International Research Journal of Plant Science

Development of an efficient protocol from invitro Micropropagation and Somatic embryogenesis for the Mass Multiplication of Ventilago maderaspatana Gaertn- A Medicinal Plant

Deepak R. Chandra and Thoyajaksha^* Research and Development Center, Bharathiar University, Coimbatore, Tamilnadu, India
^*Corresponding Author:
Thoyajaksha, Department of Botany,Government Science College,Hassan,Karnataka, India, Email: thoyajaksha@gmail.com

Received: 19-Oct-2020 Published: 21-Nov-2020, DOI: 10.14303/2141-5447.2020.0016

Introduction

Biodiversity is the immense collection of all the species of plants, animals, insects and microorganisms residing the earth either in the aquatic or the terrestrial habitats. Global community has witnessed key changes as a result of the unfolding and globalization of revolution taking place since years (Mathur, 2013). In view of the shockingly growing world population, increasing anthropogenic actions, rapidly corroding natural ecosystem, etc the natural habitat for a prodigious number of herbs and trees are dwindling. Many of them are facing extinction (Sharma et al., 2010). An extremely conservative estimate states that this loss of plant species is between a hundred and thousand times above the expected natural extinction rate the planet is losing a minimum one potential major drug every 2 years (Phillips et al., 1994). According to the International Union for Conservation of Nature and also the World Wildlife Fund, there are between 50,000 and 80,000 flowering plant species used for medicinal purposes worldwide. Among these, about 15,000 species are threatened with extinction from over harvesting and environs destruction (Bonato et al., 2006) and 20 % of their wild resources have already been nearly exhausted with the swelling of human population and plant consumption (Chen et al., 2016).

To deal with this alarming situation, biotechnology has come as boon. Advances in plant biotechnology, particularly those associated to in vitro culture and molecular biology, have also provided influential tools to support and improve conservation and management of plant diversity. One of the techniques like plant tissue culture have been used to conserve endangered, rare, crop, ornamental, medicinal and forest species, allowing the conservation of pathogen- free material, elite plants and genetic diversity for short- medium- and long-term. in vitro conservation is especially important for vegetatively propagated and for non-orthodox seed plant species (Kasagana and Karumuri,2011; Cruz-Cruz et al., 2013). It has also opened exciting frontier in the field of agriculture and offers opportunities for the increase in productivity, profitability, stability and sustainability(Kuras et al.,2004; Rameshkumaret al.,2017). Plant tissue culture techniques have also helped in large-scale production of plants through micropropagation or clonal propagation of plant species (Mathur, 2013).

Ventilago maderaspatana Gaertn., (V. maderaspatana) is a woody liana belonging to family Rhamnaceae. It is distributed in the forest of low elevations in Southern Greece, India, Indonesia, Myanmar, and Sri Lanka. Commonly called as Red creeper (Ravidra et al., 2013; Sindhura and Eswaraiah, 2014). It shows the occurrence of vathraquinonesphyscion, isofuranonaphthaquinones, emodin, xanthrorin & xanthorin-5-methyl ether, islandiein, ventilone-c, Ventiloquinones E,G,J eleuthrin, enantiopure 1,3-dimethyl pyranonappthoquinones (Kwade et al., 2014; Sasikala and Sudhakarc, 2015). Due to its substantial amount of medicinally important bioactive compounds and various anthropogenic and natural activities, authors of this paper have witnessed gradual decrease in the population of V. maderaspatanain the region of Hassan and Mysore districts of Karnataka, India. As in such situation conservation of a plant’s genetic diversity of a region becomes important. In general, conservation involves deeds such as collection, propagation, characterization, evaluation, disease indexing and elimination, storage and distribution. The conservation of plant genetic resources has long been realised as an integral part of biodiversity conservation. The goal of conservation is to support sustainable development by protecting and using biological resources in ways that do not diminish the world’s variety of genes and species or destroy important habitats and ecosystems (Kasagana and Karumuri, 2011).

Hence, the present work is aimed to establish a micro- propagation protocol for the mass multiplication and in vitro conservation of V. maderaspatana from direct shoot multiplication, callus culture and somatic embryo culture.

