Tissue Culture and Cryopreservation Unit

Delhi, India

Tissue Culture and Cryopreservation Unit

Delhi, India
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Jacob S.R.,ICAR National Bureau of Plant Genetic Resources | Tyagi V.,Germplasm Exchange Unit | Agrawal A.,Tissue Culture and Cryopreservation Unit | Chakrabarty S.K.,ICAR National Bureau of Plant Genetic Resources | Tyagi R.K.,ICAR National Bureau of Plant Genetic Resources
PLoS ONE | Year: 2015

Food security is a global concern amongst scientists, researchers and policy makers. No country is self-sufficient to address food security issues independently as almost all countries are inter-dependent for availability of plant genetic resources (PGR) in their national crop improvement programmes. Consultative Group of International Agricultural Research (CGIAR; in short CG) centres play an important role in conserving and distributing PGR through their genebanks. CG genebanks assembled the germplasm through collecting missions and acquisition the same from national genebanks of other countries. Using the Genesys Global Portal on Plant Genetic Resources, the World Information and Early Warning System (WIEWS) on Plant Genetic Resources for Food and Agriculture and other relevant databases, we analysed the conservation status of Indian-origin PGR accessions (both cultivated and wild forms possessed by India) in CG genebanks and other national genebanks, including the United States Department of Agriculture (USDA) genebanks, which can be considered as an indicator of Indian contribution to the global germplasm collection. A total of 28,027,770 accessions are being conserved world-wide by 446 organizations represented in Genesys; of these, 3.78% (100,607) are Indian-origin accessions. Similarly, 62,920 Indian-origin accessions (8.73%) have been conserved in CG genebanks which are accessible to the global research community for utilization in their respective crop improvement programmes. A total of 60 genebanks including 11 CG genebanks have deposited 824,625 accessions of PGR in the Svalbard Global Seed Vault (SGSV) as safety duplicates; the average number of accessions deposited by each genebank is 13,744, and amongst them there are 66,339 Indian-origin accessions. In principle, India has contributed 4.85 times the number of germplasm accessions to SGSV, in comparison to the mean value (13,744) of any individual genebank including CG genebanks. More importantly, about 50% of the Indian-origin accessions deposited in SGSV are traditional varieties or landraces with defined traits which form the backbone of any crop gene pool. This paper is also attempting to correlate the global data on Indian-origin germplasm with the national germplasm export profile. The analysis from this paper is discussed with the perspective of possible implications in the access and benefit sharing regime of both the International Treaty on Plant Genetic Resources for Food and Agriculture and the newly enforced Nagoya Protocol under the Convention on Biological Diversity. © 2015 Jacob et al.


Uchendu E.E.,Oregon State University | Uchendu E.E.,University of Guelph | Muminova M.,Tashkent Institute of Chemical Technology | Gupta S.,Tissue Culture and Cryopreservation Unit | Reed B.M.,National Clonal Germplasm Repository
In Vitro Cellular and Developmental Biology - Plant | Year: 2010

Regrowth of plants after cryopreservation varies, and resulting regrowth ranges from poor to excellent. Oxidative stress is a potential cause of damage in plant tissues. Antioxidants and anti-stress compounds may improve regrowth by preventing or repairing the damage. Lipoic acid (LA), glutathione (GSH), glycine betaine (GB), and polyvinylpyrrolidone (PVP) were tested during cryopreservation of shoot tips using the plant vitrification solution 2 (PVS2) protocol. Two in vitro-grown blackberry cultivars were cold acclimated and then cryopreserved in liquid nitrogen (LN). The antioxidant and anti-stress compounds were added at four critical steps of the protocol: pretreatment, loading, rinsing, and regrowth. Three out of the four compounds significantly improved regrowth of cryopreserved shoot tips. Regrowth ranged from 40% to 50% for controls to >80% for treated shoot tips. LA (4-8 mM) produced high regrowth at pretreatment, loading, and rinsing for 'Chehalem' and at all steps for 'Hull Thornless'. Recovery improved at all steps with GSH (0.16 mM) and GB (10 mM). PVP had a neutral or negative impact on regrowth. Overall addition of LA, GSH, and GB improved regrowth by ̃25% over the shoot tips cryopreserved using the regular PVS2 protocol (control). This study shows that adding non-vitamin antioxidants and anti-stress compounds during the PVS2-vitrification protocol improves regrowth of shoot cultures following cryopreservation. We recommend inclusion of antioxidants as part of standard cryopreservation protocols. © 2010 The Society for In Vitro Biology.


