Cryopreservation of citrus apices using the encapsulation dehydration ...

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Cryo-Letters 19, 177-182(1998). Published by Cryo-Letters, 7, Wootton Way, Cambridge CB3 9LX, U.K.

CRYOPRESERVATION OF CITRUS APICES USING THE ENCAPSULATIONDEHYDRATION TECHNIQUE Maria Teresa Gonzalez-Arnaol*,

Caridad Urra',

': Centro Nacional de Investigaciones Científicas (CNIC). Ave 25 y 158, Apartado 6990, Playa, La Habana, Cuba. Plant Genetic Resources Institute (IPGRI). Via delle Sette Chiese 142, 00145 Rome, Italy. -* Instituto de Investigaciones de Cítricos (IIC). Ave 7ma. e/ 30 y 32, Miramar, La Habana, Cuba.

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Abstract Cryopreservation of apices sampled on in vitro plantlets of Poncirus trifoliata (L.) Raf. was achieved using the encapsulation-dehydration technique. The highest survival rates after cryopreservation (up to 50 %) were obtained when encapsulated apices were pregrown for 3 d either directly in liqÙid medium containing 0.5M sucrose or in media with progressively increasing sucrose concentration from 0.3 to 0.75M, desiccated to 20-25'31 moisture content and frozen slowly (O.S"C.min-') from +2O"C to -40°C before immersion in liquid nitrogen. Recovery of whole plantlets from cryopreserved apices took place directly, without transitory callus formation. Key words: Cryopreservation, citrus, apices, encapsulation-dehydration. *

Introduction Seeds of many citrus spt i e s display recalcitrant or intermediate storage behaviour, and therefore cannot be stored dry at low temperature. In addition, the seeds produced are heterozygous and particular gene combinations cannot be conserved. Some cultivars are seedless and are thus propagated vegetatively. Citrus genetic resources are thus currently conserved as whole plants in field genebanks where they remain exposed to pests, diseases and other natural hazards such as drought, weather damage, human error and vandalism (20). Nor are they in a condition that is readily conducive to germplasm exchange. Field genebanks are costly to maintain and, as a consequence, are prone to economic decisions that may limit the level of replication of accessions. the quality of maintenance and even their very survival in times of economic stringency. Even under the best circumstances, field genebanks require considerable inputs in the form of land (often needing multiple sites to allow for rotation), labour, management and materials.

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The only current alternative for long-term conservation of problem crops is cryopreservation. Cryopreservation techniques have been developed for more than 1O0 species cultivated under various forms, including cell suspensions, calluses, apices, somatic and zygotic embryos (2).

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Cryopreservation of citrus germplasm has been performed using seeds (S), ovules (l), embryogenic axes (15), somatic embryos (7), embryogenic calluses and cell suspensions (3, 4, 11, 16, 17, 18). However, juvenile material only can be conserved, which iequires several years before flowering and fruit production. Apical meristems represent the material of choice fo:: citrus germplasm conservation, since plants regenerated from apices of adult cultivars will not present juvenility characteristics and will be true to type, in contrast to plants'produced from any other type of material (10). The encapsulation-dehydration technique has led to successful .results with apices of numerous temperate and tropical plant species (2).

In this paper we report the first successful application of cryopreservation to citrus apices using the encapsulation-dehydration technique. Materials and Methods Plant material

The plant material consisted of apices sampled on in vitro plantlets of Poncirus @.$oliata (L.) Raf. which were obtained from seeds germinated in vitro, which were kindly provided by IIC.

In vitro culture Plantlets were cultivated on semi-solid MS medium (9) supplemented with 50 g.L-' sucrose, 0.5 mg.L'' benzylaminopurine (BAP), 0.25 mg.L-' indole butyric acid (IBA), 40 mg.L-' adenine, 750 mg.L" malt extract and 10 g.L-' agar. They were maintained at 26°C under 8 h light/ 16 h dark photoperiod under a light intensity of 40 pmo1.m -2.s-1and subcultured every 45 d. Cryopreservation experiments For cryopreservation experiments, apices (size: 0.5-1 mm) were excised from in vitro plants 20 d after the last subculture and left overnight on standard medium for recovery. Apices were then encapsulated in alginate (3%) beads and precultured in liquid medium with various sucrose concentrations (0.3 to 1M) for different durations (1 to 10 d). In some cases, the sucrose concentration in the preculture medium was increased progressively by daily transfer of apices to medium with higher siicrose concentration, from 0.3 up to 1M. Encapsulated apices were then dehydrated at room temperature down to 20-25% moisture content (MC, fresh weight basis) under the sterile air current of the laminar flow cabinet and transferred to sterile 2ml polypropylene cryotubes. Samples submitted to preculture with progressive increase in sucrose concentration were desiccated under the same conditions down to a series of different MCs, from 36 to 15%.

