Cryopreservation of potato shoot tips by encapsulationdehydration

Potato Research 39 (1996) 69-78

Cryopreservation of potato shoot tips by encapsulation- dehydration

S. BOUAFIA1, N. JELTI1, G. LAIRy2, A. BLANC l, E. BONNEL2 AND J. DEREUDDRE1

1Laboratoire de Cryobiologie V6g6tale, Universit6 P. et M. Curie, 12 rue Cuvier, 75230 Paris cedex 05 2Germicopa, Kerguivarch, 29119 Chateauneuf du Faou

Accepted for publication: 9 February 1996

Additional keywords: Solanum, preculture, liquid nitrogen

Abbreviations: BA, 6-benzylaminopurine; GA 3, gibberellic acid, IN, liquid nitrogen; NAA, naphthaleneacetic acid.

Summary

Potato shoot tips excised from 2-week-old in vitro nodal cuttings were cryopreserved after encapsulation in alginate beads. Encapsulated shoot tips were first precultured in sucrose- enriched media, dried over silica gel, and rapidly cooled in liquid nitrogen. After slow rewarming in air at room temperature, alginate beads were transferred to solid culture medium for shoot tip recovery. After cooling in liquid nitrogen, shoot yield depended on preculture duration, sucrose concentration and water content of beads. Survival rates above 60% were obtained for each cultivar tested.

Introduction

Storage of diploid and tetraploid tuber-bearing Solanum for selection is time- consuming and expensive. Preservation of in vitro cultured potatoes for germplasm maintenance has been proposed using minimal growth regimes (Westcott, 1981a) and growth retardants (Westcott, 1981b). However, medium-term storage conditions may not ensure good genetic stability of in vitro micropropagated material. Ribosomal RNA of Solanum tuberosum cv. D6sir6e subjected to slow growth in the presence of mannitol may be altered (Harding, 1991), and long-term tissue culture reduced the ability of shoot tips from cvs D6sir6e and Golden Wonder potatoes to survive deep freezing in liquid nitrogen (Harding et al., 1991). Storage at the temperature of liquid nitrogen is therefore to be preferred for long-term preservation of potato germplasm. Storage in liquid nitrogen (LN) does not induce ploidy changes in dihaploids of Solanum tuberosum (Ward et al., 1993) or alter the ability of plants derived from cryopreserved shoot tips to produce tubers (Bajaj, 1978: Harding & Benson, 1994). The duration of'storage in LN does not affect the survival rate of potato shoot tips (Bajaj, 1985).

The first reports of successful shoot recovery after cryopreservation of potato shoot tips were published by Bajaj (1977), who used a cryoprotective solution containing dimethylsulfoxide (DMSO), glycerol and sucrose, and by Grout & Henshaw (1978)

Potato Research 39 (1996) 69

S. BOUAFIA, N. JELTI, G. LAIRY, A. BLANC, E. B O N N E L AND J. D E R E U D D R E

who used DMSO alone as cryoprotectant. Survival was achieved only after ultrarapid freezing in LN of shoot tips placed on the tip of a sterile needle. Low survival rates (20-26%) involving regrowth of shoot tips or callus formation were obtained. Shoots may regenerate from callus and there are some concerns regarding the genetic stability of cryopreserved meristems (Henshaw et al., 1985).

Cryopreservation was improved with conventional procedures: shoot tips excised from seedlings (Towill, 1984), sprouts of tubers (Manzhulin et al., 1984), or in vitro plantlets (Towill, 1983) were first incubated in cryoprotective medium and prefrozen to about -40 *C. After cooling in LN, they were rapidly thawed. Survival was greatly improved by preculture of shoot tips for 1-2 days on solid culture medium supplemented or not with DMSO (Towill, 1981a: Henshaw et al., 1985), In any case, shoot survival and recovery depended on the presence of growth regulators in post- treatment media (Towill, 1981b) and pre- and post-freeze light regimes (Benson et al., 1989). However, conventional methods are time-consuming, require sophisticated freezing equipment, and involve specific adaptations of cryoprotective solutions and cooling rates to optimize the results.

