Cryopreservation Techniques for Conservation of Tropical Horticultural Species Using Various Explants

  • S. K. MalikEmail author
  • R. Chaudhury


Ex situ conservation of tropical horticultural species is difficult using traditional conservation approaches as most of these produce nonorthodox seeds and many of these are vegetatively propagated. Clonally/vegetatively propagated tropical woody species are rather recalcitrant to in vitro establishment necessitating the need for alternate conservation methods. Cryopreservation of such difficult-to-store species has been attempted using diverse explants with invariably good success. Encouraging results while attempting cryopreservation of these species have further promoted application of vitrification-based cryotechniques to diverse plant germplasm during last two to three decades. Development of suitable cryopreservation protocols and long-term cryobanking using cryoprotocols are the two fundamental aspects of successful plant genetic resources conservation. Strengthening of basic research on cryopreservation of plant tissues, defined policy for prioritization of cryobanking of germplasm and need of quality human resource in this area are the issues which need further attention. Present chapter highlights the present status of cryopreservation of tropical horticulture species using diverse techniques and challenges in long-term cryobanking.


Cryobanking Vitrification Seeds Embryos Embryonic axes Dormant buds Pollen Genomic resources 


  1. Anonymous (2015) ICAR-National Bureau of Plant Genetic Resources (Annual report). New Delhi: NBPGR.Google Scholar
  2. Anonymous. (2017). ICAR-National Bureau of plant genetic resources (annual report). New Delhi: NBPGR.Google Scholar
  3. Benelli, C., De Carlo, A., & Engelmann, F. (2013). Recent advances in the cryopreservation of shoot-derived germplasm of economically important fruit trees of Actinidia, Diospyros, Malus,Olea,Prunus,Pyrus and Vitis. Biotechnology Advances, 31, 175–185. Scholar
  4. Berjak, P., & Pammenter, N. W. (2008). From Avicennia to Zizania: Seed recalcitrance in perspective. Annals of Botany, 101, 213–228.CrossRefGoogle Scholar
  5. Bhat, S. R., Bhat, K. V., & Chandel, K. P. S. (1994). Studies on germination and cryopreservation of Musa balbisiana seed. Seed Science and Technology, 22, 637–640.Google Scholar
  6. Chandel, K. P. S., Chaudhury, R., Radhamani, J., & Malik, S. K. (1995). Desiccation and freezing sensitivity in recalcitrant seeds of tea, cocoa and jackfruit. Ann Botany, 76, 235–240.CrossRefGoogle Scholar
  7. Chaudhury, R. (2000). Cryopreservation of seeds, embryo, embryonic axes and pollen at National Cryobank at NBPGR. In : Cryopreservation of tropical plant germplasm (Current research progress and application) (pp. 457–459). Rome: JIRCAS/IPGRI.Google Scholar
  8. Chaudhury, R., & Malik, S. K. (1999). Cryopreservation and in vitro technology for conservation of recalcitrant seeds of some tree species, pp. 119–131. In M. Marzalina, K. C. Khoo, N. Jayanthi, F. Y. Tsan, & B. Krishnapillay (Eds.), Proceedings of IUFRO Seed Symposium 1998 ‘Recalcitrant seeds’, held at FRIM, Kuala Lumpur, Malaysia.Google Scholar
  9. Chaudhury, R., & Malik, S. K. (2004). Desiccation and freezing sensitivity during seed development in jackfruit. Seed Science and Technology, 32, 785–795.CrossRefGoogle Scholar
  10. Chaudhury, R., & Malik, S. K. (2014). Implementing cryotechniques for plant germplasm: Storing seeds, embryonic axes, pollen and dormant buds. Acta Horticulturae, (1039), 273–280.Google Scholar
