Trees are fundamental to ecosystems, supporting biodiversity, regulating climate, storing carbon and providing vital resources. Several species are under severe threat from deforestation, habitat destruction, urban expansion and climate change, endangering both the species and the ecosystem services they provide. Traditional conservation methods, such as seed genebanks and field genebanks, face challenges like genetic degradation and high maintenance costs. Cryopreservation offers a promising alternative, preserving genetic material at ultra-low temperatures, halting biological processes and minimizing storage space needs. This method is especially effective for species with recalcitrant seeds or difficulty in propagation. Integrating techniques like somatic embryogenesis and organogenesis further enhances cryopreservation. Somatic embryogenesis enables the development of embryos from somatic cells, facilitating the regeneration of trees from cryopreserved tissues. Similarly, organogenesis promotes the development of in vitro cultures for species that are otherwise challenging to conserve, allowing for cryopreservation of in vitro explants. The combination of these techniques not only preserves genetic material but also ensures the regeneration of viable plants, providing a comprehensive approach to tree conservation. This review highlights the critical role of cryopreservation in preserving tree diversity, with a focus on somatic embryogenesis and organogenesis, exploring current practices, challenges and future directions.

India is a treasure trove of biological diversity with its plant genetic resources playing a crucial role in the crop improvement serving as the foundation for the country’s sustainable food and nutritional security. India’s in vitro genebank (IVG) is part of the National Genebank at the Indian Council of Agricultural Research-National Bureau of Plant Genetic Resources (ICAR-NBPGR). The IVG houses distinctive multi-crop repository that utilizes several tissue culture techniques for short- to medium-term storage in in vitro active genebank (IVAG) and cryoconservation approaches for long-term storage in in vitro base genebank (IVBG). In IVAG, germplasm is conserved under normal and slow growth conditions with a subculture period of 1–36 months depending on the species/genotype and conservation approach. Currently, the IVAG holds 2,038 germplasm accessions (69 genera and 171 species) from six crop groups, viz. (a) tropical fruit crops (449), (b) temperate and minor tropical fruit crops (408), (c) tuber crops (530), (d) bulbous and ornamental crops (187), (e) medicinal and aromatic plants (232), and (f) spices and industrial crops (232). For long-term conservation, in vitro produced explants of various species are cryopreserved at the IVBG in liquid nitrogen. Utilizing various cryoconservation procedures, 347 accessions from several crop groups have been successfully conserved in the IVBG. Over the past four decades, in vitro conservation has been accomplished by the above mentioned cutting-edge techniques. This report highlights the efforts and achievements of the National Genebank in conserving horticultural genetic resources through in vitro and cryoconservation.

Cryopreservation serves as an invaluable technique for safeguarding the genetic diversity of plants and various organisms, while also facilitating fundamental biological research. Despite notable advancements in this field, the cryopreservation of certain cell types and tissues remains challenging, particularly those that exhibit sensitivity to low temperatures. Two-celled pollen is a promising model system for the study of cryopreservation. By exploring the cryopreservation of two-celled pollen, deeper insights can be gained into the cellular and molecular mechanisms of cryoinjury and recovery. This knowledge can be used to develop new and improved cryopreservation protocols for a wider range of cell types and tissues. It is relatively simple, consisting of only two cells, and it is relatively easy to cryopreserve and culture. In addition to its potential for improving cryopreservation technologies, the study of two-celled pollen cryopreservation can also shed light on fundamental biological processes such as cell division, development, and stress tolerance. By unlocking the mysteries of two-celled pollen cryopreservation, we can gain a deeper understanding of nature’s inner workings. This article reviews examples of studies that have successfully used two-celled pollen cryopreservation, highlighting key findings and discoveries enabled by this technique as case studies.

Plant genetic diversity (PGD) plays a significant role in attaining food, nutrition and economic security. Conservation of these valuable resources is essential in the current scenario of climate change and sixth mass extinction. Since the early twentieth century, conservation of PGD has been carried out by using in situ and ex situ methods. Advances in biotechnology have opened new avenues in the fields of PGD management, with several tools applied in varied activities viz., collecting, quarantine, multiplication, conservation, characterization and evaluation, exchange and utilization. Since the past three decades, significant progress has been made in the use of biotechnology for conserving 'difficult-to-conserve' species such as clonally propagated crops, rare and endangered species and crop wild relatives. Several protocols have been developed for in vitro collecting, micropropagation, normal growth, slow growth and cryopreservation in varied species. The present chapter provides the current status of application of various biotechnological tools for the conservation of PGD.