Materials and Methods

Seed germination test

Before the initiation of micropropagation procedures, the seeds V. maderaspatana were tested for their efficiency in germination by seed germination test. To test the seed viability, seeds of were germinated using methods such as Between paper method, Top of the paper method and Sand method prescribed by Ministry of Agriculture and Farmers welfare, available in Seed testing Manual (2017). Seeds sown as per the treatments were incubated in germinator maintained at required levels of temperature with 12-hour light and dark each, until completion of germination. Observation and Datarecorded at the end of 6^th week. For each type of treatment 12 replicates were maintained.

Seeds were also germinated on solidified Murashige and Skoog (MS) (Murashige and Skoog, 1962) medium supplemented with different concentrations and various combinations of BAP and KN. The cultures were kept for incubation under lab conditions at ± 22°C for 6 weeks. Observation and Data recorded at the end of 6th week. For each type of treatment 12 replicates were maintained.

Plant material and tissue cultureconditions

Healthy and mature V. maderaspatana plants explants, stem segments (3-5cm in height) are obtained from actively growing young shoots during the month of March from different locations at Western Ghats of Karnataka (Table 1). The samples were brought to tissue culture lab in Government Science College, Hassan, in sterile bags and processed immediately to reduce risk of contamination. Tender branches were kept in running tap water for 45mins. Few drops of Tween-20 were added followed by fungicide treatment with 0.3% (w/v) Bavistin (Carbendazim 50%, WP, BASF India) a systemic fungicide for 2hr. The branches were cut to give 1.5 – 2cm long shoot tip or nodal explants. These explants were disinfected with 0.6% (w/v) mercuric chloride for 7min for nodal explants and 5min for shoot tip explants, followed by thorough rinsing in sterile distilled water for at least four to five times. Explants were trimmed further before inoculation into the culture medium. The pH of the Woody Plant Medium (WPM) (Lloyd and McCown, 1981) was adjusted to 5.8. 50ml aliquots of molten medium were dispensed into screw capped glass bottles (10.5cm x 8.5cm) prior to autoclaving at 15psi and 121°C for 20mins.

Table 1. Survey and sample collection from different regions of Karnataka for Micropropagation of V. maderaspatana

Shoot initiation and multiplication

Multiple shoots were initiated from nodal explants on WPM supplemented with 2.0mg/l 6-benzylaminopurine (BAP), Kinetin (KN) and Thidiazuron (TDZ) along with 0.3% (w/v) Activated charcoal (LR, Ranbaxy, India) or soluble Polyvinylpyrrolidone – 40 (0.1% w/v; Sigma Chemical Co. St. Louis, MO USA). The pH was adjusted to 5.8 before autoclaving at 121°C for 20 minutes. Cultures are incubated at ±22° C. After the 5^th week, in vitro developed shoots were subcultured along with the original nodal explant on to WPM with BAP (0.5mg) and Naphthalene Acetic Acid (NAA) (1.0mg/l) for shoot proliferation and elongation in nodal explants.

Micro shoots obtained from second subculture measuring between 1.5 – 4.0cm with at least one node were transferred to WPM with 0.3% activated charcoal or ex-vitro rooted by treatment with 10.0mg/l Indole-3-Butyric Acid (IBA) for 30 min. and immediately transferred into bottles and plastic pots containing sterile soil-rite. The pots were covered with polythene bags having punched holes. The ex vitro treated shoot were maintained in the growth chamber at 80% RH. After 5 weeks the number of roots per shoot and their length was recorded and rooted plants were shifted to polytunnel.

Callus induction and Somatic Embryogenesis

Callus was initiated from the shoot tip explants on Petri dishes with clear lids containing WPM-solid medium supplemented with different concentrations and various combinations of BAP, 2, 4-D and TDZ plant hormone. The Petri dishes were then sealed along the rims with two layers of parafilm. 30 g of sucrose and 8% (w/v) agar was adjusted to pH 5.8prior to the autoclaving at 121°C for 20 min. The cultures were placed in dark for 3 weeks at 20 ± 2°C and then transferred to the diffused light (16 h photoperiod, PPFD at 30 μmol m−2 s−1) provided by cool-white fluorescent lamps under same culture conditions. Data for somatic embryo formation were taken after four weeks of culture. Selected calli were subcultured on same medium for one month. Highly embryogenic callus with small preglobular somatic embryos were maintained and multiplied in WPM medium with reduced TDZ.