  11. Chaudhury, R., & Malik, S. K. (2016). Expanding applications of cryobanking: Meeting challenges for effective long-term storage. Indian Journal of Plant Genetic Resources, 29(3), 303–306.CrossRefGoogle Scholar
  12. Chaudhury, R., Mandal, B. B. and Malik SK (2000) Development of cryopreservation techniques for the long-term conservation of recalcitrant seed and vegetatively propagated plant species in India. IPGRI-NBPGR Project Report.Google Scholar
  13. Chaudhury, R., Malik, S. K., & Rajan, S. (2010). An improved pollen collection and cryopreservation method for highly recalcitrant tropical fruit species of mango (Mangifera indica L.) and Litchi (Litchi chinensis Sonn.). CryoLetters, 31, 268–278.PubMedGoogle Scholar
  14. Chaudhury, R., Malik, S. K., Joshi, J., & Shivanna, K. R. (2011). Cryopreservation of pollen for germplasm storage and for utilization in breeding programmes. Journal of Palynology, 1, 205.Google Scholar
  15. Dulloo, M. E., Hunter, D., & Borelli, T. (2010). Ex Situ and In Situ conservation of agricultural biodiversity: Major advances and research needs. Notulae Botanicae Horti Agrobotanici Cluj, 38(2) special issue, 123–135.Google Scholar
  16. Engelmann, F. (2011). Use of biotechnologies for the conservation of plant biodiversity. In Vitro Cellular & Developmental Biology—Plant, 47, 5–16.CrossRefGoogle Scholar
  17. Ganeshan, S., & Rajasekharan, P. E. (2000). Currents status of pollen cryopreservation research: Relevance to tropical horticulture. In F. Engelmann & H. Takagi (Eds.), Cryopreservation of tropical plant germplasm : Current research progress and application (pp. 360–365). Rome: JIRCAS/ IPGRI.Google Scholar
  18. Gonzalez-Arnao, M. T., Panta, A., Roca, W. M., Escobar, R. H., & Engelmann, F. (2008). Development and large scale application of cryopreservation techniques for shoot and somatic embryo cultures of tropical crops. Plant Cell, Tissue and Organ Culture, 92(1), 1–13. Scholar
  19. Hofmann, P., & Steiner, A. M. (1989). An updated list of recalcitrant seeds. Landwirtschaftliche Forschung, 42, 310–323.Google Scholar
  20. Hong, T. D., & Ellis, R. H. (1996). A Protocol to Determining Seed Storage Behaviour (Technical Bulletin no. 1, edited by J. M. M. Engels & J. Toll, p. 62). Rome: IPGRI.Google Scholar
  21. Jaiswal, V. S. (2003). Organogenic and embryogenic regeneration and thier role in in vitro conservation of tropical fruit species. In R. Chaudhury, R. Pandey, S. K. Malik, & B. Mal (Eds.), In vitro conservation and cryopreservation of tropical fruit species (pp. 101–113). New Delhi: IPGRI Office for South Asia/NBPGR.Google Scholar
  22. Keller, E. R. J., Senula, A., Zanke, C., Grube, P., & Kaczmarczyk, A. (2011). Cryopreservation and in vitro culture- state of the art as conservation strategy for genebanks. Acta Horticulturae, (918), 99–111.Google Scholar
  23. Kim, H. H., Lee, J. K., Hwang, H. S., & Engelmann, F. (2007). Cryopreservation of garlic germplasm collections using the droplet-vitrification technique. CryoLetters, 28, 471–482.PubMedGoogle Scholar
  24. Kulus, D., & Zalewska, M. (2014). Cryopreservation as a tool used in long-term storage of ornamental species-a review. Scientia Horticulturae, 168, 88–107.CrossRefGoogle Scholar
  25. Malik, S. K., & Chaudhury, R. (2006). The cryopreservation of embryonic axes of two wild and endangered Citrus species. Plant Genetic Resources: Characterization & Utilization, 4, 204–209.CrossRefGoogle Scholar