A new procedure for cryopreservation of potato shoot tips, in which prefreezing dehydration is replaced by dehydration at room temperature, has been referred to as encapsulation-dehydration (Fabre & Dereuddre, 1990). Shoot tips are trapped in alginate beads, precultured in sucrose-enriched medium, air dehydrated, and directly cooled in LN. When applied to Solanttm phureja, encapsulation-dehydration gave low and irregular survival rates, and was unsuccessful with other species and cultivars. Further research was therefore necessary to improve survival and shoot recovery after cooling in LN. The aim of the present study was to optimize and simplify the encapsulation-dehydration procedure for potato shoot tips and to apply it to other clones.

Materials and methods

Plant material. Three hybrid clones of diploid potatoes (Solanum phureja L. cvs Si22 and T39: S. Phureja x chacoense cv. 88D221) and two cultivars of tetraploid potatoes (S. tuberosum cvs Charly and Hertha) were selected. Virus-free in vitro plantlets of potato were propagated on solid standard medium devoid of growth regulators and containing Tendille & Lecerf (1974) macroelements, Murashige & Skoog (1962) micronutrients, Morel & Wetmore (1951) vitamins, Na-Fe-EDTA, 30 g/1, sucrose and 8 g/l agar (medium 1). The pH was adjusted to 5.8 with 0.1 N NaOH before addition of agar and autoclaving. Nodal cuttings produced approximately 10-cm shoots which were used for cryopreservation and micropropagation. Cultures were grown at a temperature of 21_+1 ~ with a photoperiod of 16 h/day and an irradiance of 25-30 lamoles PAR/m2/s.

Cryopreservation. For cryopreservation, axillary and terminal shoot tips (0.5 mm) were taken from nodal cuttings, cultured in Petri dishes on standard culture medium (medium 1) for 3, 7, 14 and 21 days and separately treated. Shoot tips were maintained for several hours before encapsulation on medium 2A containing Morel

70 Potato Research 39 (1996)

CRYOPRESERVATION OF POTATO SHOOT TIPS

& Muller (1964) macronutrients, Heller (1953) micronutrients, Morel & Wetmore (1951) vitamins, Na-Fe-EDTA, 0.1 M sucrose, and 8 g/l agar.

For encapsulation, shoot tips were first suspended in calcium-free medium 2A containing 3% (w/v) sodium alginate (low viscosity, Sigma). The mixture was dripped into liquid medium 2A containing 100 mM calcium chloride to form calcium alginate beads.

Beads of about 3-4 mm in diameter and containing one or two shoot tips were precultured in sucrose-enriched liquid medium 2A in Erlenmeyer flasks on a rotary shaker (100 rpm). Stepwise increase in sucrose in the preculture medium was achieved in two days by gradual addition of sucrose to successively concentrations of 0.3 M, 0.5 M, 0.75 M and 1 M. Beads were then maintained in sucrose-enriched (1 M) medium for 1 to 7 days. Direct preculture was also performed. Encapsulated shoot tips were directly placed in solutions containing different sucrose concentrations (0.1 M, 0.3 M, 0.5 M, 0.75 M, 1 M, 1.25 M or 1.5 M) for 2 days.

After preculture, shoot tip-containing beads were rapidly surface-dried on sterile filter paper and transferred onto disks over silica gel in airtight boxes for 4.50 h, at a constant temperature of 20 ~ (_+! ~ to obtain 0.20-0.22 g water/g dry weight. Dry weight was determined by drying 20 beads devoid of shoot tips in an oven (85 ~ to constant weight.

For cooling, dehydrated beads were transferred to cryotubes and directly immersed in LN where they were stored for at least 1 h. Cryotubes were removed from LN and slowly rewarmed in air at room temperature. Encapsulated shoot tips were plated for 1 week on medium 2B supplemented with growth regulators: 10-5 g/l BA, 10 -6 g/1 NAA and 5.10-3 g/1 GA 3, and transferred to medium 2C supplemented only with 10 -7 g/l GA 3 to avoid callus formation.

Expression of results. Results were expressed as survival in 1-3 independent experiments with 20 to 25 shoot tips per treatment. Survival was estimated as a percentage of treated shoot tips giving shoots without callus formation, 3 weeks after rewarming and subculture. The controls included in the study consisted of i) encapsulated, non-precultured shoot tips, ii) precultured shoot tips, and iii) dehydrated shoot tips.

Results

Initial experiments were performed with Solanum phureja cv. Si22.