Freezing was performed either rapidly by direct immersion of the cryotubes in liquid nitrogen or slowly, by cooling at O.S"C.min-' from +20"C to -40°C before immersion in liquid nitrogen, using a programmable freezer (Bio-Cool, JTS Systems, USA). Samples were kept for at least 1 h at -196 "C. For thawing, the cryotubes were placed under the air current of the laminar flow cabinet for 2-3 min. Beads were then transferred in Petri dishes to standard semi-solid medium. Apices were cultured for the first week in the dark, then transferred under standard lighting conditions.

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Fifteen to 20 apices were employed per experimental condition, and experiments were replicated 2 to 3 times. The results presented correspond to the average of these replications standard deviation. Survival was evaluated after 1 month by counting the number of apices which had developed into shoots.

Results The survival rate of encapsulated apices after pregrowth varied depending on the sucrose content in the preculture medium (Table 1). Preculture for 1 d with 0.75 and 1M sucrose was detrimental to survival. By contrast, apices could withstand extended preculture durations (up to 10 d) in media with lower sucrose concentrations (0.3 and 0.5M).

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Table 1. Effect of preculture duration and sucrose concentration on the survival ("h)of encapsulated citrus apices.

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Preculture duration (davs)

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-' Sucrose concentration (M) I 0.5 I 0.75 I

0.3 88y.O

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80k3.6 82rt2.6 85rt2.0 82k3.0

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9027.0

90-1-4.4

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-:not tested Preculture durations longer than 2 d resulted in survival rates of around 80% after desiccation (Table 2). After slow freezing, survival was achieved for all pregrowth durations experimented, whereas after rapid freezing, survival was noted for 3 and 4 d of pregrowth only. The highest survival rate with slow and rapid freezing W ~ Sachieved after 3 and 4 d of preculture, respectively. Table 2. Effect of preculture duration in medium with 0.5M sucrose on the survival (%) of apices after preculture (P), dehydration down to 20-25% MC (D), rapid (RF) or slow (SF) freezing.

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Preculture involving progressive' increase in sucrose concentration increased the tolerance to high sucrose levels in comparison with direct preculture in medium with the same final sucrose concentration. A 1M final sucrose concentration in the preculture medium was detrimental to survival (Table 3). Survival of cryopreserved apices was higher after slow freezing than after rapid freezing.

Table 3. Effect of progressive increase in sucrose concentration during preculture on the survival ("3) of apices after preculture (P), dehydration (D), rapid (RF) or slow (SF) freezing.

Survival of desiccated apices decreased in line with decreasing bead MC (Table 4). After cryopreservation, the highest survival was obtained after slow freezing, with beads dehydrated down to 23 or 28% MC.

Table 4. Effect of bead moisture content on the survival (%) of apices after dehydration, rapid or slow freezing. Apices were submitted to a progressive increase in sucrose concentration during preculture (0.3M/24h -t0.5M/24h + 0.75M/24h). Bead moisture content (%) Treatment Dehydration Rapid freezing Slow freezing

36 95d.6 O

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28 23 17 15 90rt6.2 88k4.6 50k5.6 20rt4.6 1525.3 20-1-2.7 O O 40~6.2 40~4.6 20-1-4.0 O

For recovery, control and cryopreserved apices were cultured on standard semi-solid medium. It was not necessary to extract the apices from the beads since they broke the alginate capsule without difficulty and developed into new plants without transitory callus formation. Apices which did not survive became totally black or remained white.

Discussion The cryopreservation protocol established for apices of Poncirus trifoliata rootstock comprised a preculture for 3 d in liquid medium with 0.5M sucrose or in medium with progressively increasing sucrose concentration (up to 0.75M), desiccation to 20-25% MC followed by slow freezing. Citrus apices displayed high sensitivity to sucrose since exposure to a concentration of 0.75M sucrose during preculture was tolerated only after progressive increase in sucrose concentration. Apices of several species such as grape (14), sugarbeet (19) or coffee (6)

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also require progressive sucrose increase during preculture whereas other species such as sugarcane (12) can withstand direct pregrowth in high sucrose concentrations. Slow freezing achieved better results than rapid freezing, suggesting that not all freezable water had been extracted from beaddapices during desiccation down to 20-25%, and that furtfier freeze-induced dehydration during slow prefreezing was necessary to achieve optimal survival. Similar results were noted with encapsulated grape apices for which slow freezing was necessary to achieve optimal survival (12). Growth recovery of cryopreserved citrus apices occurred directly without callus formation. It allows us to assume that most cells of the apical region were only slightly or not at all damaged during the cryopreservation process, as was observed by histocytological examination with sugarcane apices cryopreserved using the encapsulation-dehydration technique (5). Up to now, all previous attempts tob cryopreserve citrus apices had been unsuccessful (13). However, although further research is needed to improve the protocol established, these preliminary results demonstrate that it is possible to freeze apices from citrus juvenile plants. Cryopreservation of citrus apices from adult cultivars is currently under investigation using the same experimental approach,

Acknowledgements This work was performed in the framework of the IPGRT-funded project N“ 96/098 entitled “Development of cryopreservation techniques for the long-term conservation of Citrus germplasm in Cuba”. The authors thank Dr. N. Duran-Vila (Instituto Valenciano de Investigaciones Agrarias, Valencia, Spain) for helpful comments on the manuscript.