Root initiation

To induce roots, elongated shoots were excised and transferred into woody plant rooting media containing different concentrations and various combination of Indole Acetic Acid (IAA), Indole Butyric Acid (IBA) and Naphthalene Acetic Acid (NAA) plant hormone (Ayan et al., 2005). in vitro rooted plantlets were washed with distilled water to remove traces of medium followed by treatment with Bavistin solution (0.3% w/v) for 5 min. Plants were gently placed into screw capped bottles containing sterile soil-rite andweremaintainedingrowthchamberfor5 weeksat 80% RH.

Hardening and acclimatization

Well Regenerated plants were transferred into pots containing autoclaved sterile soil rite, Vermiculate, Coir peat, peat moss, Sand, Soil and a mixture of Sand and Soil for acclimatization. After 2-3 weeks they were potted into autoclaved soil mixture containing Soil rite, Vermiculate and Sand in the ratio of 1:1:1 under greenhouse condition to develop mature plants. Irrigation was done using normal tap water and were grown in greenhouse for a period of 3 – 4 months before moving to the originalhabitat.

Reintroduction of micropropagated plants to wild and Morphological examination

Nearly 100 micropropagated plants of V. maderaspatana were reintroduced in the area of Neerahalla (Hassan district, Karnataka), (Table 1) with the help of local people and our staff. The growth performance of reintroduced plants of Ventilago maderaspatana was recorded. For morphological examination, a set of 25 randomly tagged individuals among the 100 reintroduced plants that were established in the field and were selected of reintroduced plants. The morphological characters like height of the plant, inter-nodal length, number of leaves, leaf length and leaf breadth were recorded at the intervals of 6 months after transplanting. Different parameters like survival rate, height of plants, inter-nodal length, number of new shoots, leaf length and leaf breadth were recorded.

Statistical analysis

All results obtained were analysed for statistical significance using the one way The Analysis of Variance (ANOVA) and variations among means were compared using Post Hoc Duncan’s Multiple Range test at P≤0.05 was considered significant. [SPSS version 11.5.0 (SPSS Inc., Chicago, IL)]. Data were expressed as mean±SEM.

Result

Seed germination

Attempts were made to germinate seeds under laboratory condition. Techniques like Between Paper Method, Top of Paper Method and Sand Method were unsuccessful. Significantly lower germination of seeds was also found under in vitro condition. The seed explants were cultured on MS medium. As a control MS free medium showed a plant survival percentage of 66.6% while medium supplemented with BAP at 1.0 mg/l and 2.0 mg/l showed 58.3 % of plant survival and KN at 2.0 mg/l showed 75.0 % of plant survival. Media supplemented with both BAP and KN at 2.0 mg/l each showed highest plant survival rate (83.3%) in comparison with MS media supplemented with BAP and KN separately (Table 2).

Table 2. Effect of cytokinins on in vitro seed germination of V. maderaspatana after 6 weeks

Shoot initiation and multiplication

Nodal explants obtained from healthy mother plants of different locality were used for culture establishment. Shoot multiplications were obtained successfully from the nodal explants of V. maderaspatanaon WPM supplemented with different concentration and combination of BAP, KN and TDZ (Figure 1). The influence of cytokinins on multiple shoot formation from Nodal explants is summarized in (Table 3). Multiple shoot initiation was observed in different hormonal treatment after 4 weeks of culture. Maximum shoots were obtained in the WPM medium supplemented with TDZ associated with the highest percentage (83.3%) of responding explants, number (6.83) of lateral buds, length (4.75 cm) of shoot buds and number (4.30) of nodes. Whereas the Kinetin showed lower number of shoot formation. TDZ was better when compared to BAP and combination of BAP+KN for shoot proliferation

