Tissue Culture and Cryopreservation Unit

Conservation of economically important plants and their wild relatives using in vitro conservation and cryopreservation techniques

The Tissue Culture and Cryopreservation Unit (TCCU) was established in 1986 [erstwhile National Facility for Plant Tissue Culture Repository (NFPTCR), a project funded by the Department of Biotechnology] to carry out research on in vitro conservation and cryopreservation of difficult-to-conserve crops and species, leading to the development of techniques for their short-, medium-, and long-term germplasm conservation. Targeted crops and species comprise economically important agri-horticultural crops which are vegetatively/clonally propagated, rare/threatened/endangered species, medicinal plants, non-orthodox seeded species and a few prioritised orthodox seeded germplasm, including crop wild relatives (CWR). Propagules used for conservation range from meristems, shoots tips, nodal cuttings, dormant buds, seeds, embryos (somatic/zygotic), embryonic axes, pollen and cell suspensions, depending on the crop as well as method and objective of conservation. Germplasm is conserved for short- to medium-term as in vitro cultures in the In Vitro Genebank (IVGB) and for long-term in the Cryogenebank. These genebanks act as complimentary conservation strategies to the more conventional methods of germplasm conservation in seed genebanks and field genebanks.

Objectives

• Development of new or improved protocols for in vitro conservation and cryopreservation for crops belonging to fruits, spices, tuberous, bulbous, ornamental, medicinal, aromatic and threatened species, crops/species with non-orthodox seeds.
• Conservation of germplasm using biotechnological techniques.
• Monitoring conserved germplasm for viability, health and genetic stability.
• Human resource development in the area of in vitro conservation and cryopreservation.

• For details on cryopreserved germplasm visit Cryodatabase
• More than 800 accessions of various crops including Allium sp., Bacopa monnieri, Coleus forskohlii, Curcuma spp. dahlia, Dioscorea spp, Fragaria sp., gladiolus, Ipomoea batatas, Musa sp., Picrorhiza spp, Pogostemon patchouli, Vaccinium sp. and Zingiber spp. supplied as in vitro cultures to various users for field trials and evaluation of germplasm, research and breeding purposes, under Material Transfer Agreement (MTA)

Protocols for Micropropagation/In Vitro Mulitplication

• Rapid clonal multiplication leading to regeneration of plantlets through tissue culture is an important prerequisite for in vitro conservation.
• Micropropagation/in vitro multiplication protocols have been developed or refined using varied explants (shoot tips, nodal cuttings, floral meristems, seeds segments) and regeneration systems (axillary bud proliferation, organogenesis, somatic embryogenesis), Important crops and species include:  
  • Fruits: Banana (Musa spp.), almond (Prunus armenica), kiwi (Actinidia chinensis), mangosteen (Garcinia indica and G. cambogia), pear (Pyrus communis), mulberry (Morus spp.), Rubus spp.,woodapple (Aegle marmelos),
  • Spices and Industrial crops: ginger Black pepper, betel vine etc. (Piper spp.), cardamom (Elettaria cardamomum), ginger (Zingiber officinale), jojoba (Simmondia chinensis), turmeric (Curcuma spp.), vanilla (Vanilla planifolia)
  • Tubers: Asian yams (Dioscorea esculenta, D. pentaphylla, D. hispida and D. wallichii), giant taro (Alocasia indica), Chinese potato (Coleus parviflorus), sweet potato (Ipomoea batatas), taro (Colocasia esculenta),
  • Bulbs and Ornamentals: Chinese chives (Allium tuberosum, A. bakeri), Dahlia sp.
  • MAP and RET spp.: Bacopa monnieri, Coleus forskohlii, Curculigo orchoides, Gentiana kuroo, Kaempferia spp., Pogostemon patchouli, Rauvolfia canescens and Tylophora indica.

Protocols for In Vitro Conservation (Medium-term Conservation in IVGB)

Various slow growth strategies such as low temperature incubation, use of osmotic agents, growth retardants, nutritional or hormonal manipulations etc. have been evaluated for different crop species.

