culture of shoot tips in Me,SOsupplemented medium for 2 days prior to freezing has resulted in increased survival (7, 8). Our results also indicate a %-day culture on 5% Me,SO-supplemented medium is a good choice for cryopreservation.
f
OW 10
- 20 Termlnal
DISCUSSION
To determine optimum conditions for cryopreservation, the combined effects of days of culture on cryoprotectantsupplemented medium and terminal freezing temperature on survival of pea shoot
Freezing
- 30 Temperature
40 ( OC)
1. Interaction between terminal freezing temperature and exposure to liquid nitrogen. For both the shoot tips exposed to liquid nitrogen and the shoot tips not exposed to liquid nitrogen, the percentages reported are the averages of survival for all shoot tips tested at each terminal temperature. Methods are as described under Materials and Methods. FIG.
CRYOPRESERVATION
There was less survival for both shorter and longer days of culture although the results for 4 days of culture were not significantly different from those for 2 days. If Me,SO is providing some colligative support (IO), then it may be that there is an optimum time for maximum penetration of Me+0 into the shoot apex (12) without a degree of toxicity that can result after a period of growth on medium containing Me,SO (5). Another possible role of Me,SO in the culture medium used as a prefreezing treatment may be to partially dehydrate the cells and thus minimize the risk of intracellular ice formation during freezing (7), with 2 days representing the optimum dehydration period. The actual mechanism by which Me,SO increases protection during culture warrants investigation, since there is some evidence that Me$O may be involved in generating a variety of genetic and/or epigenetic changes (5). If Me,SO does cause genetic changes under these conditions of culture, and if its effect is primarily osmotic, it may be that increased concentrations of other compounds, such as sucrose, may be effectively substituted, providing these compounds are not deleterious at the concentrations used. Fabre and Dereuddre (4) found that a culture phase with sucrose prior to freezing, at 0.5 M concentration, was required for cryopreservation of carnation shoot apical meristems. Thus, perhaps higher concentrations of sucrose could be substituted for the Me,SO. In contrast to the above results, days of culture on cryoprotectant-supplemented medium did not influence survival of shoot tips not exposed to liquid nitrogen. This is also contradictory to results found for potato shoot tips. Towill (16) found that freshly harvested Solanum etuberosum shoot tips cooled to -40°C and then rewarmed did not survive, but that a 2-day culture prior to freezing allowed high survival. The 5 x 4 x 2 factorial design and the larger number of trials in our experiments
OF
PEA
SHOOT
TIPS
291
might explain these differences. Thus, at least for days of culture, results for shoot tips not exposed to liquid nitrogen should not be used alone to evaluate or predict optimum conditions for cryopreservation. Further, since the interaction between days of culture and exposure to liquid nitrogen was not statistically significant, some factor (such as degree of penetration or amount of dehydration as discussed above) other than exposure to liquid nitrogen was responsible for the observed differences. Terminal freezing temperature has been found to be a factor in optimizing conditions for cryopreservation. Strategies include using a specific cooling rate and removing samples at various terminal freezing temperatures (13, 17) or holding the samples at one or more terminal temperatures for a period (2, 9, 18). It is presumed that protective dehydration is acquired as a result of both approaches (6). Our results indicate that terminal freezing temperature did not influence survival for shoot tips exposed to liquid nitrogen. In addition, there was a significant interaction between terminal freezing temperature and liquid nitrogen. The interaction was more marked at - 10 and - 20°C than at - 30 and - 40°C. These results suggest that the dehydration level at - 10 and - 20°C might be very close to those at - 30 or -40°C. Even though there is not always a simple relationship between liquid water content and survival after storage in liquid nitrogen (18), one possible explanation is that the cooling rate used (1.3”CYmin) was not sufficiently slow or accurate enough to allow protective dehydration. Kartha et al. (7) obtained high survival at 0.6”C/min, whereas survival decreased at both 0.5 and O.TC/min. Thus, decreased viability after exposure to liquid nitrogen in our case might be a result of intracellular ice crystals. In contrast, optimum survival of shoot tips not exposed to liquid nitrogen was at - 10 and - 20°C. Survival decreased at