Kumar S.,Tissue Culture and Cryopreservation Unit | Nair K.N.,CSIR - Central Electrochemical Research Institute | Jena S.N.,CSIR - Central Electrochemical Research Institute
Genetic Resources and Crop Evolution | Year: 2013

Molecular differentiation in 24 accessions representing 19 taxa of Indian Citrus has been examined through sequence analysis of Internal Transcribed Spacer (ITS) region of nrDNA. Sequence length in the 24 accessions of Citrus taxa ranged from 512 to 665 bp (ITS1 & ITS2 partial and 5.8S complete sequence). The ITS sequences were very rich in G+C content ranging from 61.40 to 66.60% with an average of 64.2%. Genetic distance within Citrus group ranged from 0 to 13.4% with an average of 4.6%, showing moderate rate of nucleotide divergence. The phylogeny was inferred using the Maximum parsimony (MP) and Neighbor-Joining (NJ) methods. Both MP and NJ trees separated all the 24 accessions of Citrus into six distinct clusters. The disposition of all the accessions of Citrus in separate clusters in ITS-derived dendrograms was partly in accordance with the morpho-taxonomic affinities of the target taxa. This study supports the concept of Citrus medica (citron), C. reticulata (mandarin), and C. maxima (pummelo) as the basic species of the genus. However, ITS marker could not find any clear cut differentiation between subgenera Citrus and Papeda as proposed in Swingle's Citrus classification system. The present study also supports the distinctiveness of C. indica (Indian wild orange), C. latipes (Khasi papeda) and C. hystrix (Melanesian papeda) as true species, besides elucidating the probable hybrid origin and relationships among the cultivated species/biotypes, such as Citrus ×aurantiifolia (sour lime) C. ×limon (lemon), C. ×taitensis (Indian rough lemon), C. limettioides (sweet lime), C. ×aurantium (including sour and sweet oranges and grapefruit), and other indigenous varieties of Indian origin: C. megaloxycarpa (sour pummelo), C. karna (karna orange), C. pseudolimon (Hill lemon), 'Memang athur', 'Pummelo-lemon' and 'Kathairi nimbu'. © 2012 Springer Science+Business Media Dordrecht.


Gupta S.,Tissue Culture and Cryopreservation Unit
Acta Horticulturae | Year: 2011

In vitro grown shoot tips of Morus alba, M. indica, M. sinensis and Pyrus cossonii Rehder were cryopreserved by the encapsulation-dehydration technique. In Morus spp. the shoot tips were excised from 2 week cold acclimated in vitro plantlets. Encapsulated shoot tips were pretreated in 0.1, 0.3, 0.5 or 0.75 M sucrose for 20 h; desiccated for 6 h under laminar air flow, plunged in liquid nitrogen and rapidly warmed. The combination of osmotic dehydration in 0.75 M sucrose and air dehydration for 6 h produced 50-80% regrowth. However, cryopreserved shoot tips howed 30-35% regrowth when dehydrated to ~20% moisture content. The encapsulation dehydration method was suitable for mulberry cryopreserved shoot tips with reasonable recovery in M. indica (35%), M. alba (35%) and M. sinensis (30%). In P. cossonii Rehder, the in vitro plantlets were cold acclimated for 1 or 3 weeks. Encapsulated shoot tips were pretreated in 0.75 M sucrose for 20 h. The beads were desiccated for 4 h on silica gel, then plunged in liquid nitrogen and rapidly warmed. Osmotic dehydration produced 60% regrowth. Cold acclimation was effective in regrowth of cryopreserved shoot tips. Three week cold acclimated shoot tips resulted in 40% recovery, while there was no regrowth in non- or 1 week cold acclimated shoot tips after liquid nitrogen treatment.


