Overview of In vitro Strategies to Conserve Rare and Endangered Plant Species in

India is one of the seventeen mega-diverse countries in the world. The maximum diversity in India has been observed in the Eastern Himalaya, Eastern part of Western Himalaya and the Western Ghats with its endemism. However, due to intrinsic and extrinsic factors, there has been a reduction in the number of species that are threatened, rare and endangered. Therefore, to sustain the populations of these species, immediate action of conservation is required. Biotechnological approaches have been recognized as an efficient method to conserve the rare and endangered plant species. In the present study, a Google search was carried out along with other scientific databases such as PubMed, SciFinder, Web of Science, and Google Scholar to accomplish a scientific literature search and analysis. In this review, we have described a few potential in vitro methods to protect rare and endangered flowering plants such as tissue culture using different explants, nuclear-DNA assay, cold storage techniques and gene banks. These various strategies are known to have a significant role in the production of a number of plantlets with their quality traits which considerably shows the scope to accomplish the research gap.


Introduction
The diversity of flowering plants in India is predominantly intensified in 4 biodiversity hotspots, namely Eastern Himalayas, Northeast India and Andaman Islands (Indo-Burma), Nicobar Island (Sundaland) and Western Ghats, out of 34 biodiversity hotspots in the world (Arisdason and Lakshminarasimhan, 2020). In India, about 28% of plants are endemic to the country among which dicotyledonous dominate by 720 genera with 2984 taxa (74%) and monocotyledons are represented by 255 genera with 1061 taxa (26%) (Arisdason and Lakshminarasimhan, 2020). Along with the dominancy, the major flowering families contributing to 7% of the world's documentation are Poaceae, Orchidaceae, Leguminosae, Asteraceae, Rubiaceae, Cyperaceae, Euphorbiaceae and Acanthaceae (Ghosh et al. 2017).
These floristically significant areas exhibit an exceptional concentration of endemic species nonetheless experiencing the threats which lead to the higher occurrence of threatened plant species. There are extrinsic factors (Figure 1) such as degradation of habitat, loss of pollinators, overexploitation, introduction of invasive species, global warming, pollution etc. creates the ground for the endangered status of the species however intrinsic factors like poor seed setting and germination, habitat specificity, specialized pollinators, mutualism, mode of dispersal etc. leads to the same (Ravikanth et al. 2018).

Figure 1 Threats which lead to the occurrence of threatened plant species
Thus, there is a need to develop rapid micropropagation protocols to overcome the constraints of the same to conserve the endangered flora (Nagesh et al. 2010). The conservation can be done by using in-situ and/or ex-situ approaches although in-situ strategy may not be sufficient to conserve and/or to preserve such huge bio resources of endangered flora because of intrinsic or extrinsic factors. Thus, the application of in vitro methods would be helpful to recover and re-establish endangered plant species (Swamy et al. 2018). To accomplish the purpose of conservation of biodiversity, sustainable use of its components and equitable sharing of the benefits arising out of the utilization of biological resources India framed a National Policy and strategy on Biodiversity in 1999, as per the Convention on Biological Diversity (CBD) and built up National Biodiversity Action Plan (NBAP) which was broadly aligned to the global Strategic Plan for Biodiversity 2011-2020. India developed 12 National Biodiversity Targets correlated to the 20 Aichi Biodiversity Targets by the Conference of Parties to the CBD in 2010 at Nagoya, Japan through extensive stakeholder consultations and public outreach (NBAP, 2008).
According to the United Nations Food and Agricultural Organization (FAO), the global population would become near about 9.1 billion in 2050, thus there will be increase in the demand of food production up to 70% which ultimately raise the agricultural land uses and consequently leads to the global challenge of conserving biodiversity. Therefore, the International Treaty on Plant Genetic Resources for Food and Agriculture declared the contribution of farmers towards the diversity of crops, accessing and sharing plant genetic materials (Pathak and Abido, 2014).

Methods of Study
To study the various conservation strategies employed in India by in vitro methods, a thorough scientific literature search and analysis was carried out. For this, various search strategies have been prepared depending on the scientific database such as PubMed, SciFinder, Web of Science, and Google Scholar. These strategies were further run to collect, screen and analyzed a few relevant records ( Figure 2).

