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Cryopreservation of cold-acclimated shoot tips of pear in vitro cultures after encapsulation-dehydration
CRYOBIOLOGY
29, 691-700 (1992)
Cryopreservation of Cold-Acclimated Shoot Tips of Pear in Vitro Cultures after Encapsulation-Dehydration C. SCOTTEZ,*+’ E. CHEVREAU,S N. GODARD,*$ Y. ARNAUD,P M . DURON,$ AND J. DEREUDDRE*,’ *Laboratoire de Cryobiologie Ve’gttale Vniversite’ Paris VI, F 75005 Paris, France and Laboratoire de Physiologie des Organes Vkgttaux apr?s Rkolte CNRS, F 92190 Meudon, France; WTIFL, Centre de Balandran, F 30127 Bellegarde, France; PLaboratoire de Physiologie du Dtveloppement des Plantes, Vniversite Paris VI, F 75005 Paris, France; and SINRA, Station d’Amt!lioration des EspLces Fruit&es et Ornementales, F 49070 BeaucouzC, France
Cryopreservation of axillary shoot-tips of pear in vitro cultures (Pyrus communis L. cv Beurrt? Hardy) was performed after encapsulation in alginate beads. Encapsulated shoot-tips were first precultured in medium enriched with sucrose and then dried in a sterile air flow and cooled in liquid nitrogen. After slow rewarming in air at room temperature, alginate beads were transferred to solid culture medium for 1 week before removal of shoot-tips from beads and subculture onto fresh medium. Shoot recovery from cryopreserved shoot-tips was greatly improved by 8-12 weeks of cold acclimation at 0°C of donor in vitro cultures. The best results (80% shoot recovery) were obtained using 0.75 M sucrose for preculture and 4-h dehydration (giving 2 0 % residual water). The resistance of encapsulated and dehydrated shoot-tips to liquid nitrogen did not depend on cooling rate. Apical shoot-tips about 3 mm in length with several axillary buds were also cryopreserved successfully (47% shoot recovery). 0 1992 Academic Press, Inc.
have been obtained with shoot tips from cold-acclimated in vitro cultures (3, 22) using conventional techniques. Conventional techniques for cryopreservation of plant cells and organs generally use cryoprotective solutions and two-step cooling. They involve osmotic equilibration of plant cells and organs in m e d ium containing cryoprotectants, freeze-induced cell dehydration prior to immersion in liquid nitrogen, and rapid thawing. Survival is strongly dependent on several factors: the composition of cryoprotective m e d ia, the cooling rate of programmed prefreezing, the temperature of prefreezing before immersion in liquid nitrogen, and rapid thawing. An alternative to freeze-induced cell dehydration before immersion in liquid nitrogen has been proposed in a preliminary report (4). The new process involves encapsulation of shoot tips in alginate beads and Received December 3,199l; accepted May 14, 1992. their dehydration at room temperature be’ To whom correspondence should be addressed at Cryobiologie V&g&ale, Universite Paris VI, 12 rue Cu- fore cooling in liquid nitrogen. The aim of this paper is to study the efvier, F75005 Paris, France
Germplasm of fruit tree cultivars can be preserved in field collection but this is relatively costly and material may be exposed to pathogens. Storage in liquid nitrogen can ensure long-term preservation of material under conditions of good genetic and physiological stability if regrowth occurs by reactivation of the apical d o m e and avoids callus formation and adventitious organogenesis (27, 31, 32). Cryopreservation of apple and pear shoot tips was first applied to dormant vegetative buds in situ (10, 16, 24). The first attempts at cryopreservation of shoot tips taken from in vitro cultures were performed in 1985 (12). A cold-acclimation period was required to obtain survival which took the form of callus, without shoot recovery. More recently, high shoot recovery rates
691 001 l-2240/92 $5.00 Copyright B I!292 by Academic Press, Inc. All rights of reproduction in any form reserved.
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SCOTTEZ
fects of different factors on the resistance of Beurre Hardy encapsulated shoot tips to dehydration and cooling in liquid nitrogen. MATERIALS
AND
METHODS
In vitro cultures of Pyrus communis L. cv Beurre Hardy shoot were initiated in 1985. For in vitro propagation, cuttings were taken from 5-week-old in vitro shoots cultured on “cutting” medium containing macro and micro elements of Lepoivre (19), 0.55 nG’t4 mesoinositol, 1.2 mM thiamineHCl, 2.22 l&f 6-benzylaminopurine, and 0.088 M sucrose. The pH was adjusted to 5.8 before addition of 6.5 g/liter agar. Micropropagated cultures were grown at a temperature of 23 ? 1°C with a photoperiod of 16 h/day and an irradiance of 25-30 kmolPAR/m2/s. Cold acclimation of in vitro shoots was performed at 0°C with constant illumination, for 1 to 20 weeks. For deacclimation, in vitro shoots cold-acclimated at 0°C for 8 weeks were transferred for 1 to 15 days into the culture chamber. For cryopreservation, two kinds of explant were used: axillary and apical shoot tips. Axillary shoot tips (0.5 to 1 mm in length) consisted of the apical dome, two to four primordial leaves, and a large tissue base. Apical shoot tips of about 2.5 to 3 mm in length were devoid of large leaves; they included part of the stem axis and several axillary meristems. Both kinds of shoot tips were cultured on “apex” medium which differed from cutting medium by the use of Murashige and Skoog microelements (17) and the presence of nicotinic acid (4.06 n&f), pyridoxine hydrochloride (2.43 mM), and glycine (5.33 mM). Beads containing shoot tips were prepared according to the technique of Redenbaugh et al. (20). Shoot tips were suspended in calcium-free apex medium supplemented with 3% Na-alginate solution and the mixture was dripped into apex medium containing 100 mM calcium chloride. Beads containing one to three shoot tips
ET
AL.
