Germplasm conservation of economically important medicinal plant Nyctanthes arbor-tristis L. through encapsulation technique and maintenance under slow growth condition

An efficient encapsulation and slow growth conservation protocol was developed for Nyctanthes arbor-tristis L. an antiviral medicinal plant of the family Oleaceae. A gel matrix with 3% sodium alginate and 100 mM calcium chloride (CaCl2⋅2H2O) was found best for the encapsulation of nodal segments. The viability and shoot development potential of encapsulated nodal segments was optimized. Encapsulated nodal segments stored at 4 °C and 24 °C remained viable for up to 90 days and showed shoot development potential 42.89 ± 6.04% and 33.53 ± 7.15% respectively. Nodal segments maintained under slow growth conditions up to 180 days on one-eighth strength Murashige and Skoog (MS) medium supplemented with 0.5% sucrose was suitable for satisfactory viability (40.28 ± 2.04%), while further addition of 0.5 mg/l abscisic acid supported 40.36 ± 1.01% viability of the nodal segments. The best rooting response was achieved on half-strength MS medium supplemented with 4 mg/l indole-3-acetic acid. The field survival of rooted plants was 65%. The clonal fidelity of in-vitro derived plantlets was studied with start codon targeted primer profile, which showed the same banding mobility patterns as the source parent plant. The maximum banding profile was monomorphic and consistent, confirming the clonal stability of regenerated plants. The method developed will permit the in-vitro conservation of this species and facilitate an easy exchange of plant germplasm.


Introduction
Medicinal and aromatic plants are widely used in the traditional medicinal systems. The summation of over exploitation, long-term exposure to environmental stresses and current climate change affects the survival of valuable medicinal and aromatic plant species and place them at the risk of extinction (Joshi et al. 2019). Their conservation is hence urgently required and tissue culture techniques have focused on practical implementation and provide immediate strategies to conserve the medicinal plant species under controlled conditions (Singh et al. 2010).
Encapsulation technology offers an efficient means for conservation of desired plant species (Reddy et al. 2012). Encapsulated vegetative propagules can be used as an alternative to seeds for propagation (Chand and Singh 2004). Encapsulation also provides a viable tool for in-vitro germplasm conservation. During encapsulation embryonic or non-embryonic tissues are capable of conversion into plantlets either in-vitro or ex-vitro (Rihan et al. 2017). Earlier, the concept of encapsulated propagules was traditionally based on the encapsulation of somatic embryos which can be handled like a real seed for transport, storage and sowing (Rihan et al. 2017). However, in recent years the encapsulation of nodal explant was employed as a suitable alternative for germplasm maintenance (Prakash et al. 2018). This method is used for encapsulation of propagules in a wide range of species including ornamental (Hatzilazarou et al. 2020) and medicinal plants (Gupta et al. 2014). The recovery of plants through encapsulated propagules has been reported in other medicinally important plant species like Sterculia urens (Subhashini et al. 2014) and Urginea altissima (L.f.) Baker (Baskaran et al.2018).
Encapsulation technology is a convenient method for conservation of plant genetic resources without apparent risk of genetic instability using minimum space, less labour and maintenance costs and it is easy in transference (Rai et al. 2009). Short-term storage of germplasm can be achieved using encapsulated propagules at low temperature (Parveen and Shahzad 2014). An alternate method for storage of germplasm is to maintain the explants under slow growth conditions based on the manipulation of culture conditions and culture media which allow cultures to remain viable under slow growth rate (Zayova et al. 2017). Conservation under minimal growth conditions can be achieved by reducing the incubation temperature and modifying the culture medium composition (Tyagi et al. 2009). These storage techniques are applicable to a wide range of medicinal plant species and extend the ordinary subculture duration from a few weeks to several months.
Nyctanthes arbor-tristis is a valuable medicinal plant of the family Oleaceae. It is a woody, perennial deciduous small tree having tetragonal twigs and propagated through seeds. It is native to sub-tropical Himalayas of Nepal and southern parts of India along with South East Asian countries such as Thailand, Malaysia and Indonesia on rocky ground in dry hillsides and undergrowth in dry-deciduous forest (Agrawal and Pal 2013). The flower of N. arbor-tristis is axillary or terminal consisting of groups of two to seven flowers having two sub-sessile anthers inserted near the mouth of cylindrical orange red centered white corolla tube, with bifid cylindric style and has good fragrance (Mishra et al. 2016). Flowering generally occurs from late September to December, it blooms late in the evening and floral scents attract the insects for pollination (Raizada and Nangia 1989). The plant has a broad spectrum medicinal properties like ameliorative (Mishra et al. 2018) and antiviral (Gupta et al. 2005) activities. Due to over exploitation coupled with poor seed viability and germination, it is depleted in natural habitats (Sagar and Singh 2004). For germplasm conservation, Jahan and Anis (2015) reported storage of encapsulated nodal explants of N. arbor-tristis at low temperature (4 °C) for limited periods up to 6 weeks only. Against this background, it was required to develop a suitable protocol for the conservation of this species. To our knowledge, this is the first report on conservation of N. arbor-tristis by storage of encapsulated nodal segments and maintaining the nodal segments under slow growth conditions for a prolonged duration.
The objective of this study was to conserve the encapsulated nodal segments and to optimize the efficacy of their conversion into complete plantlets. Thus, we standardized short to medium term storage of encapsulated nodal segments and developed an approach to maintain the nodal segments under slow growth conditions. Recovery of the plants from stored nodal segments was optimized, and the clonal fidelity of in-vitro derived plants was established by start codon targeted (SCoT) molecular markers. SCoT markers are reproducible markers based on short conserved region in plant genes surrounding the ATG translation start or initiation codon (Collard and Mackill 2009).