Biotechnological approaches
United Nations Environment Programme (UNEP) organized a body "Convention on Biological Diversity" (CBD) to develop a protocol to conserve the biological diversity. There are two methods of conservation of flora viz. in-situ and ex-situ conservation. In-situ conservation deals with the conservation of genetic variation in its natural habitat i.e. at original location itself, while ex-situ involves conservation of the species outside the native habitat. Most of the medicinal plant species became endangered because of the overexploitation of the species and are tried to be protected in-situ method, but this method is not sufficient and therefore ex-situ conservation in vitro techniques or biotechnological approaches are becoming increasingly important in the conservation (Jain et al. 2012).
Biotechnological approaches are very important in the rapid multiplication and conservation of the critical genotype species especially for the species having problems in germination of seeds, specialized pollinators requirement, mutualism, mode of dispersal, growth form, extremely reduced population etc. (Swamy et al. 2018). Conservation of endangered plant species involves preservation and maintenance of species with the use of biotechnological tools such as in vitro propagation, molecular conservation strategy, cryopreservation and gene bank ( The conservation of endangered flora using plant tissue culture techniques is highly accepted biotechnological approaches, which provides the possibilities of rapid multiplication of the species and conserve genetic material (Paunescu, 2009).

In vitro propagation
Plant tissue culture is a rapid and effective in vitro technique to regenerate or multiply the plants at large scale (Pathak and Abido 2014). Generally, tissue culture is done through the various explants like shoot tip, nodal segments, leaves, rhizome, roots, seeds etc. In vitro culture method is a powerful tool for multiplication, conservation and management of endangered species irrespective of season (Deb et al. 2018). In vitro protocols developed for endangered species in India highlights the significance of ex-situ conservation where reproduction through conventional methods is difficult to carry out and with very low population. Thus, tissue culture technology is used in the conservation of species (Table 1)  Due to various intrinsic as well as extrinsic factors like immense flower and seed abortion, poor seed germination, viability of seeds, overexploitation and habitat degradation the plant species such as Eremostachys superba and Shorea tumbuggaia have been added under the category of endangered medicinal plant. Therefore, immediate action towards the regeneration and conservation has been taken through the in vitro propagation strategies which could be significant advantages over conventional propagation methods (Sunnichan and Shivanna 1998; Shukla and Sharma 2017).

Somatic Embryogenesis
Somatic embryogenesis is the process of formation of embryo like structure from somatic tissue. The somatic embryo may be produced either directly on the explant or indirectly from callus or cell suspension culture. For the first time, Haccius (1978) defined somatic embryogenesis as a non-sexual developmental process, which produces a bipolar embryo from somatic tissue. The first report of plantlet regeneration via in vitro somatic embryogenesis was reported in Daucus carota (Reinert, 1958;Sharma et al.2010).
Somatic embryogenesis (SE) is the most significant biotechnological tool for the rapid propagation of endangered plants. Mature seed embryos callus was used to analyse the extracted proteins from globular, heart/torpedo-shaped, and maturing embryo stages resolved in the 2-DE gels revealed increased protein expression in the developmental stages of the somatic embryos of

Nuclear DNA Assay
Genetic information is an important tool in the preparation of conservation strategies of endangered plants. Nuclear DNA assay of endangered plant species (Table 2) with multiple DNA markers plays an important role in the implications for genetic enhancement and ex-situ conservation (Kumar et al. 2014). The molecular adaptation of matK gene in Nymphaea tetragona has been studied for the purpose of conservation strategy planning. It has been observed that the adaptive changes at the molecular level associated with varying ecological conditions. Therefore, as the threat to the existing species is anthropogenic, the conservation of N. tetragona can be targeted by probable translocation to another site (Dkhar et al. 2011).
The various molecular technologies have been used to conserve and maintain the endangered plant species. Random amplified polymorphic DNA (RAPD) has been widely used in the evaluation of genetic relationship among different plant species (Khan et al., 2009). However, in case of use of Inter simple sequence repeat (ISSR) for the assessment of genetic diversity the distribution of nucleotide repeats throughout the genome could be consider and therefore becomes more potent to identify. ISSR markers are more reliable compared to RAPD because of its easy handling and more reproducibility (Wu et al., 2010). Valeriana jatamansi from the Himalayas has been studied for its isolated population. In this study it was observed that ISSR markers generated the maximum level polymorphism (89.0%) followed by RAPD (85.8%) and AFLP (67.7%) markers. Therefore, ISSR fingerprinting was more effective than the RAPD assay, in study of V. Jatamansi (Kumar et al. 2014). Commiphora wightii shows a huge population difference therefore crossing the populations may lead to risk of out breeding depression, which can be recognized as interruption of local adaptation (Haque et al.2010). Dysoxylum malabaricum, an endangered plant in the Western Ghats, faces the pressure due to the overharvesting and developmental activities have resulted in fragmentation and low size of population. Ultimately which leads to the loss of genetic variability hence results in to the inbreeding depression and loss of alleles (Ravikanth et al. 2018).
The study of Nepenthes khasiana Hook.f, a carnivorous plant from North-East India has been done by using multi-locus analysis. The study reveals that RAPD analysis showed more genetic polymorphism than that of ISSR fingerprinting and more genetic similarity in pooled RAPD-ISSR. As maximum morphological variety in context to the discriminated population the species needs an attention for conservation measures (Bhau et al. 2009).
Insufficient number of proper pollinators, fruit development and symbiotic seed germination with mycorrhiza were the reasons responsible for the endangered status of orchid Anoectochilus elatus found in the Eastern and the Western Ghats of Tamil Nadu, India. Therefore, an efficient micropropagation protocol has been standardized by Ahamed et al. (2017) for eco-restoration programmes. For shooting, MS medium with 1.5 mg/L TDZ and 50 mg/L peptone proved to be significant while for in vitro rooting MS medium supplemented with 3 g/L activated charcoal (AC) found to be efficient. The genetic stability of micropropagated plantlets were analyzed through ISSRs molecular markers and showed 2.38% polymorphism and 97.61% monomorphism with true-to-type to mother plant. 45 µM IBA showed the maximum shooting and rooting respectively. RAPD analysis and phytochemical analysis was used to confirm the clonal stability of micropropagated plants.1032 amplicons were generated which was monomorphic and true-to-type mother plant which was further confirmed by similarity matrix by Nei's coefficient and phenogram by UPGMA analysis.