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References 1. Bajaj, YPS (1984) Current Science 53, 1215-1216. * 2. Engelmann, F (1997) In Biotechnology and Plant Genetic resources: Conservation and Use, B.V. Ford-Lloyd, J.H. Newbuny, J.A. Callow, Eds., C B I , pp. 119-162. 3. Engelmann, F, Dambier, D, Ollitrault, P (1994) Cryo-Letters 15.53-58. 4. Engelmann, F, Aguilar, ME, Dambier, D, Cabasson, C, Michaux-Ferrière, N, Ollitrault, P (1994) Fruits 49, 23-30. 5. González-Amao, MT, Engelmann, F, Huet, C. Urra, C (1993) Cryo-Letters 14, 303308. 6. Mari, S, Engelmann, F, ChabriIlange, N, Huet. C, Michaux-Femère, N (1995) CryoLetters 16,289-298. 7. Marin, ML, Duran-Vila, N (1988) Plant Cell Tissue Organ Culture 14,51-57. 8. Mumford, PM, Grout, BWW (1979) Seed Science and Technology 7,407-410. 9. Murashige, T, Skoog, F (1962) Physiologia Plantarum 15,473-497. 10. Navarro, L, Ortiz, Jh4, Juárez, J (1985) HortScience 20,214-215. 11. Niino, T and Sakai, A (1992) Plant Science 87, 199-206. 12. Paulet, F, Engelmann. F, Glaszmann, JC (1993) Plant Cell Reports 12,525-529. 13. Pérez, RM (1995) Tesis doctoral. Universidad Politécnica de Valencia, Spain. 14. Plessis, P, Leddet, C, Dereuddre, J (1991) Comptes Rendus de Z’Académie des . Sciences, Paris, 313, Série III. 373-380. :

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15. Radhamani, J, Chandel, KPS (1992) Plant Cell Reports 11,'204-206. 16. Sakai, A, Kobayashi, S, Oiyama, I (1990)Plant Cell Reports 9,30-33. 17. Sakai,A, Kobayashi, S, Oiyama, I (1991)PlantPhysioZogy 137,465-470. 18. Sakai, A, Kobayashi, S, Oiyama, I (1991)PlantScience 74,243-248. 19. Vandenbussche, B, De Proft, MP (1996) Cryo-Letters 17, 137-140. 20. Withers, LA, Engels JMM (1990) IBPGR Newsletterfor Asia, the Pacific and Oceania 3, ' 1-2.

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VOLUME 19, No.3

MAY /JUNE 1998

Lette

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ISSN O 143-2044

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CQNTENTS Announcing the Publication of Supplement Volume 1 Cryopreservation and the Conservation of Biodiversity

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Conferences and Courses

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Enhanced energy metabolism at hypothermia following addition of a prostacyclin derivative in porcine liver K. Kumar Changani, B .R. Davidson, J.D. Bell, S.D. Taylor-Robinson and B.J. Fuller (Accepted March 7, 1998)

Cryopreservation of sugarcane embryogenic callus using a simplified freezing process M.E. Martínez-Montero, M.T. González-bao, C. Borroto-Nordelo, C. Puentes-Diáz and E Engelmann (Accepted April 8, 1998)

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Vitrification of mature mouse oocytes in dimethylsulphoxide: improved results following the addition of polyethylene glycol but not dextran L. O'Neil, S.J. Paynter, B.J. Fuller and R.W. Shaw (Accepted March 7, 1998) 141 Cryopreservation of transformed Papaver somnijerum cells C. Gazeau, H. Elleuch, A. David and C. Morisset (Accepted March 7, 1998)

Physical observations on rapid freezing of tumor cells to - 40°C using differential scanning calorimetry S.El-Shakhs, S.Shimi, E.E. Benson, L. Newman and A. Cushieri (Accepted March 16, 1998) 159

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Cryopreservation of citrus apices using the encapsulation-dehydration technique M. T. González-Amao, F. Engelmann, C. Urra, M. Morenza and A. Rios (Accepted April 8, 1998) 177 A comparison of ATP recovery or phosphomonoesterhnorganicphosphate ratio to assess metabolic activity in liver at hypothermia: a 31Pnmr study K. Kumar Changani, B.R. Davidson, J.D. Bell, S.D. Taylor-Robinson and B.J. Fuller (Accepted April 21,1998) 183

Haemoglobin (ErythrogerTM) improves post-thaw growth of cryopreserved rice cells K. Azhakanandam, P. Anthony, 189 M.R. Davey and K.C. Lowe