The advancement of Lathyrus through genetic enhancement and cultivar development faces challenges due to limited knowledge about their reproductive biology. Thus, the present study systematically investigates the reproductive biology, phenology, receptivity of stigma, germination and viability of pollen in Lathyrus sativus L. and L. cicera L. Experiments were carried out using 20 accessions of L. sativus and three accessions of L. cicera. Anther dehiscence and stigma receptivity were determined through microscopic observation and hydrogen peroxide test, respectively. Pollen viability was assessed through in vitro pollen germination and fluorescein diacetate (FDA) test. Flowers were found to be bisexual and protogynous, with self-pollination favored by the cleistogamous nature of flowers. Five flower-development and three pod-development stages were classified in both species. The timing of all stages differed significantly among the accessions studied. Stigma receptivity was observed to occur 2–4 days before anther dehiscence and persisted until flower wilting. The study reveals that L. sativus and L. cicera are primarily self-pollinated, underscoring the critical timing of emasculation for successful manual crossing in breeding, ultimately enhancing pod and seed set, and facilitating plant breeders in optimizing hybridization programs.

BACKGROUND: Long-term storage of cowpea pollen is important for the fertilization of spatially or temporally isolated female parents, especially during cowpea crop improvement and wide hybridization programs. OBJECTIVE: Experiments were conducted to determine pollen longevity at different storage temperatures and to develop a cryopreservation protocol for pollen of different cowpea accessions. MATERIALS AND METHODS: The investigation was carried out at the Research Farm of ICAR-NBPGR, New Delhi, India, during the kharif (rainy) season of 2022. Pollen viability was studied after storage for 1, 3, 6, 12 and 24 h, 1 and 2 weeks and 1, 3 and 5 months at three different temperatures (4, -20 and -196 degrees C). RESULTS: Fresh pollen viability ranged from 78 to 91 %. The optimal pollen moisture content was 12-14 % and the optimal air desiccation period under the laminar air flow chamber (22 +/- 1 degrees C) was 5 min for subsequent preservation at -196 degrees C. Pollen viability was lost completely at 4 and -20 degrees C after 1 and 2 weeks of storage, respectively. Pollen stored in liquid nitrogen (-196 degrees C) retained vaiblity similar to that of fresh pollen for > 5 months storage. Pollination using cryostored pollen resulted in normal fertilization. CONCLUSION: This finding opens a gateway for cowpea haploid germplasm conservation and wide hybridization programs.

The plant genetic resources (PGR) of a country are vital for its sustainable agriculture and food security. The importance of trees has been recognized by humanity since ancient times. However, changing climate and genetic erosion are causing rapid loss of diversity indicating an urgent need to conserve and maintain genetic resources for food security. India is the second largest producer of fruits and vegetables in the world. An analysis of the status of diversity, distribution, and conservation of fruit genetic resources (FGR) in India was carried out to determine the opportunities and challenges in the management of fruit germplasm for sustainable food security. The objectives of this article include (i) tracing fruit crop diversity and distribution in India (ii) discussing the potential of wild relatives of indigenous fruit crops; (iii) reviewing conservation strategies in the Indian and global context (iv) current conservation status of fruit crops (iv) analysing constraints in germplasm conservation and utilization; (v) and proposing new ways to safeguard FGR. Methods employed include database retrieval, literature review, and communication with key informants. India is home to several fruit species of global importance including mango, banana, and some citrus, and holds approximately 148 crop wild relatives (CWR) of fruits. The first gene sanctuary of wild citrus species was established in the Garo Hills of Meghalaya and efforts are being made to also establish gene sanctuaries for Musa species and mango. Over 13,000 accessions of fruit crops are conserved in field gene banks of seven regional stations of the Indian Council for Agricultural Research- National Bureau of Plant Genetic Resources (ICAR-NBPGR) and 10 National Active Germplasm Sites working under the National Agricultural Research System. The ICAR-NBPGR maintains some 4,000 accessions in its cryobank as seed, embryo, and embryonic axes as explants; some 800 accessions in the in vitro repository; and about 100 accessions in the in vitro base gene bank belonging to tropical, temperate, and minor fruits. By contrast to field crops, the conservation of fruit crops poses unique challenges; this is because these collections comprise only 6