Stepwise preculture Duration of cutting reactivation. Tolerance of dehydration of axillary shoot tips (time 0 of reactivation) excised from 2-month-old in vitro plants was 58% (Fig. 1A). After 3 days of reactivation, survival of shoot tips (which must be considered as terminal) decreased to 32%. It increased during the following days of reactivation, peaked (69%) after 14 days of reactivation, and decreased to about 27% after 3 weeks of subculture. Similar changes in tolerance of cooling occurred during reactivation of axillary shoot tips (Fig. 1B). Survival transitorily decreased from 33% to 18%,


S. BOUAFIA, N. JELTI, G. LAIRY, A. BLANC, E. BONNEL AND J. DEREUDDRE

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o~ v 60 t~ >

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0 5 10 15 20 0 5 10 15 20 A B duration of culture (d)

Fig. 1. Tolerance by apical (V) and axillary (A) shoot tips of 4.5-h dehydration (A) and subsequent cooling in liquid nitrogen (B) as a function of nodal cutting culture duration (d). Results are expressed as % survival of treated shoot tips. Vertical bars represent standard deviation.

peaked (51%) after 2 weeks of culture, and decreased to 25% at the end of the reactivation period.

For axillary shoot tips excised from cuttings, experiments were performed only after 2 and 3 weeks of cutting reactivation, the times required to obtain differentiation. Best results before and after cooling in LN were obtained after 2 weeks of reactivation (77% and 63%, respectively). A shoot reactivation period of 2 weeks was selected for further experiments.

Preculture duration. After preculturing with 1 M sucrose, 100% survival was generally obtained with encapsulated shoot tips that were not subjected to dehydration or freezing, whatever the preculture duration.

After dehydration, the proportion of shoot tips that resumed growth increased from 43% to 75% during the first three days of preculture with 1 M sucrose, and decreased to 22-23% after 5 and 7 days of preculture with 1 M sucrose (Fig.2). Similar changes occurred when shoot tips were subsequently cooled in LN: best survival (58%) was obtained after 2 days of preculture with 1 M sucrose.

Direct preculture Sucrose concentration. After 2-day preculture, most precultured shoot tips survived and resumed growth in all sucrose concentrations (data not shown). After dehydration for 4.5 h, residual water content in beads depended slightly on sucrose concentration and ranged from 0.2 to 0.22 g of water per g of dry weight (Fig. 3). Less than 10% of the control encapsulated axillary buds survived dehydration, and no


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Fig. 2. Tolerance by axillary shoot tips of 4.5-h dehydration (�9 and subsequent cooling in liquid nitrogen (0) as a function of preculture duration in 1 M sucrose. Results are expressed as % survival of treated shoot tips. Vertical bars represent standard deviation.

dehydra ted control shoot tips survived after cooling in LN. Tolerance by shoot tips of dehydrat ion increased with sucrose concentra t ion in preculture medium, peaked (88%) at 0.75 M and progressively decreased to less than 3% when the sucrose concentra t ion was raised to 1.5 M. After cooling in LN, best results (75%) were obtained with 0.75 M sucrose.

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Fig. 3. Tolerance by axillary shoot tips of 4.5-h dehydration (�9 and subsequent cooling in liquid nitrogen (A) as a function of sucrose concentration (0.1 M to 1.5 M) in preculture medium. Results are expressed as % survival of treated shoot tips. Vertical bars represent standard deviation. (O), residual water content in beads after dehydration (in g of water per g of dry weight).


S. B O U A F I A , N . J E L T I , G. L A I R Y , A. B L A N C , E. B O N N E L A N D J. D E R E U D D R E

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Fig. 4. Tolerance by axillary shoot tips of 4.5-h dehydration (�9 and subsequent cooling in liquid nitrogen (0) as a function of preculture duration (days) with 0.75 M sucrose. Results are expressed as % survival of treated shoot tips. Vertical bars represent standard deviation.

Preculture duration with O. 75 M sucrose. Encapsulated buds were rapidly cooled in LN using direct transfer to 0.75 M sucrose and various periods of preculture (1 to 7 days). Best tolerance of dehydration was obtained after 2-3 days of preculture (87 % and 90% survival, respectively) (Fig. 4). After cooling in LN, high survival rates (78% and 72%) were achieved after 2 and 3 clays of precuIture. Survival of dehydration and of cooling in LN decreased to about 45% when preculture duration was increased to 7 days.