  26. Malik, S. K., Chaudhury, R., & Kalia, R. K. (2003). Seed storage behaviour and cryopreservation of tropical fruit species. In R. Chaudhury, R. Pandey, S. K. Malik, & B. Mal (Eds.), In vitro conservation and cryopreservation of tropical fruit species (pp. 175–190). New Delhi: IPGRI Office for South Asia/NBPGR.Google Scholar
  27. Malik, S. K., Chaudhury, R., Dhariwal, O. P., & Bhandari, D. C. (2010). Genetic resources of tropical underutilized fruits in India (p. 168). New Delhi: NBPGR.Google Scholar
  28. Malik, S. K., Chaudhury, R., Kalia, R. K., & Dullo, E. (2011). Seed storage characteristics and cryopreservation of genetic resources of tropical underutilized fruits in India. Acta Horticulturae, (918), 189–198.Google Scholar
  29. Malik, S. K., Chaudhury, R., & Pritchard, H. W. (2012a). Long -term, large scale banking of Citrus species embryos: Comparisons between cryopreservation and and other seed banking temperatures. CryoLetters, 33(6), 453–464.PubMedGoogle Scholar
  30. Malik, S. K., Chaudhury, R., Kumar, S., Dhariwal, O. P., & Bhandari, D. C. (2012b). Citrus genetic resources in India: Present status and management (p. 184). New Delhi: National Bureau of Plant Genetic Resources.Google Scholar
  31. Malik, S. K., Chaudhury, R., & Singh, I. P. (2015). Management of Citrus genetic resources in India: Approaches, applications and achievements. Acta Horticulturae, 1101, 39–46. Scholar
  32. Malik, S. K., Chaudhury, R., Srivastava, V., & Singh, S. (2017). Genetic resources of Syzygium cumini in India: Present status and management. In K. N. Nair (Ed.), The genus Syzygium: Syzygium cumini and other underutilized species (pp. 195–214). Boca Raton: CRC Press, Taylor & Francis Group.CrossRefGoogle Scholar
  33. Niino, T., Yamamoto, S., Fukui, K., Castillo Martínez, C. R., Valle Arizaga, M., Matsumoto, T., & Engelmann, F. (2013). Dehydration improves cryopreservation of mat rush (Juncus decipiens Nakai) basal stem buds on cryo-plates. CryoLetters, 34, 549–560.PubMedGoogle Scholar
  34. Normah, M. N. (2000). Cryopreservation of tropical plant germplasm (Current research progress and application) (pp. 287–290). Rome: JIRCAS/IPGRI.Google Scholar
  35. Normah, M. N., Malik, S. K., Chaudhury, R., Salma, I., & Makeen, M. A. (2013). Conservation of tropical fruit genetic resources. In M. N. Normah, H. F. Chin, & B. M. Reed (Eds.), Conservation of tropical plant species (pp. 137–170). New York: Springer.CrossRefGoogle Scholar
  36. Panis, B., Piette, B., & Swennen, R. (2005). Droplet vitrification of apical meristems: A cryopreservation protocol applicable to all Musaceae. Plant Science, 168, 45–55.CrossRefGoogle Scholar
  37. Pritchard, H. W. (2004). Classification of seed storage ‘types’ for ex situ conservation in relation to temperature and moisture. In E. O. Guerrant H. K. Jr & M. Maunder (Eds.), Ex situ plant conservation: Supporting species survival in the wild (pp. 139–161). Covelo: Island Press.Google Scholar
  38. Pritchard, H. W. (2015). Plant cryopreservation-scale, scope and hope. In R. Chaudhury, S. K. Malik, & K. C. Bansal (Eds.), Cryobanking of bioresources –Cross linking plant, animal, fish, microbe and medical sciences (pp. 11–19). Book of abstracts of international conference on, “Low temperature science and biotechnological advances”, 27–30th April, 2015, New Delhi, India.Google Scholar