Medicinal plants possessing antiallergic properties are extensively used in several medicine systems across different countries. However, it is crucial to conserve these species ex situ in field genebanks (FGs), botanical gardens, seed genebanks, in vitro genebanks, and cryogenebanks because many of them are threatened in their native environments. While species producing orthodox seeds can be conserved in seed genebanks, others which are clonally or vegetatively propagated, produce recalcitrant or intermediate seeds, or belong to threatened and wild species pose challenges in collecting sufficient seeds. For such species, maintaining in FGs, clonal repositories, botanical gardens, and arboretums comes with uncertainties. Tissue culture methods therefore present a viable choice for their conservation. In the past four decades, biotechnological tools (in vitro) have been used for commercial propagation, evaluation, utilization, and repatriation of these species into their natural habitats. Then the present chapter presents a detailed overview of the progress and current state of in vitro techniques for safeguarding these species. The in vitro Genebanks facilitate short- to medium-term conservation under normal growth conditions in standard culture rooms, and slow growth conservation strategies in the in vitro Active Genebank (IVAG). Furthermore, long-term conservation is achieved through cryoconservation of in vitro-derived explants in the in vitro Base Genebank (IVBG). These techniques ensure the conservation of medicinal plants with antiallergic properties, safeguarding their genetic diversity and potential for future use.

Plant genetic resources are the most valuable and essential basic raw materials to meet the current and future needs of crop improvement programs to enhance the genetic base of a crop. With erosion playing a crucial role in diminishing diversity, different conservation methods come to play a massive role in preserving these resources. Thus, in the present paper, the authors tried to provide a gist about the on-farm conservation activities practiced in India and the importance of on-farm diversity in terms of economic importance. Different key words like on-farm conservation, landraces, custodian farmers, on-farm diversity were used to collect the different articles on on-farm conservation. Research articles from different states and regions around the country were gathered to collate the data and knowledge. In managing the key farm resources, the role of community seed banks, kitchen gardens, custodian farmers, their communities and organizations has been highlighted, along with a few case studies of on-farm conservation activities across the sites in the country. With the collection and documentation of on-farm material for genebanks and databases, it may provide easy access to on-farm material for potential users in the future. Providing incentives, identifying key problems, and providing necessary facilities may sustain on-farm conservation.

The Lens culinaris Medik is a cultivated annual diploid (2n = 2x = 14) self-pollinated species. The genetic base of the cultivated lentil is narrow compared to its wild relatives. Crop wild relatives have enormous untapped genes for several desirable traits viz., high yield, biotic and abiotic stresses, and nutrition. Though efforts have been made to transfer desirable genes from wild lentils belonging to different gene pools to cultivated lentil backgrounds, success has been hampered due to crossability barriers in many cases. However, modern biotechnological tools are needed to use for exploiting the potential of distantly related wild relatives having desirable traits. The existing variability among cultivated lentil germplasm has been exploited to reach a desirable level of productivity. However, to attain further breakthroughs in increasing yield and improving stability in future cultivars, new sources of genes/alleles need to be identified and incorporated into cultivated varieties. These efforts can help to develop multiple stress-tolerant, climate-resilient, and low-input requirement varieties that will ultimately contribute towards sustainable agriculture production.

Medicinal plants have long been integral to diverse traditional medicine systems, with many exhibiting remarkable antiallergic properties. However, several valuable herbs face threats from overexploitation, unregulated harvesting, unsustainable cultivation practices, and climate change impacts. This growing awareness of the need to conserve these plants has led to increased focus on ex situ conservation strategies, including field genebanks, botanical gardens, seed genebanks, in vitro genebanks, and cryogenebanks. While field genebanks and botanical gardens expose species to potential risks like pest infestations and natural disasters, seed conservation in seed genebanks and in vitro genebanks is effective for certain species. However, for exceptional plant species characterized by limited seed production, challenging seed collection, and seeds that cannot withstand conventional drying and storage in seed genebanks, cryogenebank conservation emerges as the most viable option. Various cryopreservation techniques, encompassing the preservation of whole seeds, zygotic embryos, embryonic axes, and dormant buds, offer the potential to store these species theoretically indefinitely in cryogenebanks. This chapter provides a comprehensive overview of research on cryopreservation methods for seeds and other explants of medicinal plant species exhibiting antiallergic properties. Additionally, several successful case studies are presented.