Preparation and source of explant
Immature fresh green seeds of N. arbor-tristis were sampled from ayurvedic garden of Institute of Medical Sciences (IMS), Banaras Hindu University, Varanasi, Uttar Pradesh, India (NL25°16ʹ23ʺ and EL82°59ʹ50ʺ) in the month of January. A voucher specimen (DG/15/124) was preserved in herbarium library, Department of Dravyaguna, IMS, BHU for further reference. The seeds were sterilized by washing under running tap water and followed by agitating in 1% cetrimide solution for 5 min, 1 3 then they were treated with 0.1% (w/v) HgCl 2 (Hi-media, Mumbai, India) solution for 5 min and washed (six times) with sterile-distilled water. Murashige and Skoog (1962) nutrient medium(15 ml) supplemented with 3% sucrose (w/v) (Hi-media, Mumbai, India) and 0.8% (w/v) agar (Hi-media, Mumbai, India) was poured into 30 ml culture tubes and autoclaved (1.06 kg cm −2 ) at 121 °C for 15 min. Immature embryos excised from sterilized seeds were aseptically inoculated on growth regulator free full-strength MS medium. The cultures were kept under a 16 h/8 h light/dark photoperiod at a light intensity of 50 µmol m −2 s −1 irradiance from cool-white fluorescent tubes (Philips, India) at 24 ± 2 °C. After 28 days of culture, the nodal segments (4-8 mm long) were excised from actively growing young seedlings and served as explant source for encapsulation and for slow growth conservation.

Encapsulation of nodal segments
For encapsulation, solutions of different concentrations of sodium alginate (SA) (1.0, 2.0, 3.0, 4.0% w/v) (HiMedia-Mumbai, India) and calcium chloride (CaCl 2 . 2H 2 O) (HiMedia-Mumbai, India) (25, 50, 75 and 100 mM) were prepared using liquid MS medium. The pH of gel matrix (SA) and complexing agent (Calcium chloride) was adjusted at 5.8 and autoclaved at 121 °C at 1.06 kg cm −2 for 15 min. For optimization of encapsulation protocol, (1) nodal segments (4-8 mm long) mixed in different concentrations (1, 2, 3 and 4%) of SA were poured gently with sterile pipette tips (7 mm) in the solution of 100 mM calcium chloride and (2) nodal segments mixed in 3% SA were poured gently with sterile pipette tips in different concentrations (25, 50, 75, 100 mM) of calcium chloride solution. Nodal segments containing droplets were left for 20 min with occasional shaking in the calcium chloride solution to achieve complete polymerization and proper cross-linking of SA. Thereafter, nodal segments encapsulated in isodiametric, easy-to-handle beads were selected, washed 3-4 times with autoclaved distilled water, and dried on sterile filter paper in Petri-dishes (Borosil, India) to remove the excess of water. These encapsulated nodal segments, considered as encapsulated seeds, were used for further study.