  39. Pritchard, H. W., Nadarajan, J., Ballesteros, D., Thammasiri, K., Prasongsom, S., Malik, S. K., Chaudhury, R., Kim, H. H., Line, L., Lie, W. Q., Yange, X. Y., & Popova, E. (2017). Cryobiotechnology of tropical seeds – scale, scope and hope. I International Symposium on Tropical and Subtropical Ornamentals. Horticulturae1167.ISHS2017.
  40. Rao, A., Chaudhury, R., Malik, S. K., Kumar, S., Radhakrishanan, R., & Qadri, S. M. H. (2009). Mulberry biodiversity conservation through cryopreservation. In Vitro Cellular & Developmental Biology –Plant, 45(6), 639.CrossRefGoogle Scholar
  41. Rohini, M. R., Malik, S. K., Choudhary, R., Kaur, S., Uchoi, A., & Chaudhury, R. (2016). Storage behavior and cryopreservation studies in Indian rough lemon (Citrus jambhiri): A promising rootstock for long-term conservation. Turkish Journal of Agriculture and Forestry, 40, 865–873. TÜBİTAK.CrossRefGoogle Scholar
  42. Sakai, A., & Engelmann, F. (2007). Vitrification, encapsulation-vitrification and droplet- vitrification: A review. CryoLetters, 28, 151–172.PubMedGoogle Scholar
  43. Schäfer-Menuhr, A. (1996). Refinement of cryopreservation techniques for potato. Final report for the period September 1, 1991–August 31, 1996 (pp. 1–41). International Plant Genetic Resources Institute, Rome, Italy.Google Scholar
  44. Seaton, P., Kendon, J. P., Pritchard, H. W., Puspitaningtyas, D. M., & Marks, T. R. (2013). Orchid conservation: The next ten years. Lankesteriana, 13, 93–101.Google Scholar
  45. Sudarmonowati, E. (2000). Cryopreservation of tropical plant germplasm (Current research progress and application) (pp. 291–296). Rome: JIRCAS/IPGRI.Google Scholar
  46. Thammasiri, K. (1999). Cryopreservation of embryonic axes of jackfruit. Cryo-Letters, 20, 21–28.Google Scholar
  47. Tweddle, J. C., Dickie, J. B., Baskin, C. C., & Baskin, J. M. (2003). Ecological aspects of seed desiccation sensitivity. Journal of Ecology, 91, 294–304.CrossRefGoogle Scholar
  48. Vineesh, P. S., Skaria, R., Mukunthakumar, S., Padmesh, P., & Decruse, S. W. (2015). Seed germination and cryostorage of Musa acuminata subsp. Burmannica from Western Ghats. South African Journal of Botany, 100, 158–163.CrossRefGoogle Scholar
  49. Volk, G. M., Shepherd, A., & Bonnart, A. M. (2014). Strategies for improved efficiency when implementing plant vitrification techniques. Acta Horticulturae, 1039, 85–90.CrossRefGoogle Scholar
  50. Walters, C., Berjak, P., Pammenter, N., Kennedy, K., & Raven, P. (2013). Preservation of recalcitrant seeds. Science, 339, 915–916.CrossRefGoogle Scholar
  51. Wesley-Smith, J., Vertucci, C. W., Berjak, P., Pammenter, N. W., & Crane, J. (1992). Cryopreservation of desiccation-sensitive axes of Camellia sinensis in relation to dehydration, freezing rate and the thermal properties of tissue water. Journal of Plant Physiology, 140, 596–604.CrossRefGoogle Scholar
  52. Wu, Y.-J., Huang, X.-L., Chen, Q.-Z., Li, X.-J., & Engelmann, F. (2007). Induction and cryopreservation of embryogenic cultures from nucelli and immature cotyledon cuts of mango (Mangifera indica L. var Zihua). Plant Cell Reports, 26, 161–168.CrossRefGoogle Scholar
  53. Yamamoto, S., Rafique, T., Priyantha, W. S., Fukui, K., Matsumoto, T., & Niino, T. (2011). Development of a cryopreservation procedure using aluminium cryo-plates. CryoLetters, 32, 256–265.PubMedGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2019

Authors and Affiliations

  1. 1.ICAR-National Bureau of Plant Genetic ResourcesNew DelhiIndia

Personalised recommendations