were precultured for 18 h in apex medium supplemented with sucrose at different concentrations (0.1 to 2 M). Dehydration was carried out by placing encapsulated shoot tips in uncovered sterile petri dishes in a sterile air flow at ambient temperature and humidity (23 ? l”C, 5565% RH) for 0 to 6 h. Beads were then transferred to cryotubes for cooling in liquid nitrogen for 1 h. Two cooling procedures were used: rapid cooling by direct immersion in liquid nitrogen and two-step freezing by progressive cooling (0.5 to 20Wmin) from +20 to -80°C before immersion in liquid nitrogen. After slow rewarming in air at room temperature, beads were transferred for 1 week to petri dishes containing solid apex medium. Apices were then extracted from the beads and subcultured on new apex culture medium, under the same conditions as the mother shoots. The results from two to four independent experiments (20 to 30 shoot tips per condition) were expressed as percentage of shoot recovery: apices which resumed growth and sprouted new shoots 8 weeks after removal from the alginate matrix. The water content of the beads was determined by drying at 100°C to constant weight. It is expressed with respect to fresh weight. RESULTS
All control axillary shoot tips excised from nonacclimated and cold-acclimated in vitro shoots and subcultured on apex medium remained green (surviving shoot tips) and 70 to 95% resumed growth, producing new shoots by direct development of the apical dome. Similar proportions were also obtained from control alginate-encapsulated shoot tips. Effect of Dehydration The first experiments were performed with axillary shoot tips excised from in vitro shoots after 8 weeks of cold acclimation. After encapsulation in alginate beads
PEAR CRYOPRESERVATION
AFTER
shoot tips were precultured overnight with 0.75 M sucrose (Fig. 1). The water content of beads decreased during preculture from 93 to 77% and continued to decrease during dehydration reaching 21 and 18% after 4 and 6 h of dehydration, respectively. The percentage shoot recovery in dehydrated controls decreased progressively during dehydration from 88 to 65% after 4 h. Shoot recovery after exposure to liquid nitrogen was zero without dehydration but increased after dehydration for more than 2 h, reaching a maximum (79%) after 4 h and subsequently declining to 58% after 6 h. A 4-h dehydration was used in further research on the effects of other factors. Effect of Sucrose Concentration
The effect of sucrose concentration during overnight preculture on resistance of shoot tips to cooling in liquid nitrogen was studied using shoot tips dissected from cultures exposed to 8 weeks of cold acclimation (Fig. 2). After 4 h of dehydration, the water content of the beads decreased to 22% after preculture with 0.1 M sucrose and 16% after preculture with 2 M sucrose.
A\ -T;
Dllratlon of dehydrahn
(hours)
Effects of the duration of dehydration on the percentage of shoot recovery from shoot tips after dehydration for 0 to 6 h (0) and subsequent direct cooling in liquid nitrogen (0) and on the water content of beads (W). Shoot tips were excised from 8 weeks coldacclimated shoots and were precultured with 0.75 M sucrose. Vertical bars represent confidence intervals (P = 5%).
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FIG. 2. Effects of sucrose concentration (IN) in preculture medium on the percentage of shoot recovery from shoot tips without dehydration after 4 h of dehydration (O), and after subsequent direct cooling in liquid nitrogen (0) and on the water content of beads (W). Shoot tips were excised from plantlets coldacclimated for 8 weeks. Vertical bars represent confidence intervals (P = 5%).
(A),
The sucrose concentration in the preculture medium had little effect on the regrowth of control shoot tips. The resistance of shoot tips to dehydration and to subsequent cooling in liquid nitrogen was greatest after preculture with sucrose concentrations between 0.5 and 1 M. The best results after cooling in liquid nitrogen were obtained with 0.75 M sucrose (20% residual water). They differed significantly from results obtained with other sucrose concentrations (x2 test, P < 0.1). The sucrose concentration of 0.75 M was used for further experiments. The value of 20% (+-1) residual water, which appeared to give the best results after preculture with 0.75 M sucrose, was also found for other concentrations of sucrose, (Figs. 3A and 3B). With 0.1 M sucrose, the highest recovery rate (7%) was obtained after 5 h of dehydration; with 2 M sucrose it was obtained after 2 h of dehydration (31%). The water contents of the beads were respectively 20 and 19%. Effect of Cooling Rate
To study the effect of cooling rate (Fig. 4), shoot tips were excised from shoots
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,1oc
ET AL.
It was 63% after rapid cooling in liquid nitrogen. After two-step cooling, shoot recovery rates ranged between 48 and 62%. Whatever the cooling rate, no significant difference in regrowth rates (x2 test; P = 5%) was noted. Resumption of Growth and Development
20
0
After cooling in liquid nitrogen, primordial leaves of axillary shoot tips (about 0.5 to 1 mm in length) remained alive (Fig. 5A). Two weeks after rewarming, elongation of leaves occurred (Fig. 5B) and reactivation of the meristematic dome led to differentiation of new primordia and subsequent formation of a bud (Fig. 5C). After removal from alginate beads (4 mm in diameter) and transfer onto new solid apex medium, apices developed into shoots (Figs. SD and 5E). Apical shoot tips (about 2.5 to 3 mm in length) were also used for cryopreservation. Encapsulation of these shoot tips led
0
Duration
of dehydration
(hours)
FIG. 3. Effects of 0.1 M (A) and 2 M (B) sucrose
concentration in the preculture medium on the percentage of shoot recovery from shoot tips after 0 to 6 h of dehydration (0) and subsequent direct cooling in liquid nitrogen (0) and on the water content of beads (W). Shoot tips were excised from plantlets coldacclimated for 8 weeks. Vertical bars represent cotidence intervals (P = 5%).
cold-acclimated for 8 weeks, precultured with 0.75 M sucrose, and dehydrated for 4 h. Two-step cooling and rapid cooling by direct immersion in liquid nitrogen (200”C/min) were performed. For two-step cooling shoot tips were progressively cooled from 0 to - 80°C at different cooling rates before immersion in liquid nitrogen. The temperature of -80°C was a few degrees below the glass transition temperature of dehydrated beads (data not shown). In these experiments, the percentage shoot recovery after 4 h of dehydration was 54%.