Shoot development potential of encapsulated nodal segments on BAP supplemented medium
For optimization of efficient shoot development potential, encapsulated nodal segments were inoculated on MS medium supplemented with different concentrations (0.0-10.0 mg/l) of 6-Benzylaminopurine (BAP). The medium without BAP was used as control. Data of shoot development potential of the encapsulated nodal segments were recorded at the end of 4 weeks of culture in terms of percent shoot development potential, number and length (cm) of shoots.

Short-term storage of encapsulated nodal segments and shoots development potential
Erlenmeyer flasks (Borosil, India) containing solid MS medium without sucrose were used for the storage of encapsulated nodal segments, at 4 °C and 24 °C for different periods (30, 60, 90 and 180 days) under dark conditions. At the end of storage duration, encapsulated nodal segments were transferred for shoot development to MS medium with 3% sucrose (w/v), 0.8% (w/v) agar and 5.0 mg/l BAP. The percent shoots development potential of encapsulated nodal segments and the number of shoots per encapsulated bead were recorded at the end of four weeks from culture initiation.

Maintenance of nodal segments under slow growth conditions and recovery of shoots
Nodal segments were cultured on full-strength, half (1/2), quarter (1/4) and one-eighth (1/8th) strength MS medium supplemented with 0.5, 1.5 and 3.0% sucrose. Cultures were incubated for 30, 90 and 180 days at 24 ± 2 °C under a 16 h/8 h light/dark regime, under the illuminance of 50 µmol m −2 s −1 from white fluorescent tubes (Phillips, India). After 30, 90 and 180 days storage, the number of shoot /explant and length of shoots (cm) were recorded. To confirm the shoot recovery potential of nodal segments after 180 days storage, they were sub-cultured on shoot recovery medium (Full-strength MS medium + 3% sucrose + 5.0 mg/l BAP) for re-growth. Data on the number shoots/explant and length of shoots were recorded at the end of four weeks from culture initiation.

Effect of abscisic acid (ABA) on slow growth storage of nodal segments and recovery of shoots
To study the effect of abscisic acid (ABA), nodal segments were cultured on 1/8th strength MS medium containing 0.5% sucrose and supplemented with four concentrations (0.0, 0.5, 1.0 and 2.0 mg/l) of ABA. ABA was filter sterilized through 0.45 µm Millipore filter before adding to the sterilized medium. Cultures were incubated for 30, 90 and 180 days at 24 ± 2 °C under a 16 h/8 h light/dark regime, with the illuminance of 50 µmol m −2 s −1 from white fluorescent tubes (Phillips, India). After storage, the number of shoots/ 1 3 node and lengths of shoots were recorded. To confirm the shoot recovery potential of nodal segments after 180 days storage, nodal segments were transferred onto shoot recovery medium (full-strength MS medium supplemented with 3% sucrose and 5.0 mg/l BAP) for regrowth. Data based on regrowth, the responding frequency (%), mean number shoot/explant and mean length of shoots was recorded at the end of four weeks from culture initiation.

In-vitro rooting, hardening and acclimatization of plantlets
For in-vitro rooting, green, actively growing healthy shoots (4-5 cm) developed from germinating encapsulated nodal segments or slow growth conserved nodal segments on shoot recovery medium were used. Individual shoots were cultured on half-strength MS medium supplemented with 1, 2, 4, 6 and 8 mg/l Indole-3-acetic acid (IAA), and medium without IAA was used as control. The root induction frequency (%), number of roots/shoot and roots length were noted after four weeks from culture initiation. The complete plantlets with 1 cm long roots were removed from rooting medium, washed gently with sterile distilled water to remove attached media on roots, and transferred to plastic cups containing a 1:1:1 mixture of sterilized commercial soil-rite (Kel Perlite, Vishwas Nagar, Karnataka, India), garden soil and sand. The cups were covered with clear polythene bags to maintain high humidity and irrigated daily with 1-2 ml of sterilized MS salt solution upto six days. The plants were maintained in a culture room at 24 ± 2 °C and 16-h/day illuminance of 20 µmol m −2 s −1 from cool-white fluorescent tubes. After two weeks bags were partially opened. The acclimatized juvenile plantlets were transferred to the potting mixture above and kept in a greenhouse with controlled temperature (27 °C) and natural sunlight conditions in to the open field.