• Use of minimal media in Acorus calamus, Actinidia spp., Aegle marmelos, Allium spp., Aristolochia indica, Artocarpus lakoocha, Cholorophytum borivilianum, Centella asiatica, Curcuma spp., Colocasia esculenta Centella asiatica, Curcuma spp., Dioscorea spp., Ipomoea batatas, Kaempferia spp., Malus spp., Mentha spp., Morus spp., Piper spp., Plumbago spp., Pogostemon patchouli, Pyrus spp., Rubus spp., Tylophora indica,
• Low temperature incubation (4-20^oC) in general found effective in prolonging subculture interval to 6-24 months in many species, especially those belonging to Allium, Fragaria, Gentiana, Musa, Picrorhiza, Rauvolfia, Swertia and Valeriana.
• Induction of storage organs in Alliums, ginger, turmeric, yams, taro, Kaempferia spp. significantly increased storage period up to 20-24 months at 25^oC. In vitro produced rhizomes, bulblets or micro- tubers could also be used for direct field planting with high success.
• Minor modifications of the media extended shelf life of cultures in Alocasia, Coleus Piper, Rauvolfia, Simmondsia chinensis, etc. to 12-24 months at 25^oC. In yams, small increase in cytokinin concentration helped extend survival of cultures beyond 14 months at 25^o C. Mannitol at low levels prolonged subculture period to 16 months at 25^oC in sweet potato, while mineral oil overlay in Bacopa monnieri cultures increased storage from 6 to 24 months.
• Protocols developed by using low-cost media worked effectively in banana, ginger, turmeric, galangal, without any loss of regeneration potential.

Protocols for Cryopreservation (Long-term Conservation in Cryogenebank)

• Cryopreservation protocols developed/refined using shoot tip/meristem explants derived from in vitro cultures in Allium hookeri, A. scorodoprasum, A. ramosum, A. chinense A. sativum, A tuberosum, Bacopa monnieri, Curcuma longa, Dioscorea bulbifera, D. deltoidea, Dahlia, Elettaria cardamomum, Gentiana kurroo, Malus, Musa, Prunus, Picrorhiza, Rauvolfia serpentina, Rubus and Vaccinium.
• Studies on seed viability, moisture content, desiccation and freezing sensitivity conducted on cheura (Diploknema butyracea), jamun (Syzygium cuminii), phalsa (Grewia asiatica), wild apricot (Prunus armeniaca), Pilu (Salvadora oleoides), S. persica, Alangium salvifolium, Murraya paniculata, M. koeingii, Capparis decidua and grape fruit (Citrus paradisi), trifoliate orange (Poncirus trifoliata) and its hybrids, Citrus species, viz., Citrus sinensis, C. reticulata, C. aurantium, Citrumelo (Poncirus trifoliata × C. paradisi) and several other citrus spp and underutilized fruits. Seeds of Diploknema butyracea, Syzygium cuminii, Citrus jhambiri, Poncirus trifoliata were found to be recalcitrant while others showed intermediate storage behaviour.
• Basic cryobiological studies on morphological, physiological and biochemical parameters of difficult-to-store germplasm of intermediate and recalcitrant seed species (> 37 genera of 70 species) carried out in order to determine factors responsible for degree of recalcitrance.
• Base collection with sizeable indigenous variability established for Citrus spp. (32 species), Buchanania lanzan, Capparis sp., Manilkara hexandra, Piper nigrum, Juglans regia, Prunus amygdalus, Jatropha curcas, Pongamia pinnata, Prunus armeniaca, Morus spp. and Malus cvs. (vegetative buds).
• Vast genetic wealth of Citrus existing in Northeast and Northwest India which includes rare, endangered, endemic species and important genotypes of other indigenous Citrus species have been cryopreserved by developing suitable cryopreservation protocols ensuring long-term safe conservation. Methods of desiccation-freezing, encapsulation and vitrification have been successfully applied in various Citrus species and cultivars. Comprehensive cryobase collection of more than 600 accessions of Indian Citrus germplasm has been established in cryogenebank that comprises rootstocks, wild, rare and endangered species such as C. indica, C. macroptera, C. megaloxycarpa, C. laptipes etc.

Monitoring Health Status of Germplasm

• Health status of accessions of diverse crops, both fresh and cryostored, are checked routinely to ensure pest-free conservation.
• All in vitro cultures screened periodically for identification and elimination of covert endogenous bacteria.
• In vitro cultures of Allium spp., Fragaria spp., Musa spp., Rubus spp., Ipomoea spp., indexed for viruses using serological and immunological techniques Direct Antigen coated-indirect-enzyme linked Immunobsorbent assay (DAC-ELISA, DAS-ELISA). PCR based techniques and electron microscopy. 
• Meristem culture technique standardized for sweet potato and Allium for recovering virus-free plants.
• Cryotherapy as a means for obtaining virus-free material attempted in germplasm of banana, garlic and strawberry, with various degree of success.