Gupta S.,Tissue Culture and Cryopreservation Unit
Acta Horticulturae | Year: 2011

India is enormously rich in temperate fruit genetic resources. In the north, the temperate Himalayas stretch from Jammu and Kashmir to the north-eastern hill region, and in the south the Nilgiri hills also harbour a vast genetic diversity of temperate fruits, including apple, pear, peach, apricot, plum, walnut, almond, pecan nut, hazelnut, chestnut, berries, and several of their wild relatives. The National Bureau of Plant Genetic Resources is the nodal organization for exchange, quarantine, collection, conservation, evaluation and documentation of plant genetic resources. Numerous germplasm accessions comprising broad genetic diversity in temperate fruits were introduced from other countries. Many introduced cultivars have been used directly for large scale cultivation, for example, the 'Red Delicious' group of cultivars in apple, etc. Diverse temperate fruit crops have been collected, characterized and evaluated; promising germplasm has been identified and utilized in crop improvement programmes. The germplasm is conserved through complementary in situ and ex situ strategies. The wild temperate fruit species are conserved in protected areas and national reserves. The National Genebank at NBPGR has a large ex situ conservation facility including field genebanks, seed genebank, in vitro multi-crop repository, and cryobank. Important temperate fruit accessions are being conserved in the field genebanks; however, germplasm remains under the threat of loss due to pestpathogen attack, natural calamities and climate change. A back-up in vitro collection includes more than 300 exotic and indigenous accessions of Actinidia, Fragaria, Malus, Morus, Prunus, Pyrus, Rubus and Vaccinium. Future strategies must include monitoring of in situ genetic diversity loss due to climate change, identification of trait-specific germplasm such as low-chilling, biotic and abiotic stress resistance, systematic conservation and efficient utilization. This review focuses on the present status of temperate fruit genetic resources, their management and future perspectives.


Gupta S.,Tissue Culture and Cryopreservation Unit
Acta Horticulturae | Year: 2011

India is one of the most significant and unique countries in the world from the point of view of fruit genetic resources and fruit diversity. Over 300 species of fruits, including temperate, subtropical and tropical, are growing in the country. Among the pome fruits, in addition to cultivated fruits like apple and pear, a wide range of wild, temperate pome fruits occur in the Indian Himalayas. These include several species of Malus, Pyrus, Sorbus, Cydonia, Cotoneaster, Crataegus, Pyracantha, Diospyrus and Docynia. Several of these species are being maintained in field genebanks or orchards. However, with changing climatic conditions, especially in the Himalayas, some of the temperate field genebanks must shift to new locations with more favourable conditions for growth and reproduction. In situ sites need to be monitored for loss and migration of germplasm. A formal unit must be established in the institutes to monitor and analyze climate change impacts in this area especially to track the loss and movement of diversity. With the growing purchasing power and curiosity of Indian consumers, there are emerging opportunities to strengthening the commercialization of underutilized pome fruits with high nutritional potential and taste appeal. Poor production and postharvest handling procedures practiced in the region lead to heavy losses of traditional pome fruit products, while weak infrastructure handicaps marketing prospects. Solutions to these problems lie in the establishment of organized systems of production and suitable postharvest handling procedures, including controlled atmosphere storage facilities, refrigerated transport system, good roads, etc. Exploitation of underutilized pome fruit crops will not only benefit India, but also cater to the increasing demand for exotic products in the international market.


Chaudhury R.,Tissue Culture and Cryopreservation Unit | Malik S.K.,Tissue Culture and Cryopreservation Unit | Rajan S.,Central Institute for Subtropical Horticulture
Cryo-Letters | Year: 2010

An improved method for pollen collection from freshly dehiscing anthers of mango (Mangifera indica L.) and litchi (Litchi chinensis Sonn.) using the organic solvent cyclohexane has been devised. Using this method pollen quantity sufficient for large scale pollinations could be collected and stored for future use. Transport of pollen in viable conditions over long distances, from site of collection (field genebank) to cryolab was successfully devised for both these fruit species. Cryopreservation was successfully applied to achieve long-term pollen storage over periods of up to four years. Pollen viability was tested using in vitro germination, the fluorochromatic reaction (FCR) method and by fruit set following field pollination. On retesting, four year cryostored pollen of different mango and litchi varieties showed high percentage viability as good as fresh control pollens. Pollens of more than 180 cultivars of mango and 19 cultivars of litchi have been stored in the cryogenebank using the technology developed, thus facilitating breeding programmes over the long-term.