Application to other clones The two procedures (direct and progressive precultures) were applied to cryopreservation of three dihaploid and two tetraploid clones. For direct preculture, shoot tips were precultured for 2 days at two sucrose concentrations: 0.5 M and 0.75 M. For stepwise preculture, the procedure previously described was used (two-day increase in sucrose concentration), together with two days in 1 M sucrose. Results are presented on Table 1. Shoot survival was clone-dependent and influenced by preculture.

After progressive increase in sucrose concentration up to 1 M, shoot tip tolerance of dehydration differed significantly according to clones. Best results (67% survival) were obtained with S. phureja. With other cultivars tested, only diploid clones had survival rates higher than 50%, whereas low survival of dehydration was observed with tetraploid clones selected for this study. Differences in dehydration tolerance were not due to the ability of shoot tips to tolerate preculture: all were viable after preculture. Similar differences occurred after cooling in LN: dihaploid clones appeared more tolerant to deep freezing than tetraploid clones.



Table 1. Effects of preculture on survival (% of treated shoot tips) of different cultivars to dehydration (DH) and to cooling in liquid nitrogen (LN) with indication of standard deviation. In progressive preculture, sucrose concentration was increased to 1 M in 2 days. In direct preculture, shoot tips were directly transferred in medium containing 0.5 M or 0.75 M sucrose. Shoot-tips were maintained in the three media for 2 days. Results were obtained from 3 independent experiments (Si22) and 1 to 3 experiments for other cultivars (values in brackets represent number of experiments).

Cultivar Stepwise preculture Direct preculture

1 M 0.5 M 0.75 M

DH LN DH LN DH LN

Si22 (2x) 74_+9(2) 68_+8(31 72_+7OI 59__.6(31 87_+5(3) 78_+7(3) T39(2x) 59(11 42-+11(3) 58_+3(2) 55+__7(21 86+_812) 69_+912) 88D221 (2x) 54(1) 36_+7(3) 6001 57(I) 90(11 640) Charly (4x) 28_+5(2) 12__.3(3) 64(I) 71 ___6(2) 61(I) 50_+12(2) Hertha (4x) 38+_1(2) 26+_4(3) 62(I) 58(11 70(I) 64_+8(21

Direct preculture with lower sucrose concentrations (0.5 or 0.75 M) led to better survival for every cultivar tested. More than 60% of shoot tips survived dehydration. Best results were obtained with diploid clones. With the exception of cv. Charly, best results after cooling in LN were obtained with 0.75 M sucrose. For every cultivar tested, shoot recovery from cryopreserved shoot tips was equal to or higher than 50%. With the exception of Si22, the tolerance of which was high after progressive preculture, improvement in survival was significant.

Shoot recovery Recovery proceeded via leaf expansion and apical meristem regrowth with subsequent formation of leaf primordia. This was obtained by using hormone-free medium for preculture and by shortening to 1 week the post-culture period on medium supplemented with BA and N A A , encapsulated shoot tips then being transferred to medium 2C with G A 3 alone as hormone. With potato, extraction of shoot tips from the beads did not appear to be necessary.

Discussion

We propose an improved procedure for cryopreservation of Solanum shoot tips. This consists of encapsulation of axillary shoot tips in alginate beads, preculture with sucrose-enriched preculture medium, air-drying, rapid cooling in LN, slow rewarming, and.subculture on standard culture medium. This simple procedure does not require sophisticated equipment or refinements to give high survival rates. Shoot recovery is also easily achieved. Another advantage of the encapsulation- dehydration procedure is that preculture with sucrose, a widely employed cryoprotective substance, is combined with desiccation as cryoprotective treatment, thus avoiding the mixtures of sugars (sucrose), polyols (sorbitol, mannitol), DMSO,


S. BOUAFIA, N. JELTI, G. LAIRY, A. BLANC, E. B O N N E L AND J. D E R E U D D R E

glycerol, proline, ethylene glycol, polyethylene glycol, and bovine serum albumin (BSA) generally used in conventional and vitrification procedures. Direct regrowth of shoot tips without significant callus formation was obtained after cooling in LN by using a specific sequence of hormonal additions to the different media used during preculture and post-treatment.

High survival rates after dehydration and subsequent cooling in LN of potato shoot tips was obtained using axillary shoot tips. During the development of the stem from axillary shoot tips (obtained by cutting), survival rates decreased transiently, and then gradually increased over the following days. A similar phenomenon has been seen in carnation (Dereuddre et al., 1988), thus underlining the importance of the physiological or morphological state of the plant material in LN tolerance of plant organs.