Table 3. Effect of cytokinins on multiple shoot formation from nodal explant of V. maderaspatana after 6 weeks

Callus induction and Somatic Embryogenesis

Callus was initiated from the shoot tip explants on WPM supplemented with different concentrations and various combinations of BAP, IBA, 2, 4-D, NAA and TDZ (Figure 2). Within 10 - 12 days the shoot-tip explants enlarged and developed calluses at the surface of shoot-tip explants in most of media tested, subsequently covered the whole surface of explants within 4 weeks. The response of explants varied with the different concentrations and the various combinations of plant hormones (Table 4). NAA showed the formation of nodular callus and 2, 4-D showed fragile callus formation. While IBA, showed the formation of root at different concentrations respectively. The combinations of NAA and BAP at different concentration also showed the formation of nodular callus formation. Embryonic callus formation was occurred in WPM containing the combination of NAA and TDZ. Maximum explant response found with the concentration NAA (0.5mg/l) and TDZ (0.5mg/l).

Table 4: Effect of different concentrations and various combination of BAP, 2,4-D and TDZ on the Somatic embryos and Callus formation form leaf explants cultured on WPM-solid medium for four weeks.

Embryogenic callus and Nodular calluswere slow-growing, compact, light brown or dark green. Some of these turned brown and died. Non-embryonic callus or fragile callus was fast growing, light yellow and loose. Subsequent subculture of these embryos from embryonic callusin the same medium for one month resulted in their multiplication and transferring them to a callus multiplication medium with lower concentration of TDZ for one month resulted in maturation of somatic embryos. Embryogenic callus with small pre-globular somatic embryos were maintained
and multiplied in WPM medium with reduced TDZ. The regenerability of the somatic embryos was clearly visible as green tiny shoot primordia were observed within 3-4 weeks of transfer and after a week the green shoots emerged out (Figure 2). Some of the somatic embryos developed into shoot only, while most developed into shoots and root. Plantlets with 5-10 cm long from somatic embryos regeneration media were transferred to rooting medium.

Rooting of plantlets

Rooting was induced in shoots grown on WPM supplemented with auxins (Table 5). Roots emerged after 4 weeks of culture initiation. WPM medium supplied with NAA produced calluses and tufted roots at the bases of shoots. Combination of IBA and IAA exhibited lower effect in the root formation. MS media supplemented with IBA, root formation was better in the lower concentration. While media supplemented with 0.5 mg/l IAA showed least number of root formation. WPM medium supplemented with 0.50 mg/l IBA was the most effective one, associated with the highest percentage (91.5%) of shoots developed roots, the highest number (4.08) of roots per shoot and the tallest (8.45cm) roots (Figure 3).

Table-5: Effect of auxins on rooting of microshoots  of V. maderaspatana, Observations were made after 6 weeks of culture

Acclimatization and Transfer of plantlets to field

Regenerated plants were transferred into pots containing soil rite, vermiculate, coir peat, peat moss, sand and the mixture of sand and soil for acclimatization. Regenerated plants grown well under both growth chambers and mist chamber (Polyhouse) conditions. The survival rate during the hardening was high in Soil rite (92.65%) and in the mixture of Sand and soil (69.3%) in comparison with other substrates (Table 6). Survived and normally grown plants were, subsequently transferred to greenhouse conditions (Figure 3).

Table 6. Effect of different substrate on hardening of plantlets of V. maderaspatana, observations were made after 6 weeks of transformation

Morphological examination

The morphological observation of 25 randomly tagged individual of reintroduced plants showed similar performance in morphological characters like height of the plant, inter-nodal length, number of leaves, leaf length and leaf breadth (Table 7).