CT
05
1 Coohng
5 rate
10
20
”
200
(‘C/mm)
FIG. 4. Effects of cooling rate on shoot recove
ry
from shoot tips excised from plantlets cold-acclimated for 8 weeks, precultured with 0.75 M sucrose, and dehydrated for 4 h. CT, control of dehydration; 2OO’C, rapid cooling in liquid nitrogen; 0.5, 1, 5, 10, and 2WC, cooling rates used for two-step cooling from 0 to - 80°C. Vertical bars represent confidence intervals (P = 5%).
FIG. 5. Development of encapsulated axillary shoot tips excised from cold-acclimated in vitro cultures, 7, 15, and 20 days (A, B, and C, respectively) after rewarming and shoot recovery after 7 weeks of subculture on new medium (D and E). ef, surviving primordial leaves; bars represent 1 mm. 695
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to the formation of larger beads (5 mm in diameter), which were dehydrated more slowly and needed more prolonged dehydration to achieve liquid nitrogen resistance. After 6 h of dehydration, the water content of the beads remained high (27%). However, the percentage shoot recovery was about 46%. With these explants, regrowth also occurred directly by reactivation of apices and the stem remained green (Figs. 6A and 6B). A new shoot was formed inside the beads during the first month after rewarming. Effect of Cold Acclimation Shoot recovery was studied as a function of the duration of cold acclimation. The experimental conditions were those used in previous experiments: preculture with 0.75 M sucrose, 4 h of dehydration, and direct immersion in liquid nitrogen. Cold acclimation lasted 0 to 20 weeks. Cold treatment had no effect on percentage shoot recovery
from control shoot tips (Fig. 7). After dehydration, shoot recovery increased rapidly from 8% (unacclimated shoot tips) to 60% after 1 week of cold acclimation. This percentage of recovery continued to increase progressively during the following weeks of cold treatment (65 and 86% after 8 and 12 weeks, respectively) and then decreased to ca. 65%. After cooling in liquid nitrogen, changes in shoot recovery were similar to those of resistance to dehydration. During the first 2 weeks of cold acclimation, the percentage shoot recovery increased sharply from 2% (without cold treatment) to 44%, reaching 82% by 12 weeks, and then decreased slightly during the following weeks. Effect of Deacclimation The percentage shoot recovery from control encapsulated shoot tips was not altered during deacclimation (Fig. 8). The resistance of shoot tips to 4 h of dehydration
FIG. 6. Development of cryopreserved encapsulated apical shoot tips excised from coldacclimated in vitro cultures 7 days (A) and 15 days (B) after rewarming; bars represent 1 mm.
PEAR
CRYOPRESERVATION
AFTER
70 60 50
t
40 I
10 I
0 14
1
-LA-U 0
4 Durahon
6 12 of cold-hardenmg
16 20 (weeks)
FIG. 7. Effect of cold acclimation of in vitro shoots on percentage of shoot recovery from shoot tips dehydrated for 4 h (0) and after subsequent cooling in liquid nitrogen (0); control shoot tips (A). Vertical bars represent confidence intervals (P = 5%).
decreased sharply during the first 3 days of culture under standard conditions (42%)) and then remained unchanged. The percentage shoot recovery after cooling in liquid nitrogen decreased during deacclimation reaching 0 after 15 weeks.
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ENCAPSULATION-DEHYDRATION
tips (4). It has been successively applied to shoot tips of other cultivars of pear and apple (unpublished results), So/unum (6), carnation (unpublished results), and grape (18). This procedure also allows survival of somatic embryos (2, 5). In our experiments, dehydration was performed with encapsulated shoot tips by evaporation of water in a sterile air flow. It involved cold acclimation of donor in vitro cultures, encapsulation of shoot tips in alginate beads, overnight preculture with a high concentration of sucrose, dehydration at room temperature, rapid or two-step cooling in liquid nitrogen, and slow rewarming. When cryopreservation is performed in liquid cryoprotective medium, either after conventional cryopreservation or after vitrification, survival of plant cells remains highly dependent on cooling rates during prefreezing to - 30 or - 40°C. In conventional procedures, cooling rates of 1”Clmin or lower to - 30 or - 40°C were important to obtain high survival of meristematic tissues. In the vitrification procedure, high survival only occurred if cells were cooled
DISCUSSION 90
In conventional cryopreservation procedures, intracellular ice crystallization can be avoided if dehydration resulting from extracellular ice formation is great enough to allow vitrification of the cytoplasm and of the remaining amorphous residue of the cryoprotective medium at the prefreezing temperature. Removal of freezable water from the cells may also be performed by placing cells and organs in extremely concentrated solutions of permeating and/or nonpermeating cryoprotectants (13, 14, 25, 30). This technique, which is referred to as “vitrification,” involves direct immersion in liquid nitrogen and rapid rewarming. Another procedure, which may be referred to as encapsulation-dehydration, was proposed in a preliminary report for pear shoot
60 70
I
60 t 50 40
1
30 20
I t
0
3 Duratlon
6 9 of dehardenmg
12 (days)
15
FIG. 8. Effect of the duration of deacclimation of in vitro shoots on the percentage of shoot recovery from
shoot tips dehydrated for 4 h (0) and directly cooled in liquid nitrogen (0); control shoot tips (A). Vertical bars represent confidence intervals (P = 5%).