292
MCADAMS,
RATNASABAPATHI,
- 30°C and again at - 40°C. This decreased survival below - 20°C may be due in part to the degree of cryoprotection afforded by the concentrations of Me,SO and sucrose used in our experiments (5% Me,SO and 2% sucrose). Sakai and Yoshida (14) found cabbage cells were protected to about -20°C using 0.5 M solutions of Me,SO or sucrose, yet these same compounds gave protection down to - 30°C at 1 A4 concentrations. In addition, a 1 M glucose solution protected cabbage cells to -50°C (14). Thus the use of higher concentrations (0.5 to 1 A4) of Me,SO, sucrose, or glucose, at least in the freezing solution, needs to be examined in relation to cryoprotection of shoot tips. The variations in viability observed in 75% of our data were relatively high. These variations have been attributed to contamination (17) or to differences in the physiological state of the shoot tips, the different sizes of the explants, or injuries caused by the explantation (2). However, another possible source of variation may be the use of ampoules. Cooling in ampoules may not be as homogeneous as, for example, other methods such as droplet freezing (6). Thus, the local environment of each shoot tip in the ampoule may vary. Diettrich ef al. (2) found reduced viability for Digitalis lanata shoot tips in ampoules compared to uncovered shoot tips. This may also account for at least some of the observed differences between experiments. In conclusion, a 2-day culture of 5% Me,SO-supplemented medium allows moderate survival of cryopreserved pea shoot tips. However, if cryopreservation induces modifications in genotype or phenotype, or selects from within a heterogeneous population, failing to preserve unique traits, then the benefits of this technique will be lost (19). Thus, a high percentage of normal shoot tip regrowth is important for clonal reproducibility (11). The factorial experimental design should provide a useful
AND
SMITH
means for achieving this goal by assessing the protection or damage that occurs during the initial slow cooling stage and during the transfer to liquid nitrogen. REFERENCES
1. Braun, A. Cryopreservation of sugar beet germplasm. Z%nt Cell Tissue Organ Cult. 14, 161168 (1988). 2. Diettrich, B., Wolf, T., Bormann, A., Popov, A. S., Butenko, R. G., and Luckner, M. Cryopreservation of Digitalis Zanata shoot tips. Plant. Med. 53, 359-363 (1987). 3. Dowdy, S., and Wearden, S. “Statistics for Research.” Wiley, New York, 1983. 4. Fabre, J., and Dereuddre, J. Effects of different substances (sucrose, glucose, sorbitol and mannitol) on the resistance to deep freezing in liquid nitrogen of meristems from in vitro cultured carnations (Dianthus caryophyllus L., var Eolo. C.R. Acad. Sci. Ser. 3 304, 507-510 (1987). 5. Finkle, B. J., Zavala, M. E., and Uhich, J. M. Cryoprotective compounds in the viable freezing of plant tissues. In “Cryopreservation of Plant Cells and Organs” (K. K. Kartha, Ed.), pp. 75-113. CRC Press, Boca Raton, FL, 1985. 6. Kartha, K. K. Advances in the cryopreservation technology of plant cells and organs. In “Plant Tissue and Cell Culture” (C. E. Green, D. A. Somers, W. P. Hackett, and D. D. Biesboer, Eds.), pp. 447-458. Alan R. Liss, New York, 1987. 7. Kartha, K. K., Leung, N. L., and Gamborg, 0. L. Freeze-preservation of pea meristems in liquid nitrogen and subsequent plant regeneration. Plant Sci. Lett. 15, 7-16 (1979). 8. Kartha, K. K., Leung, N. L., and Pahl, K. Cryopreservation of strawberry meristems and mass propagation of plantlets. .Z.Am. Sot. Hort. Sci. 105, 481-484 (1980). 9. McGann, L. E., and Farrant, J. Survival of tissue culture cells frozen by a two-step procedure to - 196°C. I. Holding temperature and time. Cryobiology 13, 261-268 (1976). 10. Meryman, H. T., and Williams, R. J. Basic principles of freezing injury to plant cells: Natural tolerance and approaches to cryopreservation. In “Cryopreservation of Plant Cells and Organs” (K. K. Kartha, Ed.), pp. lw7. CRC Press, Boca Raton, FL, 1985. 11. Reed, B. M., and Lagerstedt, H. B. Freeze preservation of apical meristems of Rubus in liquid nitrogen. Hort. Sci. 22, 302-303 (1987). 12. Sakai, A. Cryopreservation of shoot-tips of fruit
CRYOPRESERVATION
13.