Cryopreservation
In India, in vitro conservation and cryopreservation techniques began in the early 1980s. Plant cryopreservation comprises the plant tissue storage, generally seeds or shoot tips are stored in liquid nitrogen (LN) at -196 °C or in the vapour phase of it at -135 °C followed by the re-warming (Kaczmarczyk et al., 2012). Cryostorage plays an important role in the conservation of endangered species (Table 3), especially when species with very few seed formation or quality of seeds (Kaczmarczyk et al., 2012). The cryopreserved samples possibly take few weeks to months or year to produce the micro propagated plants depending upon the species (Sakai & Engelmann, 2007).
Vitrification technique employed in the cryopreservation of shoot tips of Picrorhiza kurroa, an endangered medicinal plant, by preculturing of shoot tips at 4ºC for 2 days on MS medium containing 5% DMSO before the dehydration in PVS2 solution for 15 min at 0ºC. After vitrification, the shoot tips are directly immersed in LN2. (Sharma and Sharma, 2003).

Synthetic seeds
Synthetic seeds are the encapsulated plant tissues like shoot tips, shoot buds, somatic embryos, cell aggregates, or the tissues which can be cultured as a seed and grown into a whole plant either in vitro or in ex vitro conditions with the potential of viability after cold storage (Magray et al. 2017;Rihan et al. 2017). Murashige (1977) was the first researcher who discussed the concept of synthetic seeds, whereas artificial carrot seeds were first produced by Kitto and Janick (1982) (Nandini B. and Giridhar P. 2019).
The advantages of artificial seeds i.e. propagules encapsulation comprises multiplication of recalcitrant and/or vegetatively propagated species. This strategy has been used in the conservation of various plant species, such as orchids in which encapsulated protocorms proved 70% viability even after more than six months of storage at 4 0 C (Paunescu, A. 2009). Synseed technology has been used especially for the plants with poor seed viability and germination, seedless fruit, as well as in the plants in which special dependance of mycorrhizal-fungal symbiosis for germination (Gantait et al. 2015). Synseed technology is not only useful for the sterile genotypes but also in the germplasm preservation of endangered and rare plant species (Nandini B. and Giridhar P. 2019).
Among the various ex situ conservation methods, synthetic seeds technology thought to be one of the convenient techniques for long-term conservation.

Gene Banks
Gene banks are the bio repositories in which the plant genetic material is preserved for the improvement of the germplasm and conservation purpose.
National Gene Bank has mainly three categories for the conservation of species

Conclusion
In recent decades several plant species in India are known to become endangered and are included under the red-listed plant category. Due to various intrinsic and extrinsic factors, biodiversity seems to be threatened. Thus, the conservation of an endangered species has become an important aspect to prevent the extinction of species. The conservation can be done by using in situ and/or ex situ methods, although in-situ strategies may not be sufficient enough to conserve the endangered species hence the application of in vitro methods would be helpful to recover and re-establish endangered plants. In India, various biotechnological approaches have been used to formulate the conservation strategies such as micro-propagation, molecular profiling, cryopreservation and gene bank which act as important tools in the conservation and improvement of mainly endangered species. The use of in vitro propagated plantlets for the reintroduction of endangered species is a crucial strategy for conservation. Along with this cryopreservation of vegetative tissues or recalcitrant seeds is also a significant option for long-term storage of germplasm. The levels of genetic diversity and population pattern help in the conservation and management of an endangered species therefore various molecular characterizations can be done to achieve the goal of formulation of a conservation strategy.
These various conservation strategies of endangered plants played a significant role to raise the number of plantlets along with their quality traits which shows the scope for future research to fulfill the existing lacunae on different aspects of the study.