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SCOTTEZ
and rewarmed rapidly to avoid recrystallization. After encapsulation-dehydration, high recovery rates of pear shoot tips were obtained after direct immersion in liquid nitrogen as well as after two-step cooling, independently of the cooling rate from 0.5 to 200”CYmin. This property may be due to the ability of encapsulating material and plant organs to vitrify during cooling and to remain vitrified during rewarming whatever the cooling and rewarming rates (5). In general, when cryopreservation was performed according to conventional procedures, only some groups of cells in the meristematic region of the shoot apex remained viable after freezing, while the leaf primordia did not (9, 26). When cryopreservation of pear shoot tips was performed in liquid medium containing 0.75 M sucrose and 5 to 15% Me,SO (3) only the meristematic tissues of the apical dome remained alive and regrowth occurred with differentiation of new leaf primordia. The encapsulation-dehydration technique allowed regrowth to occur by reactivation of the apical meristem without callus formation, suggesting that most of the cells survived after the cryogenic treatment. In the case of apical shoot tips, part of the stem axis also survived. The water content of the beads (sum of the encapsulating material and the shoot tip) that allowed best survival after cooling in liquid nitrogen was about 20%. For apical shoot tips encapsulated in larger alginate beads, a higher value of 27% appeared to be sufficient to allow their resistance to liquid nitrogen. These two values are close to those which permitted survival of Asparagus shoot tips dehydrated without encapsulation on silica gel (29) and encapsulated somatic embryos (2, 5). Thus the resistance of these explants was closely related to their ability to tolerate dehydration; it was similar to the resistance of winter buds presenting extra-organ freezing (1,23), zygotic embryos excised from recalcitrant seeds of oil palm (8), and somatic embryos (7, 15).
ET AL.
Like zygotic and somatic embryos, encapsulated shoot tips can withstand considerable dehydration in air. For shoot tips of Asparagus (29) and carnation (unpublished results), tolerance to dehydration was obtained after overnight preculture with a high concentration of sucrose. For other species like grape (18), Sohum (6), and unhardened pear in vitro shoots, overnight preculture with a high concentration of sucrose did not appear to be sufficient to induce this tolerance. Specific pretreatments appeared to be essential for these species. For species which cannot be cold-acclimated, progressive increase of sucrose concentration in preculture medium for grape shoot tips (18) or longer duration of preculture for shoot tips of Solunum (6) can be used. The role of sucrose may be to decrease the water content of the cells and to increase their dry weight (29). As in seeds, it may act as the principal agent of desiccation tolerance (11). For pear in vitro cultures which can withstand prolonged exposure to low temperatures, cold acclimation seemed essential for good survival and shoot recovery from encapsulated shoot tips. One week of cold treatment generally enhanced significantly the post-liquid nitrogen survival rates of cryopreserved shoot tips excised from in vitro cultures of different species of Rubus (21) and Pyrus (3, 22) when treated by conventional procedures. Further improvement of survival can be obtained by increasing the duration of the treatment at low temperature to several months. However, for in vitro shoots of pear, cold acclimation alone appeared to be insufficient to induce significant tolerance of shoot tips to dehydration and subsequent cooling in liquid nitrogen. Full acclimation required complementary overnight preculture of shoot tips in medium supplemented with sucrose, unlike natural cold acclimation which can induce resistance to dehydration in winter buds of hardy species of fruit trees (28).
PEAR CRYOPRESERVATION
AFTER
In conclusion, the encapsulationdehydration process described here displayed several advantages over conventional cryopreservation: easier handling of organs, simplification of cryoprotective media, elimination of costly programmed freezers, independence of survival from cooling rates, and increased size of explants surviving liquid nitrogen storage. This technique may be useful as a practical procedure to cryopreserve shoot tips and somatic embryos which are sensitive to freezing of the surrounding cryoprotective medium. ACKNOWLEDGMENTS
We thank C. Joulie and M. Gallet for their technical assistance. This work was supported by ANRT (Association Nationale pour la Recherche Technique) (convention CIFRE 21/90) and by MRES (Ministere de la Recherche et de 1’Enseignement Superieur) (convention 88 R 0751).
ENCAPSULATION-DEHYDRATION
7.
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1977. 20. Redenbaugh, K., Paasch, B. D., Nichol, J. W., Kossler, M. E., Viss, P. R., and Walker, K. A. Somatic seeds: Encapsulation of asexual plant embryos. Biotechnology 4, 797-801 (1986).
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21. Reed, B. M. Cold acclimation as a method to improve survival of cryopreserved Rubus meristems. Cryo Lett. 9, 166167 (1988). 22. Reed, B. M. Survival of in vitro-grown apical meristems of Pyrus following cryopreservation. HortScience 25, 11 l-l 13 (1990). 23. Sakai, A. Freezing avoidance mechanism of primordial shoots of conifer buds. Plant Cell Physiol. 20, 1381-1390 (1979). 24. Sakai’, A., and Nishiyama, Y. Cryopreservation of winter vegetative buds of hardy fruit trees in liquid nitrogen. HortScience 13,225-227 (1978). 25. SakaL, A., Kobayashi, S., and Ooyama, I. Survival by vitrification of nucellar cells of Navel orange (Citrus sinensis var. brasiliensis Tanaka) cooled to - 196°C. .Z. Plant Physiol. 137, 465470 (1991). 26. Seibert, M., and Wetherbee, P. J. Increased survival and differentiation of frozen herbaceous plant organ cultures through cold treatment. Plant Physiol. 59, 1043-1046 (1977).