14. 15.
16.
trees and herbaceous plants. In “Cryopreservation of Plant Cells and Organs” (K. K. Karth, Ed.), pp. 135-158. CRC Press, BocaRaton, FL, 1985. Sakai, A., Yamakawa, M., and Sakata, D. Development of a whole plant from an excised strawberry runner apex frozen to - 196°C. Low Temp. Sci. Ser. B 36, 31-38 (1978). Sakai, A., and Yoshida, S. The role of sugar and related compounds in variations of freezing resistance. Cryobiology 5, 160-174 (1968). Siebert, M., and Wetherbee, P. J. Increased survival and differentiation of frozen herbaceous plant organ cultures through cold treatment. Plant Pkysiol. 59, 1043-1046 (1977). Towill, L. E. Solarium etuberosum: A model for
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studying the cryobiology of shoot-tips in the tuber-bearing Solanum species. Plant Sci. Lett. 20, 315-324 (1981). 17. Towill, L. E. Improved survival afer cryogenic exposure of shoot tips derived from in vitro plantlet cultures of potato. Cryobiology 20, 567-573 (1983). 18. Tyler, N., Stushnoff, C., and Gusta, L. V. Freezing of water in dormant vegetative apple buds in relation to cryopreservation. Plant Pkysiol. 87, 201-205 (1988). 19. Withers, L. A. Cryopreservation of cultured plant cells and protoplasts. In “Cryopreservation of Plant Cells and Organs” (K. K. Kartha, Ed.), pp. 243-267. CRC Press, Boca Raton, FL, 1985.
28, 288-293 (1991)
Influence of Days of Culture on Cryoprotectant-Supplemented Medium and of Terminal Freezing Temperature on the Survival of Cryopreserved Pea Shoot Tips SEAN MCADAMS,* Departments
of *Biological
DORAISWAMY RATNASABAPATHI?, ROBERT A. SMITH*
Sciences and TMathematics Science, Philadelphia,
and Physics, Pennsylvania
Philadelphia 19104
AND
College
of Pharmacy
and
Germplasm preservation, via shoot tip cryopreservation, is one method used to ensure access in the future to a broad genetic base. A method commonly used to achieve this goal is slow cooling in the presence of a cryoprotectant down to - 30 or -40°C followed by immersion in liquid nitrogen. Two factors that influence survival are days of culture on cryoprotectant-supplemented medium and the terminal temperature prior to immersion in liquid nitrogen. Pea (Pisum sativum L. cv Early Alaska) shoot tips were cultured on solid B5 pea shoot medium containing 5% Me,SO. The time that the shoot tips remained on the cryoprotectant-supplemented medium prior to freezing was varied (0 to 4 days). Percentage survival was evaluated at - 10, -20, - 30, and -4O”C, with and without immersion in liquid nitrogen, using three-way factorial analyses of variance. For shoot tips not immersed in liquid nitrogen, number of days of culture did not influence survival, whereas terminal freezing temperature did have an effect. Optimum survival was at - 10 and -20°C. For shoot tips immersed in liquid nitrogen, terminal freezing temperature had no effect, whereas number of days of culture did influence survival. The largest number of survivors was obtained after 2 days of culture. In addition, the interaction between terminal freezing temperature and exposure to liquid nitrogen was significant, indicating additional damage as a result of this step alone. That is, there was decreased survival as a result of transfer from the terminal freezing temperature to liquid nitrogen, and this damage was greater at - 10 and - 20°C than at - 30 and - 40°C. These results suggest that the dehydration levels at - 10 and - 20°C might be very close to those at -30 or -40°C. 8 1~1 Academic press, I~C.