Malik S.K.,Tissue Culture and Cryopreservation Unit | Chaudhury R.,Tissue Culture and Cryopreservation Unit
Seed Science and Technology | Year: 2010

Wild apricot (Prunus armeniaca L.) is an economically important fruit crop with rich germplasm variability existing in the high altitude and cold desert areas of India. Due to the vast economic importance of this fruit species and availability of diverse genetic resources, there is an urgent need to collect, characterize and conserve the existing genetic variability of apricot for safe protection and utilization. A cryopreservation protocol for seeds, half seeds and embryonic axes has been successfully devised using desiccation-followed by fast-freezing with high recovery growth percentages. Whole seeds showed viability in the range of 82.5 to 92.5%, half seeds from 85 to 100% and embryonic axes from 98.5 to 100%. The excised embryonic axes showed the highest values of germination in both unfrozen and frozen samples. A total of 250 accessions belonging to 28 folk cultivare have been successfully cryostored as a base collection in the National Cryogenebank at NBPGR, New Delhi, India. The conserved germplasm represents the invaluable variability existing in both cultivated and wild populations of apricot present in India.


Gupta S.,Tissue Culture and Cryopreservation Unit
Acta Horticulturae | Year: 2014

A range of cryopreservation techniques for conserving plant cells and tissues have been developed and tested in the last four decades and thus significantly progressed routine storage of plant germplasm in liquid nitrogen. Protocols are available to cryopreserve in vitro derived explants such as shoot tips, meristems, somatic embryos, hairy roots, cell suspensions in various plant species. Amongst the available protocols, cryopreservation of shoot apices is the most common method for long-term ex situ conservation of clonally propagated plants, which is increasingly being used to conserve germplasm of various herbaceous and woody plant species. The commonly applied vitrification-based cryopreservation techniques include vitrification, encapsulation-dehydration, encapsulation-vitrification and dropletvitrification. Vitrification involves the application of cryoprotectant solutions that increase cell viscosity to a critical point at which water forms a meta-stable glass on exposure to ultra low temperatures while ncapsulation-dehydration involves removing cell water through osmotic and evaporative dehydration to achieve the same state. The encapsulation-dehydration procedure is based on the technology developed for the production of artificial seeds and established to cryopreserve potato shoot tips for the first time. Explants are encapsulated in alginate beads, pregrown in sucrose-enriched medium for 1-7 days, partially desiccated in the air current of a laminar airflow cabinet or with silica gel to get moisture content around 20% on the fresh weight basis, then frozen rapidly. Recovery of cryopreserved samples is generally rapid and direct, without callus formation. This technique has been applied to apices of numerous species from temperate and tropical origin for example, dicots: apple, pear, blackberry, raspberry, mulberry, Eucalyptus, Melia, Robinia, mint; monocots: Cynodon, Zoysia and Lolium grasses, sugarcane, yam, lily and banana; and to cell suspensions and somatic embryos of several species. Using encapsulation-dehydration technique is advantageous because no special equipment is needed, and because of the use of nontoxic cryoprotectant. The disadvantage include requirement of handling each bead several times, and some plants may not tolerate the high sucrose concentrations. We have cryopreserved wide range of germplasm of Morus, Pyrus, Rubus using encapsulation-dehydration technique in the In Vitro Genebank at National Bureau of Plant Genetic Resources, New Delhi, India. The paper discusses various aspects of cryobanking of in vitro-grown shoot tip explants using encapsulation-dehydration technique.


Sundararaj S.G.,Tissue Culture and Cryopreservation Unit | Agrawal A.,Tissue Culture and Cryopreservation Unit | Tyagi R.K.,Tissue Culture and Cryopreservation Unit
Scientia Horticulturae | Year: 2010

Synseeds of ginger (Zingiber officinale) were produced using aseptically proliferated 2-week old encapsulating explants (microshoots) upon complexation of 4% sodium alginate prepared in Murashige and Skoog (1962) medium (MS) and 100. mM calcium chloride. Conversion of synseeds into plantlets (conversion) was recorded as 66% and 53% on MS (3% sucrose) and on MS (3% sucrose). +. 2.5. mg/l BA media, respectively. However, shoots/synseed were significantly higher on MS (3% sucrose). +. 2.5. mg/l BA medium. For short-term storage of germplasm, sucrose-dehydrated synseeds were found better than air-dehydrated or fresh synseeds. Synseeds dehydrated in 0.25. M sucrose liquid medium for 16. h and stored in cryovials (with out medium) at 25°C for 8 weeks and 12 weeks exhibited 53% and 13% conversion, respectively, on MS (3% sucrose). +. 2.5. mg/l BA medium. Plantlets obtained from stored synseeds were hardened, established successfully ex vitro and were morphologically similar to each other as well as their mother plants. This synseed protocol could be useful for short-term storage and exchange of germplasm of ginger between national as well as international laboratories. © 2010 Elsevier B.V.

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