Table-7: Growth performance of the introduced micro propagated plants of V. maderaspatana

Discussion

The independence of the next generation of plants begins withtheseed. Ithas theembryoasthenewplant in miniature, which plays an important role as a dispersal unit and is well provided with food reserves to sustain the growing seedling until it establishes itself as a self-sufficient, autotrophic organism (Derek, 1997). Attempts to germinate seeds under laboratory condition were unsuccessful. Seeds cultured on MS free medium showed a plant survival percentage of 66.6% while media supplemented with both BAP and KN at
2.0 mg/l each showed highest plant survival rate (83.3%) in comparison with MS media supplemented with BAP and KN separately. Seed germination failure and lowergermination rate in MS free medium clearly indicates the seed dormancy of V. maderaspatana in natural conditions. This may be due to the fact that seed may be sensitive to the basic requirements and environmental factors like water, light, temperature and minerals. There has been a large volume of published work and significant progress has been made in understanding seed dormancy. However, earlier reviews concluded that it is one of the least understood phenomena in the field of seed biology (Hilhorst, 1995; Bewley, 1997) and also there have been some potential sources of confusion that have been reported in the literature (Finch- Savage and Leubner-Metzger, 2006).

Institution of reliable procedures for regeneration of plants from tissue explants and callus cultures has been helpful of success in genetic engineering techniques of plants (Minocha R and Jain SM, 2000). Although great progress has been made in this regard with woody species, standard media formulations normally used for herbaceous plants (e.g. MS and B5) did not sustenance good growth of many woody plants (McCown and Sellmer, 1987). For woody plants many researchers found that Woody Plant media formulated by Lloyd and McCown (1981), Showed good result in comparison with MS media (Wolfe et al., 1983; Barney et al., 2007; McCown et al., 1987; Thakur and Kanwar 2008). Hence, in this study WPM with different combinations and concentration of plant hormones was used for the propagation of V. maderaspatana.

The nodal explants obtained from healthy mother plants showed a low rate of shoot germination in WPM supplemented with 0.5mg/L of BAP. Whereas, 0.5mg/L TDZ supplemented media showed high rate of regeneration. In a similar study conducted on Musa spp. MS media supplemented with 5.0 mg/L BAP and 0.5 mg/L of TDZ was considered to bethe best concentration. But media supplemented with 2.0mg/L TDZ showed a reduced shoot initiation. Lee (2001) reported that TDZ at 0.91 μM induced the largest number of shoots in Banana, but TDZ at a concentration 9.1 μM inhibited elongation of shoots was and clumps of small globular buds appeared at the base of shoots. A number of similar studies disclosed that high concentration of TDZ inhibit shoot formation or results in the formation of adventitious shoots (Mithila et al., 2003; Lee, 2005; Shirani et al.,2009; Murti et al., 2012)

In callus induction and somatic embryogenesis, NAA, 2,4- D, IBA and BAP at different concentrations formed nodular callus or fragile callus, whereas a combination of NAA and TDZ at various combination and concentration formed embryonic callus. A combination of 0.5mg/L NAA and 0.5mg/L TDZ obtained highest number of explant response. Earlier studies showed that high level of TDZ triggered rapid propagation and high stress levelresulted in direct somatic embryogenesis or by callus formation in strawberry (Biswaset al., 2009; Murti et al., 2012). Murthy et al., (1998) reported that high fraction TDZ induced Callus formation via axillary shoot proliferation and low concentration induces compact-green nodular callus. Either alone or in combination with other growth regulators TDZ also induces high-frequency somatic embryogenesis in many plant species. In Ampelocissus latifolia (belongs to Vitaceae a closely related family to Rhamnaceae)Murashige and Skoog medium supplemented with BAP (0.5 mg/l) and NAA (1.0 mg/l) showed the best result for callus induction (Anand et al., 2018). In Cayratia pedata var. glabra the maximum callusing frequency and a greater number of shoot formation was resulted in the lower concentration and combination of BAP (0.5 mg/l) and NAA (0.2 mg/l) (Sharmila et al., 2020)