Soybean is an important oilseed crop, known to be fourth most cultivated crop globally, contributing to approximately 53% of total oil production. As a rainfed crop, soybean is particularly susceptible to the impacts of climate change. Climate change is expected to result in higher temperatures, elevated CO2 levels and altered rainfall pattern. As per IPCC Synthesis Report 2023, climate change may increase global temperatures by 1.5°C between 2021 and 2040 under high-emission scenarios. Without substantial mitigation efforts, the consequences could be catastrophic, leading to a 3.6-4.4°C rise in global temperatures and CO2 concentrations could rise to levels 2-4 times higher than those recorded in the past 0.8 million years, resulting in unprecedented climate changes. This climate change (elevated CO2) is found to have a positive impact on soybean seed yield (increase 32-37%) under weed-free conditions, however, under weedy condition seed yield of soybean may be reduced by 30% by C3 weeds and 45% by C4 weeds. Thus, C4 weeds are more competitive to C3 crops such as soybean under climate change condition. Elevated temperature was found to have more direct and positive impact on growth of most of the weed species, while it negatively impacted the soybean growth and yield parameters. However, interaction effect of CO2 and temperature was beneficial to both weeds and soybean. Apart from this, interaction of rainfall and temperature play a critical role in soybean productivity, where the simulation study advocates that increase in 1°C temperature with rainfall remaining constant, leads to a decline in productivity by 10-15%. Anticipating potential damage from weed to soybean is crucial for formulating effective and sustainable weed management strategies. Therefore, it is vital to address soybean-weed interactions and weed management in the context of climate change, as there has been inadequate research conducted in this area.

Piper nigrum L. (Piperaceae), commonly known as black pepper, is a globally cherished spice and a key player in the spice trade. This review article discusses the significance of black pepper, its diverse applications, and the need for its genetic resource conservation. Importantly, it provides the ‘trans situ’ approach being followed in India for safe conservation of P. nigrum germplasm. This encompasses various methods of ex situ conservation, including field genebanks, seed genebanks, in vitro genebanks, and cryo genebanks. In situ conservation efforts involving on-farm practices by ‘custodian farmers’ are also provided. The review showcases the extensive efforts in India to conserve black pepper genetic resources through collaborative initiatives among research institutions, universities, and farming communities. Research gaps in terms of in vitro cryopreservation have been identified. Overall, this article underscores the critical importance of preserving black pepper genetic diversity to safeguard its future and support ongoing agricultural, research, and breeding endeavors.

Landraces are important genetic resources that have a significant role in maintaining the long-term sustainability of traditional agro-ecosystems, food, nutrition, and livelihood security. In an effort to document landraces in the on-farm conservation context, Central Western Ghat region in India was surveyed. A total of 671 landraces belonging to 60 crops were recorded from 24 sites. The custodian farmers were found to conserve a variety of crops including vegetables, cereals and pulses, perennial fruits, spices, tuber and plantation crops. The survey indicated a difference in the prevalence of landraces across the sites. A significant difference with respect to the Shannon-diversity index, Gini-Simpson index, evenness, species richness, and abundance was observed among the different survey sites. Computation of a prevalence index indicated the need for immediate intervention in the form of collecting and ex situ conservation of landraces of some crops as a back-up to on-farm conservation. The study also identified the critical determinants of on-farm conservation, including (i) suitability to regional conditions, (ii) relevance in regional cuisine and local medicinal practices, (iii) cultural and traditional significance, and (iv) economic advantage. The information documented in this study is expected to promote the collection and conservation of landraces ex situ. The National Genebank housed at ICAR-NBPGR, New Delhi conserves around 550 accessions of landraces collected from the Central Western Ghats region surveyed in this report. Information collected from custodian farmers on specific uses will be helpful to enhance the utilization of these accessions.

Grasspea (Lathyrus sativus L.) is an underutilized pulse crop which can withstand adverse environmental conditions such as drought, heat, salinity, waterlogging, pest and diseases. To facilitate hybridization studies, pollen cryoconservation was attempted in L. sativus. Among the different media combination tested for in vitro pollen germination, BK medium with 15% sucrose was optimal. Pollen viability was tested at different storage regimes, viz. 25, 4, -20 and -196 ºC at different storage duration (1, 3, 5, 7, 9, 24 h, 1 week, 2 week and 6 months). For long-term pollen cryopreservation, an ideal MC of 14–16% was achieved by desiccating the pollen for 10 min in a laminar air flow (LAF) chamber. Negative correlation was found between pollen viability, storage duration and temperature (25, 4 and -20°C). Cryopreserved (-196°C) pollen showed significantly higher viability compared to all the other storage conditions. The standardized cryoconservation protocol was applied to 20 accessions of Lathyrus sativus for conservation up to six months without loss of pollen viability. Successful fertilization, fruit and seed set was observed in cross combinations attempted using cryoconserved pollen without any incompatibility barriers. Hence, the present protocol can be used for long-term cryoconservation of L. sativus pollen. The development of an effective long-term storage method of Lathyrus sativus pollen ensures the availability of pollen for grasspea breeding throughout the year.