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27. Stushnoff, C. Cryopreservation of apple genetic resources. Can. .Z. Plant Sci. 67, 1151-1154 (1987). 28. Tyler, N. J., and Stushnoff, C. Dehydration of dormant apple buds at different stages of cold acclimation to induce cryopreservability in different cultivars. Can. J. Plant Sci. 68, 11691176 (1988). 29. Uragami, A., Sakai’, A., and Nagai’, M. Cryopreservation of dried axillary buds from plantlets of Asparagus officinalis L. grown in vitro. Plant Cell Rep. 9, 328-331 (1990). 30. Uragami, A., SakaL, A., Nagai’, M., and Takahashi, T. Survival of cells and somatic embryos of Asparagus officinalis cryopreserved by vitrification. Plant Cell Rep. 8, 418-421 (1989). 31. Withers, L. A. Germplasm storage in plant biotechnology. In “Plant Biotechnology” (S. H. Mantel1 and H. Smith, Eds.), 187-218 (1983). 32. Withers, L. A. In-vitro conservation. Biol. J. Lin. Sot. 43, 3142 (1991).
29, 691-700 (1992)
Cryopreservation of Cold-Acclimated Shoot Tips of Pear in Vitro Cultures after Encapsulation-Dehydration C. SCOTTEZ,*+’ E. CHEVREAU,S N. GODARD,*$ Y. ARNAUD,P M . DURON,$ AND J. DEREUDDRE*,’ *Laboratoire de Cryobiologie Ve’gttale Vniversite’ Paris VI, F 75005 Paris, France and Laboratoire de Physiologie des Organes Vkgttaux apr?s Rkolte CNRS, F 92190 Meudon, France; WTIFL, Centre de Balandran, F 30127 Bellegarde, France; PLaboratoire de Physiologie du Dtveloppement des Plantes, Vniversite Paris VI, F 75005 Paris, France; and SINRA, Station d’Amt!lioration des EspLces Fruit&es et Ornementales, F 49070 BeaucouzC, France
Cryopreservation of axillary shoot-tips of pear in vitro cultures (Pyrus communis L. cv Beurrt? Hardy) was performed after encapsulation in alginate beads. Encapsulated shoot-tips were first precultured in medium enriched with sucrose and then dried in a sterile air flow and cooled in liquid nitrogen. After slow rewarming in air at room temperature, alginate beads were transferred to solid culture medium for 1 week before removal of shoot-tips from beads and subculture onto fresh medium. Shoot recovery from cryopreserved shoot-tips was greatly improved by 8-12 weeks of cold acclimation at 0°C of donor in vitro cultures. The best results (80% shoot recovery) were obtained using 0.75 M sucrose for preculture and 4-h dehydration (giving 2 0 % residual water). The resistance of encapsulated and dehydrated shoot-tips to liquid nitrogen did not depend on cooling rate. Apical shoot-tips about 3 mm in length with several axillary buds were also cryopreserved successfully (47% shoot recovery). 0 1992 Academic Press, Inc.
have been obtained with shoot tips from cold-acclimated in vitro cultures (3, 22) using conventional techniques. Conventional techniques for cryopreservation of plant cells and organs generally use cryoprotective solutions and two-step cooling. They involve osmotic equilibration of plant cells and organs in m e d ium containing cryoprotectants, freeze-induced cell dehydration prior to immersion in liquid nitrogen, and rapid thawing. Survival is strongly dependent on several factors: the composition of cryoprotective m e d ia, the cooling rate of programmed prefreezing, the temperature of prefreezing before immersion in liquid nitrogen, and rapid thawing. An alternative to freeze-induced cell dehydration before immersion in liquid nitrogen has been proposed in a preliminary report (4). The new process involves encapsulation of shoot tips in alginate beads and Received December 3,199l; accepted May 14, 1992. their dehydration at room temperature be’ To whom correspondence should be addressed at Cryobiologie V&g&ale, Universite Paris VI, 12 rue Cu- fore cooling in liquid nitrogen. The aim of this paper is to study the efvier, F75005 Paris, France
Germplasm of fruit tree cultivars can be preserved in field collection but this is relatively costly and material may be exposed to pathogens. Storage in liquid nitrogen can ensure long-term preservation of material under conditions of good genetic and physiological stability if regrowth occurs by reactivation of the apical d o m e and avoids callus formation and adventitious organogenesis (27, 31, 32). Cryopreservation of apple and pear shoot tips was first applied to dormant vegetative buds in situ (10, 16, 24). The first attempts at cryopreservation of shoot tips taken from in vitro cultures were performed in 1985 (12). A cold-acclimation period was required to obtain survival which took the form of callus, without shoot recovery. More recently, high shoot recovery rates
691 001 l-2240/92 $5.00 Copyright B I!292 by Academic Press, Inc. All rights of reproduction in any form reserved.