Rooting of micro shoots found out that IBA induced best percentage of roots. IBA at 0.5mg/L induced highest number of shoots (91.5%) and average root length was found to be 8.45 cms in length. Although average number of roots per shoot was highest in WPM supplemented with 1.0 mg/L of IBA. IAA and NAA individually at different concentration and with IBA did not show significant results. In the earliest studies of auxinic compounds in rooting and propagation assays, IBA was reported to be more effective than IAA (Preece, 2003). Larsen and Guse (1997) andKester et al., (1990) reported that Indolebutyric acid (IBA) is the most reliable rooting hormone. And is still probably the best hormone for general use because of being non-toxic to plants over a wide range of concentration levels.In many studies that the combination of cytokinins (BAP and kinetin) and auxins [NAA, indole-3-acetic acid (IAA) and indole-3- butyric acid (IBA)] produced better results compared to the use of auxins or cytokinins alone (Yamini et al., 2003; Shirin et al., 2007). But in this study a combination of 0.5mg/L IAA and 1.0mg/L BAP induced 66.6% of root. While BAP alone induced 91.5% of roots. In a similar study, Peter et al., (2007) reported that Indole butyric acid at 10 and 100 M gave 100% rooting and produced more roots per micro shoot and the quality of the rooted shoots was also superior than IAA (Peter et al., 2007).

The hardening of in vitro raised plantlets is essential for better survival and successful establishment. Direct transfer of in vitro raised plants to field/wild is not possible due to high rate of mortality. They are prone to rapid desiccation on transplanting, owing to poor development of epicuticular wax and ineffective control of stomatal function under the high humidity and low levels of illumination that obtain during culture (George and Sherrington, 1984; Sutter, 1985; Brainerd and Fuchigami, 1992). This is also because they regenerate in the culture condition that has been cosseted environment with a very high humidity, varied light and temperature condition and being protected from the attack of microbial and other agents. Direct transfer to sunlight also causes charring of leaves and wilting of the plants (Hiren et al., 2004; Lavanya et al., 2009). The ultimate success of in vitro propagation lies in the successful establishment of plants in the soil (Saxena and Dhawan, 1999). In other words, the survival percentage is determined by the hardening of the plantlets. It is therefore, necessary to accustom the plants to a drier or natural atmosphere by a process called acclimatization or hardening (Deb and Imchen, 2010). The painstaking care required during this period (Murali and Ducan, 1995)

During the transfer of in vitro cultured V. maderaspatana plantlets for hardening great care has been observed. Plantlets were transferred into pots containing soil-rite, vermiculate, coir peat, peat moss, sand and the mixture of sand and soil for acclimatization. The survival rate during the hardening was high in Soil-rite (92.6%). In ex vitro culture of Citrus aurantifolia soil-rite ensured 95% to 100% survival of plantlets (Chaturvedi et al., 2002). Generally, soil-rite is the prime medium for primary hardening in tissue culture laboratories (Cronaur and Krikorian, 1984; Zimmerman and Debergh, 1991). Soil-rite is a combination of perlite, vermiculite and peatmoss. For secondary hardening of plantlets, generally a mixture of press mud cake and sand in a ratio 1:1 is employed (Zimmerman et al., 1991). On the contrary, a study found that vermicompost containing about 20-25 % carbon was found to be a good alternative to soil-rite, for primary hardening in Musa spp (Bhalsing et al., 2001).

The morphological observation of 25 randomly tagged reintroduced individual plants found to be normal as the characters like height of the plant, inter-nodal length, number of leaves, leaf length and leaf breadth were found similar with the plants growing in the natural condition (data not shown). And genetic similarity studies steered by the authors of this paper using RAPD and ISSR markers and dendrogram generated by cluster analysis using the UPGMA methods on Jaccard’s coefficient conducted showed similarity in regenerated plantlets with mother plant (Chandra and Thoyajaksha, 2018).

Conclusion

The present work shows the complete success of protocols developed for in-vitro conservation and micropropagation of V. maderaspatana. Woody Plant Medium was used as prime medium for explant culture. A combination of KN and BAP was found to be effective in in-vitro seed germinations. It was observed that TDZ a cytokine used for the micropropogation is very effective in shoot initiation and callus induction. Among the auxins used IBA showed better root regeneration percentage. The protocols used for the present research were developed with a great efforts and care.The above procedure allows the micropropagation of Ventilago maderaspatana and the proliferation of cell mass, which could be used for studies on organic compounds of pharmaceutical interest and for research on woody climbers.