Regeneration of Cryostored Germplasm

• Periodic viability testing undertaken for cryopreserved seeds and in vitro cryopreserved meristems. Original viability retained in 5-31 years of cryostorage in species tested so far.

Testing Genetic Stability of In Vitro Conserved and Cryopreserved Germplasm

• Plants re-established in soil following various period of in vitro conservation/ cryopreservation were found to maintain their fruit characters (Musa), special chemical constituents (Coleus, Pogostemon, Bacopa monnieri), morphological characters (sweet potato, garlic, galangal) and genomic integrity (Bacopa, banana, ginger, turmeric, cardamom, yams, taro, etc.).
• Thus, conservation protocols developed at TCCU have been found to maintain genetic integrity which is of utmost importance in germplasm conservation programme anywhere in the world.

• Dr Anuradha Agrawal, nominated as Member, Committee constituted for developing a report on ‘Agricultural Policies and Action-Plans for Secure and Sustainable Agriculture’, via OM dated 12 Feb. 2019, by office of the Principal Scientific Adviser (PSA) to the Government of India (pdf).
• Dr Anuradha Agrawal elected as General Secretary, Indian Society of Plant Genetic Resources (ISPGR), New Delhi for 2019-2021.
• Dr Sangita Bansal received Distinguished Scientist Award 2019 from Hi-Tech Horticulture Society, Dec. 1, 2019.
• Dr Sandhya Gupta elected Vice Chair of Commission on Plant Genetic Resources, International Society of Horticultural Science (ISHS) for 2014-2018.
• Dr Gowthami R. received Young Scientist Award during the International Conference on Advances in Agricultural and Biodiversity Conservation for Sustainable Development (ABCD-2017), C.C.S. University, Meerut, Uttar Pradesh, Oct. 27-28, 2017.
• Dr Anuradha Agrawal elected as Treasurer, ISPGR, New Delhi for 2015-2018.
• Dr Anuradha Agrawal conferred with Panjabrao Deshmukh Outstanding Woman Scientist Award 2011 of ICAR, July 16, 2012.
• Dr Sandhya Gupta elected as Fellow, ISPGR, New Delhi, 2014.
• Dr Sandhya Gupta received Fellowship by Indian Society of Horticultural Research & Development (ISHRD), Uttarakhand, 2010.
• Dr Ruchira Pandey and Dr Neelam Sharma elected as Fellow, ISPGR, New Delhi, 2009.
• Dr Anuradha Agrawal elected as Fellow, Association for Improvement of Banana and Plantain (AIPUB), Tiruchirapalli, 2008.
• Dr Anuradha Agrawal elected as Fellow, ISPGR, New Delhi, 2008.

• Dr Era V Malhotra awarded ‘Best Oral Presentation Award’ for paper entitled ‘Characterization of MicroRNAs and their target genes in Cajanus scarabaeoides (L.) Thouars – a crop wild relative of pigeonpea’ during the 3rd Global Meet on Science and Technology, Dec. 1-3, 2019, ICAR–NRCSS, Ajmer, Rajasthan.
• Dr Era V. Malhotra awarded ‘Best Oral Presentation’ for paper presented in 3rd International Conference on Bioresource and Stress Management, Jaipur, Rajasthan, Nov. 8-10, 2017.

Research and Service

• Development of protocols for (i) in vitro multiplication/micropropagation (ii) in vitro conservation (iii) cryopreservation using diverse explants (meristems, dormant buds, seeds, embryos/embryonic axis, pollen).
• Application of protocols for in vitro conservation and cryopreservation for short-, medium- and long-term conservation of germplasm of clonally propagated crops, non-orthodox seeded species, rare/endangered/threatened and medicinal/aromatic plant species.
• Monitoring genetic stability and health status of in vitro conserved and cryopreserved germplasm.
• Germplasm augmentation from national and international sources; supply of material to users.

Teaching and Training

• Courses related to plant tissue culture, in vitro conservation and cryopreservation taught to M.Sc. and Ph.D. students of PG, School, IARI, New Delhi.
• Guidance for thesis to M.Sc. and Ph.D. students of PG, School, IARI, New Delhi, and other Universities.
• Short-term training related to plant tissue culture, in vitro conservation and cryopreservation (3-6 months) to students from various Universities.
• International and national trainings/conferences/symposia organized periodically.