Controlled freezing and low-temperature storage of isolated meristems have the potential of providing suitable and reliable means of germplasm preservation (7). However, the viable freezing of plant samples to a low temperature is still in a highly empirical state (5). Slow cooling is one method employed in cryopreservation. Slow cooling is thought to prevent intracellular ice formation and thus prevent freezing injury (6). With this technique, samples are cooled to about - 30 or - 40°C. During this time it is thought that extracellular freezing occurs and this results in a protective dehydration (6). This slow cooling is then followed by direct immersion of the samples in liquid nitrogen. However, there
is a “window” between excessive and insufficient dehydration (lo), in which damage should be minimal. Some of the factors that influence survival during slow cooling are cooling rate (8, 15>, concentration of cryoprotectant (16), culture on cryoprotectant-supplemented medium prior to freezing (1, 4, 7), terminal freezing temperature (17)) and time at the terminal freezing temperature before immersion in liquid nitrogen (2, 18). In this study, the effects of number of days of culture prior to freezing on a medium containing cryoprotectant and the terminal freezing temperature on survival of cryopreserved pea (Pisum sativum L. cv Early Alaska) shoot tips were determined. Since protection or damage may occur either during the initial slow cooling phase or during the phase of rapid cooling from the
Received April 20, 1990; accepted June 23, 1990. 288 001 l-2240/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
CRYOPRESERVATION
terminal temperature to liquid nitrogen (9), these effects were determined with and without exposure to liquid nitrogen, using three-way factorial analyses of variance.
OF
PEA
SHOOT
TIPS
289
ture for each day, with and without immersion in liquid nitrogen (five shoot tips/tube, two tubes/replication, five replications). Shoot tips, without washing, were then transferred to 16 x lOO-mm glass culture MATERIALS AND METHODS tubes containing 2.5 ml of solid pea shoot Pea seeds (P. sativum L. cv Early medium without cryoprotectant. The tubes Alaska) were disinfected and aseptically were wrapped in Parafilm and incubated for germinated in the dark for 5 days at 28°C 6 weeks under the previously mentioned (7). Shoot tips approximately 1 to 2 mm in growth chamber conditions. Only shoot length containing several leaf primordia and tips with healthy apical meristems and leaf subapical stem material were dissected primordia were judged as surviving. aseptically from the germinated peas. Five Data were analyzed using three- and twoshoot tips were cultured together in each 16 way factorial analyses of variance with an x loo-mm glass tube containing 2.5 ml of arcsine transformation (3). Fisher’s least solid pea shoot medium. Pea shoot medium significant difference (LSD) test was used consisted of B5 medium containing 0.5 t.~iW to locate optimum conditions. benzyladenine, 2% sucrose, and 0.8% agar RESULTS (7). The medium was supplemented with 5% dimethyl sulfoxide (Me,SO) for culturThe effects of days of culture on cryoproing of shoot tips prior to freezing. tectant-supplemented medium and terminal The shoot tips were placed in a growth freezing temperature, with and without imchamber with a 20°C day and 15°C night mersion in liquid nitrogen, are presented in and a 16-hr photoperiod (120 PE rnp2 Table 1. The results for shoot tips not imset- ‘) (7). The number of days shoot tips mersed in liquid nitrogen were not predicwere cultured on cryoprotectanttive of results for shoot tips that were imsupplemented medium was varied from 0 to mersed in liquid nitrogen. For shoot tips 4 days. Following this, five shoot tips were not immersed in liquid nitrogen, the numtransferred to each 15-ml polystyrene cen- ber of days of culture on cryoprotectanttrifuge tube containing 0.5 ml of freezing supplemented medium did not influence solution (pea shoot medium without the survival (P = 0.85), whereas terminal agar but with 5% Me,SO). For tips im- freezing temperature was significant (P < mersed in liquid nitrogen, 