692
SCOTTEZ
fects of different factors on the resistance of Beurre Hardy encapsulated shoot tips to dehydration and cooling in liquid nitrogen. MATERIALS
AND
METHODS
In vitro cultures of Pyrus communis L. cv Beurre Hardy shoot were initiated in 1985. For in vitro propagation, cuttings were taken from 5-week-old in vitro shoots cultured on “cutting” medium containing macro and micro elements of Lepoivre (19), 0.55 nG’t4 mesoinositol, 1.2 mM thiamineHCl, 2.22 l&f 6-benzylaminopurine, and 0.088 M sucrose. The pH was adjusted to 5.8 before addition of 6.5 g/liter agar. Micropropagated cultures were grown at a temperature of 23 ? 1°C with a photoperiod of 16 h/day and an irradiance of 25-30 kmolPAR/m2/s. Cold acclimation of in vitro shoots was performed at 0°C with constant illumination, for 1 to 20 weeks. For deacclimation, in vitro shoots cold-acclimated at 0°C for 8 weeks were transferred for 1 to 15 days into the culture chamber. For cryopreservation, two kinds of explant were used: axillary and apical shoot tips. Axillary shoot tips (0.5 to 1 mm in length) consisted of the apical dome, two to four primordial leaves, and a large tissue base. Apical shoot tips of about 2.5 to 3 mm in length were devoid of large leaves; they included part of the stem axis and several axillary meristems. Both kinds of shoot tips were cultured on “apex” medium which differed from cutting medium by the use of Murashige and Skoog microelements (17) and the presence of nicotinic acid (4.06 n&f), pyridoxine hydrochloride (2.43 mM), and glycine (5.33 mM). Beads containing shoot tips were prepared according to the technique of Redenbaugh et al. (20). Shoot tips were suspended in calcium-free apex medium supplemented with 3% Na-alginate solution and the mixture was dripped into apex medium containing 100 mM calcium chloride. Beads containing one to three shoot tips
ET
AL.
were precultured for 18 h in apex medium supplemented with sucrose at different concentrations (0.1 to 2 M). Dehydration was carried out by placing encapsulated shoot tips in uncovered sterile petri dishes in a sterile air flow at ambient temperature and humidity (23 ? l”C, 5565% RH) for 0 to 6 h. Beads were then transferred to cryotubes for cooling in liquid nitrogen for 1 h. Two cooling procedures were used: rapid cooling by direct immersion in liquid nitrogen and two-step freezing by progressive cooling (0.5 to 20Wmin) from +20 to -80°C before immersion in liquid nitrogen. After slow rewarming in air at room temperature, beads were transferred for 1 week to petri dishes containing solid apex medium. Apices were then extracted from the beads and subcultured on new apex culture medium, under the same conditions as the mother shoots. The results from two to four independent experiments (20 to 30 shoot tips per condition) were expressed as percentage of shoot recovery: apices which resumed growth and sprouted new shoots 8 weeks after removal from the alginate matrix. The water content of the beads was determined by drying at 100°C to constant weight. It is expressed with respect to fresh weight. RESULTS
All control axillary shoot tips excised from nonacclimated and cold-acclimated in vitro shoots and subcultured on apex medium remained green (surviving shoot tips) and 70 to 95% resumed growth, producing new shoots by direct development of the apical dome. Similar proportions were also obtained from control alginate-encapsulated shoot tips. Effect of Dehydration The first experiments were performed with axillary shoot tips excised from in vitro shoots after 8 weeks of cold acclimation. After encapsulation in alginate beads
PEAR CRYOPRESERVATION
AFTER
shoot tips were precultured overnight with 0.75 M sucrose (Fig. 1). The water content of beads decreased during preculture from 93 to 77% and continued to decrease during dehydration reaching 21 and 18% after 4 and 6 h of dehydration, respectively. The percentage shoot recovery in dehydrated controls decreased progressively during dehydration from 88 to 65% after 4 h. Shoot recovery after exposure to liquid nitrogen was zero without dehydration but increased after dehydration for more than 2 h, reaching a maximum (79%) after 4 h and subsequently declining to 58% after 6 h. A 4-h dehydration was used in further research on the effects of other factors. Effect of Sucrose Concentration
The effect of sucrose concentration during overnight preculture on resistance of shoot tips to cooling in liquid nitrogen was studied using shoot tips dissected from cultures exposed to 8 weeks of cold acclimation (Fig. 2). After 4 h of dehydration, the water content of the beads decreased to 22% after preculture with 0.1 M sucrose and 16% after preculture with 2 M sucrose.
A\ -T;
Dllratlon of dehydrahn
(hours)
Effects of the duration of dehydration on the percentage of shoot recovery from shoot tips after dehydration for 0 to 6 h (0) and subsequent direct cooling in liquid nitrogen (0) and on the water content of beads (W). Shoot tips were excised from 8 weeks coldacclimated shoots and were precultured with 0.75 M sucrose. Vertical bars represent confidence intervals (P = 5%).
693
ENCAPSULATION-DEHYDRATION
FIG. 2. Effects of sucrose concentration (IN) in preculture medium on the percentage of shoot recovery from shoot tips without dehydration after 4 h of dehydration (O), and after subsequent direct cooling in liquid nitrogen (0) and on the water content of beads (W). Shoot tips were excised from plantlets coldacclimated for 8 weeks. Vertical bars represent confidence intervals (P = 5%).
(A),
The sucrose concentration in the preculture medium had little effect on the regrowth of control shoot tips. The resistance of shoot tips to dehydration and to subsequent cooling in liquid nitrogen was greatest after preculture with sucrose concentrations between 0.5 and 1 M. The best results after cooling in liquid nitrogen were obtained with 0.75 M sucrose (20% residual water). They differed significantly from results obtained with other sucrose concentrations (x2 test, P < 0.1). The sucrose concentration of 0.75 M was used for further experiments. The value of 20% (+-1) residual water, which appeared to give the best results after preculture with 0.75 M sucrose, was also found for other concentrations of sucrose, (Figs. 3A and 3B). With 0.1 M sucrose, the highest recovery rate (7%) was obtained after 5 h of dehydration; with 2 M sucrose it was obtained after 2 h of dehydration (31%). The water contents of the beads were respectively 20 and 19%. Effect of Cooling Rate
To study the effect of cooling rate (Fig. 4), shoot tips were excised from shoots
694
SCOTTEZ
,1oc
ET AL.