Assessment of clonal stability
For assessment of clonal stability, leaves were taken from acclimatized plants and frozen immediately in liquid nitrogen, were used for DNA extraction. A comparison between the start codon targeted (SCoT) profile of the source parent plant and five acclimated plants was performed. Doyle and Doyle (1990) method with modification was used for the isolation of DNA. Clonal fidelity was assessed by the PCR based 14 SCoT primers (SCoT 5,6,8,9,10,11,24,25,26,27,28,29,30,31) analysis. The gel was prepared with 0.8% agarose and stained with ethidium bromide dye. The quality and quantity of isolated DNA were checked by Dyna Quant 200 Fluorimeter. The PCR reaction consists of 75 ng of template DNA, 0.5 µM primer, 2.5 mM MgCl 2 , 0.2 mM dNTP (GeNeiTM, Bangalore, India) in 25 µl reaction volume, 2U Taq DNA polymerase (GeNeiTM, Bangalore, India) along with suitable buffer (100 mM Tris-Cl; pH 9.0, 50 mM KCl, 1 percent Triton X-100). A total of 35 cycles of denaturation at 94 °C for four minutes, annealing at72 °C for 1 min, and extension at 72 °C for 8 min were used for the PCR amplification. After agarose gel electrophoretic separation, amplicons were visualized by exposing the gel through ultra-violet rays under the Gel documentation system (Syngene Gel Doc, Syngene, Synoptics Ltd., UK) for scoring the bands. To assess the homogeneity of band profiles, PCR amplification was carried out in triplicate. Data analysis was carried out by scoring well-marked, high-intensity amplified bands. The size of the amplicons was determined by comparing them with a 1 Kb DNA ladder (GeNeiTM, Bangalore, India).

Statistical analysis
All the experiments were setup in a completely randomized block design. Each experiment was repeated thrice with twenty culture tubes in each. Analysis of variance (ANOVA) was used to examine the data and Tukey's t-test was used to determine the significance of differences between the treatment means at P < 0.05 level.

Encapsulation of nodal segments, shoot development potential and short-term storage
Results revealed that lower concentrations of SA (1.0 and 2.0% w/v) were not suitable for the formation of calciumalginate beads. The lower concentrations of SA resulted in beads of undefined shape and they were difficult to handle. The shoot emergence from these beads was not observed on MS basal medium (Table S1). The higher SA concentration (4.0%) produced firm and spherical beads that affect the shoot emergence from the encapsulated nodal segments. SA at 3.0% was found most ideal for the formation of desirable beads. These beads have a maximum shoot development potential (55.73 ± 1.58%) from encapsulated nodal segments. Similarly, a low concentration of calcium chloride (25 mM) did not support good beads preparation, and no shoot growth response was observed from these beads. Calcium-alginate beads prepared using 50 and 75 mM calcium chloride support the poor shoot emergence. Beads prepared using 100 mM calcium chloride was found most suitable for the high-frequency shoot development potential (55.73 ± 1.58%) from encapsulated nodal segments (Table S1). Thus, gelling matrix of 3.0% SA and 100 mM calcium chloride was found the ideal combination for the formation of calcium-alginate beads (Fig. 1A).
To evaluate the efficient shoot development response from encapsulated nodal segments, calcium-alginate beads were cultured on MS medium supplemented with various concentrations (0.0-10.0 mg/l) of BAP (Fig. 2). The results revealed that the response of the encapsulated nodal segments varied depend on the concentration of BAP. The maximum frequency (82.91 ± 7.70%) of shoot development ( Fig. 2A), number (5.55 ± 0.26) of shoots emerged from each encapsulated node (Fig. 2B), and maximum (2.64 ± 0.21 cm) average shoots length (Fig. 2C) was recorded on MS medium supplemented with 5 mg/l BAP (Fig. 1B, C).
To assess the shoot emergence response from nodal segments encapsulated in calcium-alginate beads and stored at 4 °C and 24 °C upto 30, 60, 90 and 180 days, were subcultured on best shoot multiplication medium (MS + 5 mg/l BAP). The encapsulated nodal segments stored at 4 °C for 30 days showed the maximum frequency (80.24 ± 1.01%) of shoot development, and the number (6.07 ± 0.19) of shoots /encapsulated bead (Table 1; Fig. 1D). However, when storage duration was increased from 30 to 180 days, the viability and shoot development response of encapsulated nodal segments were gradually decreased (Table 1). Similarly,  (Table 1). The viability of encapsulated nodal segments and their shoot development response was drastically decreased with increasing the storage periods from 30 to 180 days (Table 1). In general, it was observed that encapsulated nodal segments stored at 4 °C were found more responsive for shoot development potential (Table 1). Encapsulated nodal segments stored up-to 180 days either at 4 °C or 24 °C were not responsive at all.