Dahlias are the one amongst the tuberous herbaceous plants, valued for their attractive colorful flowers with varied size cultivated due to its ornamental value as a potted plant or cut flower in many countries. Dahlia germplasm was being conserved in the in vitro Genebank of ICAR- NBPGR, New Delhi since 2005-06 under normal growth and slow growth with a regular subculturing in every 6 months and 1 year respectively, which is laborious and time-consuming. Hence, in the current study we attempted to develop an efficient dropletvitrification cryopreservation protocol for safe and long-term conservation of dahlia germplasm. Significant effect of type and position of explants, plant vitrification solution-2 (PVS2) dehydration duration, pregrowth period and loading solution treatment duration on post-thaw regrowth after cryoconservation was observed. Shoot tips, each being 1.0 mm in length and 0.5 mm in width positioned at the tip of in vitro shoots with a similar developmental stage (similar shoot length) were excised from in vitro cultures pregrown for 6 wks on B5 basal medium supplemented with 0.5 mg/l BAP, 30 mg/l silver nitrate and 3% sucrose. The excised shoot tips were precultured on MS + 0.3 M sucrose for overnight followed by loading solution (2.0 M glycerol and 0.4 M sucrose in liquid MS medium) treatment for 80 min, PVS2 dehydration for 30 min and then transferred onto PVS2 droplets on aluminum foil strips and cryostored in liquid nitrogen (LN). An average of 44.44% post-thaw regrowth was obtained in six accessions tested and no significant difference among the accessions was observed. There was no significant loss in regrowth of cryostored shoot tips after 1 year and 6 years of cryobanking. This justifies the genotype-wide and sustainability of the protocol for cryoconservation of Dahlia germplasm using droplet-vitrification cryopreservation procedure in the cryobanks. To the best of our knowledge, this is the first report on cryoconservation of Dahlia germplasm.

Background: Pigeon pea is an important dietary protein source for humans but the production was constrained by various biotic and abiotic factors. Breeding strategies were followed to improve yield and developing high yielding varieties but at the same time utilization of genetic resources have declined. Pigeon pea is native to India with huge natural genetic variability in the local germplasm and its wild relatives. So it is necessary to identify and select breeding material from germplasm with considerable genetic and morphological variability to utilize in breeding programmes. As an initial study, 200 pre-breeding lines developed were evaluated for morphological variability patterns.Methods: A total of two hundred lines selected from F4 generation of pigeon pea developed at ICAR-NBPGR were evaluated in Randomized Block design (RBD) during 2014-2015 kharif season under Indo-Swiss collaboration in Biotechnology at Agricultural College and Research Institute, Madurai (TNAU). The accessions found to be superior in seed yield than the local check APK1were forwarded to the next generation (2015-2016) for assessment of genetic variability, heritability, genetic advance and association studies.Result: Qualitative traits were evaluated and variation in leaflet shape, stem colour, pattern of streaks and base seed colour were observed. All tested lines expressed greater variability for most of the traits. Maximum coefficient of variation was observed for number of pods per plant followed by number of primary branches per plant. Selection of traits with moderate heritability coupled with high genetic advance like number of pods per plant, number of primary branches per plant could help in crop improvement program. Seed yield was positively correlated with number of seeds per pod, number of pods per plant and hundred seed weight. Potential genetic stocks and donors for high yield were selected based on hundred seed weight and seeds per pod. The accessions superior in number of pods and seed yield than check were forwarded to next generation for assessment. The identified trait-specific accessions will help in future breeding program.

This study aimed to develop a long-term pollen storage protocol for Luffa species (L. acutangula, L. cylindrica, L. echinata, and L. graveolens) and assess its potential for crop improvement. The optimal medium for in vitro pollen germination varied by species, with Brewbaker and Kwack (BK) medium with 10% sucrose suitable for L. acutangula, L. cylindrica, and L. echinata, and BK medium with 3% sucrose ideal for L. graveolens. Overestimation in staining tests compared to in vitro pollen germination was observed. The best results for cryopreservation were achieved with desiccation periods of 20, 30, and 40 min, maintaining moisture content between 14.04% and 18.55%. Pollen viability was negatively correlated with storage temperature (25, 4, and −20°C) and duration. Cryopreserved pollen at −196°C exhibited the highest viability over a prolonged period (2 months) and was comparable to fresh pollen in terms of germination, ovule fertilization, and fruit and seed set. This study presents a simple and reproducible pollen cryopreservation protocol applicable across Luffa species, facilitating long-term storage and its use in crop improvement efforts.