It was 63% after rapid cooling in liquid nitrogen. After two-step cooling, shoot recovery rates ranged between 48 and 62%. Whatever the cooling rate, no significant difference in regrowth rates (x2 test; P = 5%) was noted. Resumption of Growth and Development
20
0
After cooling in liquid nitrogen, primordial leaves of axillary shoot tips (about 0.5 to 1 mm in length) remained alive (Fig. 5A). Two weeks after rewarming, elongation of leaves occurred (Fig. 5B) and reactivation of the meristematic dome led to differentiation of new primordia and subsequent formation of a bud (Fig. 5C). After removal from alginate beads (4 mm in diameter) and transfer onto new solid apex medium, apices developed into shoots (Figs. SD and 5E). Apical shoot tips (about 2.5 to 3 mm in length) were also used for cryopreservation. Encapsulation of these shoot tips led
0
Duration
of dehydration
(hours)
FIG. 3. Effects of 0.1 M (A) and 2 M (B) sucrose
concentration in the preculture medium on the percentage of shoot recovery from shoot tips after 0 to 6 h of dehydration (0) and subsequent direct cooling in liquid nitrogen (0) and on the water content of beads (W). Shoot tips were excised from plantlets coldacclimated for 8 weeks. Vertical bars represent cotidence intervals (P = 5%).
cold-acclimated for 8 weeks, precultured with 0.75 M sucrose, and dehydrated for 4 h. Two-step cooling and rapid cooling by direct immersion in liquid nitrogen (200”C/min) were performed. For two-step cooling shoot tips were progressively cooled from 0 to - 80°C at different cooling rates before immersion in liquid nitrogen. The temperature of -80°C was a few degrees below the glass transition temperature of dehydrated beads (data not shown). In these experiments, the percentage shoot recovery after 4 h of dehydration was 54%.
CT
05
1 Coohng
5 rate
10
20
”
200
(‘C/mm)
FIG. 4. Effects of cooling rate on shoot recove
ry
from shoot tips excised from plantlets cold-acclimated for 8 weeks, precultured with 0.75 M sucrose, and dehydrated for 4 h. CT, control of dehydration; 2OO’C, rapid cooling in liquid nitrogen; 0.5, 1, 5, 10, and 2WC, cooling rates used for two-step cooling from 0 to - 80°C. Vertical bars represent confidence intervals (P = 5%).
FIG. 5. Development of encapsulated axillary shoot tips excised from cold-acclimated in vitro cultures, 7, 15, and 20 days (A, B, and C, respectively) after rewarming and shoot recovery after 7 weeks of subculture on new medium (D and E). ef, surviving primordial leaves; bars represent 1 mm. 695
696
SCOTTEZ ET AL.
to the formation of larger beads (5 mm in diameter), which were dehydrated more slowly and needed more prolonged dehydration to achieve liquid nitrogen resistance. After 6 h of dehydration, the water content of the beads remained high (27%). However, the percentage shoot recovery was about 46%. With these explants, regrowth also occurred directly by reactivation of apices and the stem remained green (Figs. 6A and 6B). A new shoot was formed inside the beads during the first month after rewarming. Effect of Cold Acclimation Shoot recovery was studied as a function of the duration of cold acclimation. The experimental conditions were those used in previous experiments: preculture with 0.75 M sucrose, 4 h of dehydration, and direct immersion in liquid nitrogen. Cold acclimation lasted 0 to 20 weeks. Cold treatment had no effect on percentage shoot recovery
from control shoot tips (Fig. 7). After dehydration, shoot recovery increased rapidly from 8% (unacclimated shoot tips) to 60% after 1 week of cold acclimation. This percentage of recovery continued to increase progressively during the following weeks of cold treatment (65 and 86% after 8 and 12 weeks, respectively) and then decreased to ca. 65%. After cooling in liquid nitrogen, changes in shoot recovery were similar to those of resistance to dehydration. During the first 2 weeks of cold acclimation, the percentage shoot recovery increased sharply from 2% (without cold treatment) to 44%, reaching 82% by 12 weeks, and then decreased slightly during the following weeks. Effect of Deacclimation The percentage shoot recovery from control encapsulated shoot tips was not altered during deacclimation (Fig. 8). The resistance of shoot tips to 4 h of dehydration
FIG. 6. Development of cryopreserved encapsulated apical shoot tips excised from coldacclimated in vitro cultures 7 days (A) and 15 days (B) after rewarming; bars represent 1 mm.
PEAR
CRYOPRESERVATION
AFTER
70 60 50
t
40 I
10 I
0 14
1
-LA-U 0
4 Durahon
6 12 of cold-hardenmg
16 20 (weeks)
FIG. 7. Effect of cold acclimation of in vitro shoots on percentage of shoot recovery from shoot tips dehydrated for 4 h (0) and after subsequent cooling in liquid nitrogen (0); control shoot tips (A). Vertical bars represent confidence intervals (P = 5%).
decreased sharply during the first 3 days of culture under standard conditions (42%)) and then remained unchanged. The percentage shoot recovery after cooling in liquid nitrogen decreased during deacclimation reaching 0 after 15 weeks.