Maintenance of nodal segments under slow growth conditions and recovery of shoots
The slow growth conservation of N. arbor-tristis was evaluated by maintaining the nodal segments on full, half (1/2), quarter (1/4) and one-eighth (1/8th) strength of MS medium supplemented with 3.0, 1.5 and 0.5% sucrose, and stored upto 30, 90 and 180 days at 24 ± 2 °C ( Table 2). The percentage viability of explants and shoot regeneration response varied significantly. It was depend on   Table 2). In general, the proliferations of axillary shoots from nodal segments were gradually decreased, when the concentration of sucrose was reduced from 3.0 to 0.5%. It also affects the length of the axillary shoots. The results revealed that when MS medium strength and sucrose concentration were reduced, the length of axillary shoots was gradually decreased. Nodal explants maintained upto 180 days on the one-eighth strength MS medium supplemented with 0.5% sucrose was found most suitable for reducing the number of shoot buds development (1.61 ± 0.28) and length (1.12 ± 0.32 cm) of axillary buds ( Fig. 1G-I). As a result, one-eighth strength MS medium supplemented with 0.5% sucrose was found most effective for slow growth conservation of N. arbor-tristis, because it enhanced the dormancy of axillary shoot buds with the reduced number of shoot growth from nodal segments.
To promote the dormancy of axillary shoot buds, the oneeighth strength MS medium with a reduced concentration of sucrose (0.5%), was supplemented with 0-2 mg/l ABA (Table 3). After 180 days of storage, nodal explants maintained on the one-eighth strength MS media supplemented with 0.5 mg/l ABA exhibited good viability (40.36 ± 1.01%) with reduced number of shoots/ node (1.32 ± 0.11) and shoot length (0.50 ± 0.14 cm) (Fig. 1J).
To evaluate the shoot re-growth response from 180 days stored nodal segments maintained either on one-eighth strength MS medium containing 0.5% sucrose or one-eighth strength MS medium supplemented with 0.5% sucrose and 0.5 mg/l ABA, further sub-cultured on optimum shoot induction medium (MS + 5 mg/l BAP). The nodal segments maintained on one-eighth strength MS medium supplemented with 0.5% sucrose showed the maximum shoot regrowth frequency of 80.26 ± 4.26%, the maximum number of shoots/node 4.67 ± 1.10, an optimum length of shoots 2.25 ± 0.45 cm on the recovery medium after four weeks from culture initiation (Fig. 1K). Similarly, the nodal segments stored on one-eighth strength MS medium supplemented with 0.5% sucrose and 0.5 mg/l ABA, when sub-culture on re-growth medium, showed a maximum responding frequency 74.54 ± 1.85%; 3.07 ± 0.38 number of shoots/

In-vitro rooting, hardening and acclimatization of plantlets
Elongated shoots (4-5 cm) were sub-cultured on a halfstrength MS medium supplemented with various concentrations (1, 2, 4, 6 and 8 mg/l) of IAA for root induction (Table 4). The shoots cultured on a half-strength MS medium without IAA were unable to induce the root formation. Results revealed that the percentage frequency (%) of root induction, number of roots/shoot and root length was depend on the concentration of IAA. The best rooting response with maximum rooting frequency, efficient number of roots/shoot, and proper root length were achieved on a half-strength MS medium supplemented with 4 mg/l IAA (Fig. 1L, M; Table 4). The higher concentration of IAA induced green compact callus development at the base of the shoots without root formation. The well-rooted plants were taken out from the culture vials, washed gently with sterile distilled water and transferred into plastic cups filled with a mixture of garden soil, soil-rite, and sand for hardening. These cups were kept for acclimatization of plants under green-house conditions (Fig. 1N, O) and further transferred into the field. These plants in the field had a survival rate 65%.