2.0-ml polypro0.05). Optimum survival was at - 10 and pylene cryovials were used. All shoot tips -20°C (P < 0.05). Survival decreased at were left in freezing solution at room tem- 30°C and again at - 40°C. For shoot tips perature for 1 hr. immersed in liquid nitrogen, terminal freezThe tubes containing the shoot tips were ing temperature did not influence survival then transferred to a Virtis refrigerated bath (P = 0.45), whereas number of days of culchamber containing 95% ethanol at 0°C. ture on cryoprotectant-supplemented meSamples were cooled to - 10, -20, - 30, dium did (P = 0.02). The largest number of or -40°C at a rate of 1.3”Umin. Half the survivors is found at 2 days of culture. Fishsamples were thawed, if necessary, immeer’s LSD test revealed that the results for 2 diately upon removal from the alcohol bath. days of culture were significantly different The other half were immersed in liquid ni- (P < 0.05) from those for all other days trogen for 30 to 60 min before thawing. except Day 4. However, the results for 4 Samples were thawed in a 50°C water bath days of culture are not significantly differuntil the ice plug melted. A total of 50 shoot ent from those for any other days of culture tips were tested at each terminal temperaexcept Day 0. Thus, the results suggest that
290
MCADAMS,
RATNASABAPATHI,
TABLE
AND
SMITH
1
Inthrence of Days of Culture on Cryoprotectant-Supplemented Medium and Terminal Freezing Temperature, with and without Immersion in Liquid Nitrogen, on Survival of Pea Shoot Tips Percentage survival at each terminal freezing temperature
Days of culture
Shoot tips not immersed in liquid nitrogen - 10°C
- 20°C
0 1
100
2 3 4
100 100 98
98 98 92 92 94
100
Shoot tips immersed in liquid nitrogen
-30°C
-40°C
30 42 48 68 54
18 24 22 22 28
-10°C
- 20°C
- 30°C
0 16 18 0 0
2 12 32 0 4
4 2 14
-40°C 0 2 12
10
10
30
14
Note. See Materials and Methods for the conditions of the experiment. Percentage survival is the number of shoot tips out of 50 tested with healthy apical meristems and leaf primordia 6 weeks after treatment.
2 days of culture on cryoprotectantsupplemented medium is the best choice. There was no interaction (at the 0.05 level of significance) between days of culture and terminal temperature or between days of culture and liquid nitrogen. However, exposure to liquid nitrogen revealed a statistically significant (P < 0.01) decrease in survival, indicating additional damage as a result of this step alone. In addition, the interaction between terminal freezing temperature and exposure to liquid nitrogen (Fig. 1) was found to be highly significant (P < 0.01). The decrease in survival from exposure to liquid nitrogen is much more marked at - 10 and - 20°C than at - 30°C. The damage due to exposure to liquid nitrogen at -40°C is slightly less. For shoot tips not exposed to liquid nitrogen at - 10 and -20°C there was less significant variation in viability observed between experiments. However, at -30 and -40°C as well as for all temperatures for shoot tips exposed to liquid nitrogen, the variation was highly significant.
tips were examined. These effects were examined with and without exposure to liquid nitrogen, since damage or protection may be a result of the initial cooling to the terminal temperature or of the transfer to liquid nitrogen and thawing (9). Culture of shoot tips in Me,SOsupplemented medium for 2 days prior to freezing has resulted in increased survival (7, 8). Our results also indicate a %-day culture on 5% Me,SO-supplemented medium is a good choice for cryopreservation.
f
OW 10
- 20 Termlnal
DISCUSSION
To determine optimum conditions for cryopreservation, the combined effects of days of culture on cryoprotectantsupplemented medium and terminal freezing temperature on survival of pea shoot
Freezing
- 30 Temperature
40 ( OC)
1. Interaction between terminal freezing temperature and exposure to liquid nitrogen. For both the shoot tips exposed to liquid nitrogen and the shoot tips not exposed to liquid nitrogen, the percentages reported are the averages of survival for all shoot tips tested at each terminal temperature. Methods are as described under Materials and Methods. FIG.