697
ENCAPSULATION-DEHYDRATION
tips (4). It has been successively applied to shoot tips of other cultivars of pear and apple (unpublished results), So/unum (6), carnation (unpublished results), and grape (18). This procedure also allows survival of somatic embryos (2, 5). In our experiments, dehydration was performed with encapsulated shoot tips by evaporation of water in a sterile air flow. It involved cold acclimation of donor in vitro cultures, encapsulation of shoot tips in alginate beads, overnight preculture with a high concentration of sucrose, dehydration at room temperature, rapid or two-step cooling in liquid nitrogen, and slow rewarming. When cryopreservation is performed in liquid cryoprotective medium, either after conventional cryopreservation or after vitrification, survival of plant cells remains highly dependent on cooling rates during prefreezing to - 30 or - 40°C. In conventional procedures, cooling rates of 1”Clmin or lower to - 30 or - 40°C were important to obtain high survival of meristematic tissues. In the vitrification procedure, high survival only occurred if cells were cooled
DISCUSSION 90
In conventional cryopreservation procedures, intracellular ice crystallization can be avoided if dehydration resulting from extracellular ice formation is great enough to allow vitrification of the cytoplasm and of the remaining amorphous residue of the cryoprotective medium at the prefreezing temperature. Removal of freezable water from the cells may also be performed by placing cells and organs in extremely concentrated solutions of permeating and/or nonpermeating cryoprotectants (13, 14, 25, 30). This technique, which is referred to as “vitrification,” involves direct immersion in liquid nitrogen and rapid rewarming. Another procedure, which may be referred to as encapsulation-dehydration, was proposed in a preliminary report for pear shoot
60 70
I
60 t 50 40
1
30 20
I t
0
3 Duratlon
6 9 of dehardenmg
12 (days)
15
FIG. 8. Effect of the duration of deacclimation of in vitro shoots on the percentage of shoot recovery from
shoot tips dehydrated for 4 h (0) and directly cooled in liquid nitrogen (0); control shoot tips (A). Vertical bars represent confidence intervals (P = 5%).
698
SCOTTEZ
and rewarmed rapidly to avoid recrystallization. After encapsulation-dehydration, high recovery rates of pear shoot tips were obtained after direct immersion in liquid nitrogen as well as after two-step cooling, independently of the cooling rate from 0.5 to 200”CYmin. This property may be due to the ability of encapsulating material and plant organs to vitrify during cooling and to remain vitrified during rewarming whatever the cooling and rewarming rates (5). In general, when cryopreservation was performed according to conventional procedures, only some groups of cells in the meristematic region of the shoot apex remained viable after freezing, while the leaf primordia did not (9, 26). When cryopreservation of pear shoot tips was performed in liquid medium containing 0.75 M sucrose and 5 to 15% Me,SO (3) only the meristematic tissues of the apical dome remained alive and regrowth occurred with differentiation of new leaf primordia. The encapsulation-dehydration technique allowed regrowth to occur by reactivation of the apical meristem without callus formation, suggesting that most of the cells survived after the cryogenic treatment. In the case of apical shoot tips, part of the stem axis also survived. The water content of the beads (sum of the encapsulating material and the shoot tip) that allowed best survival after cooling in liquid nitrogen was about 20%. For apical shoot tips encapsulated in larger alginate beads, a higher value of 27% appeared to be sufficient to allow their resistance to liquid nitrogen. These two values are close to those which permitted survival of Asparagus shoot tips dehydrated without encapsulation on silica gel (29) and encapsulated somatic embryos (2, 5). Thus the resistance of these explants was closely related to their ability to tolerate dehydration; it was similar to the resistance of winter buds presenting extra-organ freezing (1,23), zygotic embryos excised from recalcitrant seeds of oil palm (8), and somatic embryos (7, 15).
ET AL.
Like zygotic and somatic embryos, encapsulated shoot tips can withstand considerable dehydration in air. For shoot tips of Asparagus (29) and carnation (unpublished results), tolerance to dehydration was obtained after overnight preculture with a high concentration of sucrose. For other species like grape (18), Sohum (6), and unhardened pear in vitro shoots, overnight preculture with a high concentration of sucrose did not appear to be sufficient to induce this tolerance. Specific pretreatments appeared to be essential for these species. For species which cannot be cold-acclimated, progressive increase of sucrose concentration in preculture medium for grape shoot tips (18) or longer duration of preculture for shoot tips of Solunum (6) can be used. The role of sucrose may be to decrease the water content of the cells and to increase their dry weight (29). As in seeds, it may act as the principal agent of desiccation tolerance (11). For pear in vitro cultures which can withstand prolonged exposure to low temperatures, cold acclimation seemed essential for good survival and shoot recovery from encapsulated shoot tips. One week of cold treatment generally enhanced significantly the post-liquid nitrogen survival rates of cryopreserved shoot tips excised from in vitro cultures of different species of Rubus (21) and Pyrus (3, 22) when treated by conventional procedures. Further improvement of survival can be obtained by increasing the duration of the treatment at low temperature to several months. However, for in vitro shoots of pear, cold acclimation alone appeared to be insufficient to induce significant tolerance of shoot tips to dehydration and subsequent cooling in liquid nitrogen. Full acclimation required complementary overnight preculture of shoot tips in medium supplemented with sucrose, unlike natural cold acclimation which can induce resistance to dehydration in winter buds of hardy species of fruit trees (28).
PEAR CRYOPRESERVATION
AFTER
In conclusion, the encapsulationdehydration process described here displayed several advantages over conventional cryopreservation: easier handling of organs, simplification of cryoprotective media, elimination of costly programmed freezers, independence of survival from cooling rates, and increased size of explants surviving liquid nitrogen storage. This technique may be useful as a practical procedure to cryopreserve shoot tips and somatic embryos which are sensitive to freezing of the surrounding cryoprotective medium. ACKNOWLEDGMENTS
We thank C. Joulie and M. Gallet for their technical assistance. This work was supported by ANRT (Association Nationale pour la Recherche Technique) (convention CIFRE 21/90) and by MRES (Ministere de la Recherche et de 1’Enseignement Superieur) (convention 88 R 0751).
ENCAPSULATION-DEHYDRATION
7.
8.
9.
10.
11.
12.
13.
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