Assessment of clonal stability
The SCoT markers were used to determine the clonal stability of the plants transferred in to field. To validate the clonal stability, five acclimatized plants were randomly selected from the field, along with the source parent plant (P) and subjected to the genetic stability analysis. The finding suggests that, the banding profiles of recovered plants were similar to the parent plant and no genetic variation was observed (Fig. 3). A total of 78 distinct bands were scored from 14 SCoT primers. The number of the scorable bands varied between 2 to 8 per SCoT primers. A total of 444 bands were amplified using SCoT primers (Table 5). In comparison with the source parent plant, the maximum bands were consistent; uniform with no distinct differences was observed. The identical banding pattern revealed the genetic homogeneity among the recovered plants with source parent plant (P). Based on binary matrices data of the SCoT profile of presence (1) or absence (0) of bands, a genetic similarity index was analyzed and subjected to cluster analysis using the unweighted pair group and arithmetic averages (UPGMA) clustering methods. The average Jaccard's similarity coefficient was 0.92-0.99 with the mean value of 0.96 in two clustered groups were shown through UPGMA matrix-based phenogram (Fig. 4). The different numbers in dendrogram hierarchical clustering showed that 4 and 6 have most genetic closeness. The numbers 4, 6 and 5 are almost similar to each-other as well as 1, 3 and number 2 are similar to all.