CRYOPRESERVATION
There was less survival for both shorter and longer days of culture although the results for 4 days of culture were not significantly different from those for 2 days. If Me,SO is providing some colligative support (IO), then it may be that there is an optimum time for maximum penetration of Me+0 into the shoot apex (12) without a degree of toxicity that can result after a period of growth on medium containing Me,SO (5). Another possible role of Me,SO in the culture medium used as a prefreezing treatment may be to partially dehydrate the cells and thus minimize the risk of intracellular ice formation during freezing (7), with 2 days representing the optimum dehydration period. The actual mechanism by which Me,SO increases protection during culture warrants investigation, since there is some evidence that Me$O may be involved in generating a variety of genetic and/or epigenetic changes (5). If Me,SO does cause genetic changes under these conditions of culture, and if its effect is primarily osmotic, it may be that increased concentrations of other compounds, such as sucrose, may be effectively substituted, providing these compounds are not deleterious at the concentrations used. Fabre and Dereuddre (4) found that a culture phase with sucrose prior to freezing, at 0.5 M concentration, was required for cryopreservation of carnation shoot apical meristems. Thus, perhaps higher concentrations of sucrose could be substituted for the Me,SO. In contrast to the above results, days of culture on cryoprotectant-supplemented medium did not influence survival of shoot tips not exposed to liquid nitrogen. This is also contradictory to results found for potato shoot tips. Towill (16) found that freshly harvested Solanum etuberosum shoot tips cooled to -40°C and then rewarmed did not survive, but that a 2-day culture prior to freezing allowed high survival. The 5 x 4 x 2 factorial design and the larger number of trials in our experiments
OF
PEA
SHOOT
TIPS
291
might explain these differences. Thus, at least for days of culture, results for shoot tips not exposed to liquid nitrogen should not be used alone to evaluate or predict optimum conditions for cryopreservation. Further, since the interaction between days of culture and exposure to liquid nitrogen was not statistically significant, some factor (such as degree of penetration or amount of dehydration as discussed above) other than exposure to liquid nitrogen was responsible for the observed differences. Terminal freezing temperature has been found to be a factor in optimizing conditions for cryopreservation. Strategies include using a specific cooling rate and removing samples at various terminal freezing temperatures (13, 17) or holding the samples at one or more terminal temperatures for a period (2, 9, 18). It is presumed that protective dehydration is acquired as a result of both approaches (6). Our results indicate that terminal freezing temperature did not influence survival for shoot tips exposed to liquid nitrogen. In addition, there was a significant interaction between terminal freezing temperature and liquid nitrogen. The interaction was more marked at - 10 and - 20°C than at - 30 and - 40°C. These results suggest that the dehydration level at - 10 and - 20°C might be very close to those at - 30 or -40°C. Even though there is not always a simple relationship between liquid water content and survival after storage in liquid nitrogen (18), one possible explanation is that the cooling rate used (1.3”CYmin) was not sufficiently slow or accurate enough to allow protective dehydration. Kartha et al. (7) obtained high survival at 0.6”C/min, whereas survival decreased at both 0.5 and O.TC/min. Thus, decreased viability after exposure to liquid nitrogen in our case might be a result of intracellular ice crystals. In contrast, optimum survival of shoot tips not exposed to liquid nitrogen was at - 10 and - 20°C. Survival decreased at
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- 30°C and again at - 40°C. This decreased survival below - 20°C may be due in part to the degree of cryoprotection afforded by the concentrations of Me,SO and sucrose used in our experiments (5% Me,SO and 2% sucrose). Sakai and Yoshida (14) found cabbage cells were protected to about -20°C using 0.5 M solutions of Me,SO or sucrose, yet these same compounds gave protection down to - 30°C at 1 A4 concentrations. In addition, a 1 M glucose solution protected cabbage cells to -50°C (14). Thus the use of higher concentrations (0.5 to 1 A4) of Me,SO, sucrose, or glucose, at least in the freezing solution, needs to be examined in relation to cryoprotection of shoot tips. The variations in viability observed in 75% of our data were relatively high. These variations have been attributed to contamination (17) or to differences in the physiological state of the shoot tips, the different sizes of the explants, or injuries caused by the explantation (2). However, another possible source of variation may be the use of ampoules. Cooling in ampoules may not be as homogeneous as, for example, other methods such as droplet freezing (6). Thus, the local environment of each shoot tip in the ampoule may vary. Diettrich ef al. (2) found reduced viability for Digitalis lanata shoot tips in ampoules compared to uncovered shoot tips. This may also account for at least some of the observed differences between experiments. In conclusion, a 2-day culture of 5% Me,SO-supplemented medium allows moderate survival of cryopreserved pea shoot tips. However, if cryopreservation induces modifications in genotype or phenotype, or selects from within a heterogeneous population, failing to preserve unique traits, then the benefits of this technique will be lost (19). Thus, a high percentage of normal shoot tip regrowth is important for clonal reproducibility (11). The factorial experimental design should provide a useful
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means for achieving this goal by assessing the protection or damage that occurs during the initial slow cooling stage and during the transfer to liquid nitrogen. REFERENCES
1. Braun, A. Cryopreservation of sugar beet germplasm. Z%nt Cell Tissue Organ Cult. 14, 161168 (1988). 2. Diettrich, B., Wolf, T., Bormann, A., Popov, A. S., Butenko, R. G., and Luckner, M. Cryopreservation of Digitalis Zanata shoot tips. Plant. Med. 53, 359-363 (1987). 3. Dowdy, S., and Wearden, S. “Statistics for Research.” Wiley, New York, 1983. 4. Fabre, J., and Dereuddre, J. Effects of different substances (sucrose, glucose, sorbitol and mannitol) on the resistance to deep freezing in liquid nitrogen of meristems from in vitro cultured carnations (Dianthus caryophyllus L., var Eolo. C.R. Acad. Sci. Ser. 3 304, 507-510 (1987). 5. Finkle, B. J., Zavala, M. E., and Uhich, J. M. Cryoprotective compounds in the viable freezing of plant tissues. In “Cryopreservation of Plant Cells and Organs” (K. K. Kartha, Ed.), pp. 75-113. CRC Press, Boca Raton, FL, 1985. 6. Kartha, K. K. Advances in the cryopreservation technology of plant cells and organs. In “Plant Tissue and Cell Culture” (C. E. Green, D. A. Somers, W. P. Hackett, and D. D. Biesboer, Eds.), pp. 447-458. Alan R. Liss, New York, 1987. 7. Kartha, K. K., Leung, N. L., and Gamborg, 0. L. Freeze-preservation of pea meristems in liquid nitrogen and subsequent plant regeneration. Plant Sci. Lett. 15, 7-16 (1979). 8. Kartha, K. K., Leung, N. L., and Pahl, K. Cryopreservation of strawberry meristems and mass propagation of plantlets. .Z.Am. Sot. Hort. Sci. 105, 481-484 (1980). 9. McGann, L. E., and Farrant, J. Survival of tissue culture cells frozen by a two-step procedure to - 196°C. I. Holding temperature and time. Cryobiology 13, 261-268 (1976). 10. Meryman, H. T., and Williams, R. J. Basic principles of freezing injury to plant cells: Natural tolerance and approaches to cryopreservation. In “Cryopreservation of Plant Cells and Organs” (K. K. Kartha, Ed.), pp. lw7. CRC Press, Boca Raton, FL, 1985. 11. Reed, B. M., and Lagerstedt, H. B. Freeze preservation of apical meristems of Rubus in liquid nitrogen. Hort. Sci. 22, 302-303 (1987). 12. Sakai, A. Cryopreservation of shoot-tips of fruit
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trees and herbaceous plants. In “Cryopreservation of Plant Cells and Organs” (K. K. Karth, Ed.), pp. 135-158. CRC Press, BocaRaton, FL, 1985. Sakai, A., Yamakawa, M., and Sakata, D. Development of a whole plant from an excised strawberry runner apex frozen to - 196°C. Low Temp. Sci. Ser. B 36, 31-38 (1978). Sakai, A., and Yoshida, S. The role of sugar and related compounds in variations of freezing resistance. Cryobiology 5, 160-174 (1968). Siebert, M., and Wetherbee, P. J. Increased survival and differentiation of frozen herbaceous plant organ cultures through cold treatment. Plant Pkysiol. 59, 1043-1046 (1977). Towill, L. E. Solarium etuberosum: A model for
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studying the cryobiology of shoot-tips in the tuber-bearing Solanum species. Plant Sci. Lett. 20, 315-324 (1981). 17. Towill, L. E. Improved survival afer cryogenic exposure of shoot tips derived from in vitro plantlet cultures of potato. Cryobiology 20, 567-573 (1983). 18. Tyler, N., Stushnoff, C., and Gusta, L. V. Freezing of water in dormant vegetative apple buds in relation to cryopreservation. Plant Pkysiol. 87, 201-205 (1988). 19. Withers, L. A. Cryopreservation of cultured plant cells and protoplasts. In “Cryopreservation of Plant Cells and Organs” (K. K. Kartha, Ed.), pp. 243-267. CRC Press, Boca Raton, FL, 1985.