Progressive increase in sucrose concentration to 1 M, as proposed by Plessis et al. (1991, 1993) for cryopreservation of grapevine shoot tips, also gave good survival for shoot tips of Solanum phureja and, to a lesser extent, of other diploid clones. However, survival was significantly lower (less than 26%) for the tetraploid clones tested. These poor results seemed unrelated to the sensitivity of meristems of such clones to preculture at high sucrose concentration: most shoot tips survived preculture. For tetraploid clones, 2-day preculture with lower sucrose concentrations (0.5 M or 0.75 M) allowed survival rates higher than 60%.

We performed 4.5-h dehydration at 20 *C to obtain 0.20-0.22 g water/g DW in beads. This water content is known to allow tolerance of shoot tips to direct cooling in LN with (Dereuddre et al., 1990) or without (Uragami et al., 1990) encapsulation. During the drying process, sucrose concentration in alginate beads increased markedly and probably reached or exceeded the saturation point of the sucrose solution, thus resulting in glass transition during cooling and rewarming in both alginate beads and explants (Dereuddre et al., 1991).

In conclusion, the encapsulation-dehydration procedure appears to be a practical and efficient method for cryopreservation of potato shoot tips. It could be proposed for routine use if axillary shoot tips are excised from 2-week nodal subcultures. Between-clone differences could be reduced simply by adjusting the sucrose concentration in preculture medium.

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Bajaj, Y.P.S., 1978. Tuberization in potato plants regenerated from freeze-preserved meristems. Crop Improvement 5: 137-141.

Bajaj, Y.P.S., 1985. Cryopreservation of germplasm of potato (Solanum tuberosum L.) and cassava (Manihot esculenta Crantz): viability of excised meristems cryopreserved up to four years. Indian Journal of Experimental Biology 23: 285-287.

Benson, E.E., K. Harding & H. Smith, 1989. Variation in recovery of cryopreserved shoot-tips of Solarium taberosum exposed to different pre- and post-freeze light regimes. Cryo-Letters 10: 323-344.



Dereuddre, J., J. Fabre & C. Bassaglia, 1988. Resistance to freezing in liquid nitrogen of carnation (Dianthus caryophyllus L. var. Eolo) axillary shoot tips excised from different aged in vitro plantlets. Plant Cell Reports 7: 170-173.

Dereuddre, J., N. Hassen, S. Blandin & M. Kaminski, 1991. Resistance of alginate-coated somatic embryos of carrot (Daucus carota L.) to desiccation and freezing in liquid nitrogen: 2. Thermal analysis. Crvo-Letters 12: 135-148.

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Morel, G. & R.M. Wetmore, 1951. Fern callus tissue culture. American Journal o f Botany 38: 141-143.

Murashige, T. & F. Skoog, 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiology Plantarum 15: 473--497.

Plessis+ P., C. Leddet & J. Dereuddre, 1991. Rdsistance ~l la ddshydratation et h la congdlation dans I'azote liquide d'apex enrobds de vigne (Vitis vinifera L. cv. Chardonnay). Comptes Rendus AcadOmie des Sciences Paris 313: 373-380.

Plessis+ P., C. Leddet, A. Collas & J. Dereuddre, 1993. Cryopreservation of Vitis vinifera L. cv. Chardonnay shoot tips by encapsulation-dehydration: effects of pretreatment, cooling and postculture conditions. Cryo-Letters 14: 309-320.

Tendille, C. & M. Lecerf, 1974. La multiplication vdgdtative de l'asperge (Aspargus officinalis L.). Action de divers facteurs, en particulier de la nutrition mindrale, sur le ddveloppement des mdristbmes d'asperge, sur la croissance des plantules issues de ces mdrist6mes et sur la production de plantes adultes. Annales de l'AmOlioration des Plantes 24: 269-282.

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S. BOUAFIA, N. JELTI, G. LAIRY, A. BLANC, E. BONNEL AND J. DEREUDDRE

Ward, A.C.W., E.E. Benson, N.W. Blackhall, S. Cooper-Bland, W. Powell, J.B. Powell & M.R. Davey, 1993. Flow-cytometric assessments of ploidy stability in cryopreserved dihaploid Solanum tuberosum and wild Solanum species. Cryo-Letters 14: 145-152.

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Cryopreservation of potato shoot tips by encapsulationdehydration