Discussion
Germplasm conservation of elite plant species was achieved through storage of encapsulated propagules (Shilpha et al. 2021) and maintenance of explants on suitable media under slow growth conditions (Nasiruddin and Rafiul Islam 2018). The encapsulation protocol was influenced by the effective concentration of SA and calcium chloride used during the preparation of calcium-alginate beads (Saeed et al. 2018). Calcium-alginate beads varied morphologically in texture, shape and transparency. It depends on the concentration of SA and calcium chloride used during the preparation of calcium-alginate beads. The lower concentrations of SA (1-2%) and calcium chloride (25-75 mM) was not found suitable for calcium-alginate bead preparation. It may be due to a reduction in the gelling capacity. A higher concentration of SA (4%) produced firm and isodiametric beads that delayed the shoot emergence from encapsulated nodal segments. This delay in the emergence of shoots may be due to the hardness of the beads. In the case of N. arbor-tristis 3% SA and 100 mM calcium chloride were used in the preparation of calcium-alginate beads. It was highly effective for the emergence of shoots from encapsulated nodal segments. In photographs, an alphabetical letter (L) represents 1 Kb ladder, letter (P) denotes the source parent mother plant of N. arbor-tristis L., and numeric number 1 to 5 denotes bands of tissue culture recovered acclimatized hardened plants 1 3 Similar concentrations were used previously by Ahmad et al. (2012) in Ruta graveolens and by Varshney and Anis (2014) in Balanites aegyptiaca. The developments of multiple shoots from encapsulated explants depend on the species and media used during the germination of calcium-alginate beads. Singh et al. (2006) reported multiple shoots development from encapsulated shoot tips of Withania somnifera on MS medium without cytokinin, while in N. arbor-tristis MS medium supplemented with BAP supported multiple shoot development from encapsulated nodal segments. Hatzilazarou et al. (2020) found that BAP supplemented media was highly effective for the emergence of several shoots from encapsulated explants in Viburnum dentatum L. Encapsulation technology for short-term storage of encapsulated explants for germplasm conservation was achieved by the scientists (Micheli et al. 2007). Using this technology, germplasm can be stored for a few months (Bhattacharyya et al. 2018) to a year (Kaminska et al. 2018). Storage of encapsulated explants is a crucial factor that regulates the recovery of plantlets after transfer to regrowth media. In present study, the recovery of shoots from encapsulated nodal segments stored at 4 °C was found better than those stored at 24 °C. The retention of viability of encapsulated explants stored at low temperature may be due to the availability of nutrients in the gel matrix for longer period. At this temperature, the encapsulated explant slows down its metabolic activities. Hence, they remained in a quiescent state for a longer period. Thus, it will be helpful in conserving the encapsulated explants during cold storage. The encapsulated explants of Pseudostellaria heterophylla were stored at 4 °C was most favourable for the recovery of plantlets (Ma et al. 2011). Encapsulated explant stored at a higher temperature showed a significant decrease in the rate of shoot development on recovery media. It might be due to reduced respiration of plant material due to oxygen deficiencies in SA beads or the beads desiccating under lowhumidity conditions (Ahmad and Anis 2010).
Slow growth is an important biotechnological tool for medium-term conservation of germplasm under in-vitro conditions. This procedure was successfully employed for the conservation of Cynara cardunculus var scolymus L. (Tavazza et al. 2015). Slow growth is usually achieved by modifying the salt concentrations of culture media. Media supplemented with osmotic agents and growth inhibitors enhanced the slow growth periods. The modification of medium strength was found to be effective for maintaining the explant as such for longer period in pear (Ahmed and Anjum 2010) and Elettaria cardamomum (Tyagi et al. 2009). Sucrose is a major component of plant tissue culture media. It acts as a carbon energy source and osmotic agent (Yaseen et al. 2013). The variation in the sucrose concentration in the media, which acts as an osmoticum, was effective in extending the storage life of in-vitro grown tissues (Jo et al. 2009;El-Bahr 2016). Reducing the sucrose concentration in MS medium was helpful in promoting the axillary buds dormancy and maintaining the nodal segments as such up to 180 days in N. arbor-tristis. Kaminska et al. (2016) found that media supplemented with ABA was beneficial in promoting the shoot bud dormancy, which helped in maintaining the explant as such for a longer duration under in-vitro condition. ABA is an important growth inhibitor and is commonly used in in-vitro studies (Cid et al. 2008). It regulates several physiological and developmental processes in the plants (Vishwakarma et al. 2017) and its inhibitory effects could be attributed to interactions with auxin and cytokinin level in the host (Sah et al. 2016). Media supplemented with ABA was highly effective for slow growth storage of viable explant of Tetrastigma hemsleyanum (Peng et al. 2015). ABA was also found effective for the storage of nodal segments under slow growth condition in N. arbor-tristis.
The process of hardening of rooted plantlets and their transfer into the field is very crucial for their growth and survival. It is the slow adaptation of plants to their natural environment. Hardened plants, when transferred into the Fig. 4 Picture of maximum likelihood tree based on Jaccard's average similarity co-efficient originated through UPGMA matrix of tissue culture recovered acclimatized plants of N. arbor-tristis L. field, compete with abiotic and biotic stress. The ultimate survival of hardened plants in the field depends on their ability to overcome the stress induced on them during the field establishment process (Teixeira da Silva et al. 2017). Plantlet of N. arbor-tristis had a survival rate of 65% when transferred to the field. All the plants that survived in the field had no phenotypic variations.
Clonal fidelity assessment of plants recovered from stored explants is highly important. Several DNA based molecular markers were recommended to study the genetic stability of recovered plants. These molecular markers have been used to detect somaclonal variations induced in regenerates (Kalia et al. 2011). Among these markers, the SCoT marker is frequently used to detect genetic variation among recovered plants (Seth et al. 2017). The recovered plants of N. arbor-tristis showed clonal fidelity to the source parent plant, as evidenced by the SCoT marker, which revealed no polymorphism in any of the bands. The genetic relatedness was calculated and determined using the Jaccard Similarity co-efficient. The similarity coefficient revealed the in-vitro recovered plants were true-to-type and they were genetically related to the source parent plant. Start codon targeted (SCoT) DNA polymorphism is a type of DNA marker based on the conserved region flanking translation start codon 'ATG' (Collard and Mackill 2009). The assessment of clonal stability by SCoT markers has been studied in several medicinal plants like Cleome gynandra (Rathore et al. 2014) and Citrullus lanatus (Vasudevan et al. 2017) by other workers.

Conclusion
In conclusion, we have optimized the germplasm conservation protocol for N. arbor-tristis, which is an economically important antiviral medicinal plant. The germplasm in the form of encapsulated nodal segments is maintained at low temperature for 90 days. The nodal segments maintained under slow growth conditions up to six months showed good viability of explants and recovery of plantlets. ABA enhanced the dormancy of axillary buds and helped in maintaining the explant as such under slow growth conditions. The rooting of recovered shoots (4-5 cm) was achieved on half-strength MS medium supplemented with 4.0 mg/l IAA. The rooted plants were acclimatized successfully and showed 65% survival when transferred into the field. The hardened plants did not show any morphological variations from the parent plant. SCoT primers were used to assess clonal fidelity of recovered plants, which confirmed the genetic stability of these plants, and no variation was observed with respect to the parent plant. In summary, the outcome of optimized protocol provides improved scope for short-term conservation of germplasm of this economical antiviral plant. The methods open up avenues to protect this species from habitat loss.