Acknowledgement

The authors of this paper are thankful to the UGC (University Grant Commission), Government of India, for sanctioning a major research project and financial support.

References

1. Anand DC, Meena R, Patni V (2018). in vitro Callus Induction and Comparative Gc-Ms Analysis of Methanolic Extracts of Callus and Leaf Samples of Ampelocissus latifolia (Roxb.) Planch. Int. J. Pharm. Pharm. Sci. 10(9):68-72.
2. Ayan, AK, Cirak C, Kevserolu K, Sokmen A (2005). The effects of explant types and culture constituents on plant regeneration and hypericin content in Hypericum perforatum. L. Turk. J. Agric. For., 29: 197-204.
3. Barney, Lopez OA, King E (2007). Micropropagation of Cascade Huckleberry, Mountain Huckleberry, and Oval-leaf Bilberry Using Woody Plant Medium and Murashige and Skoog Medium Formulations Danny L. HortScience. 17(3):279-284.
4. Bewley JD (1997). Seed Germination and Dormancy. The Plant Cell. 9:1055-1066.
5. Bhalsing SR, Teli NP, Pawar PK, Saindane PV, Baviskar MP, Maheshwari VL (2001). Tissue Culture Grown Banana: A Cost-Effective Strategy for Hardening; Physiol. Mol. BioI. Plants. 7(2): 185-189.
6. Biswas MK, Dutt M, Roy UK, Islam R, Hossain M (2009). Development and evaluation of in vitro somaclonal variation in strawberry for improved horticultural traits. Sci. Hort. 122(3):409-416.
7. Bonato ALV, Calvo ES, Geraldi IO, Arias CAA (2006). Genetic similarity among soybean (Glycine max (L) Merrill) cultivars released in Brazil using AFLP markers. Genetics and Molecular Biology, 29(4):692-704.
8. Brainerd KE, Fuchigami, LH (1982). Stomata1 functioning of in vitro and greenhouse apple leaves in darkness, mannitol, CO, and ABA. J. Exp. Bot. 33: 388-392.
9. Chandra DR, Thoyajaksha S (2018). Evaluation of Genetic Similarity of Micropropogated Plants of Ventilago maderaspatanaGeartn. using RAPD and ISSR Markers. Eur. Exp. Biol.  8:33.
10. Chaturvedi HC, Singh SK, Sharma AK(2002). Method for regenerating viable and fertile citrus plants by tissue culture from explants, US6485975B1 United States-2002; https://patents.google.com/patent/US6485975B1/en.
11. Chen SL, Yu H, Luo HM, Wu Q, Li CF, Steinmetz A (2016). Conservation and sustainable use of medicinal plants: problems, progress, and prospects. Chin Med. 11(37):1-10.
12. Cronauer SS, Krikorian AD (1984). Multiplication of Musa from excised stem tips. Ann. Bot. 53: 321-328.
13. Cruz-Cruz CA, González-Arnao MT, Engelmann F (2013). Biotechnology and Conservation of Plant Biodiversity. Resources. 2:73-95.
14. Deb CR, Imchen T (2010). An Efficient in vitro Hardening Technique of Tissue Culture Raised Plants. Biotechnology. 9: 79-83.
15. George EF, Sherrington PD (1984). Plant propagation by tissue culture. Handbook and Directory of Commercial Laboratories. Exegetics, Eversely, UK. pp.709
16. Hilhorst HWM (1995). A critical update on seed dormancy. I. Primary dormancy. Seed Science Research. 5: 61– 73.
17. Hiren AP, Saurabh R, Subramanian M (2004). in vitro regeneration in Curculigo orchioides Gaertn. An endangered medicinal herb. Phytomorphology. 54:85–95.
    Kasagana VN, Karumuri SS (2011). Conservation of Medicinal Plants (Past, Present & Future Trends). J. Pharm. Sci. & Res. 3(8):1378-1386.
18. Kawde AB, Batra RJ, Weginwar RG, Akkewar DM, Gond GS, Aparna Y (2014). Preliminary Phytochemical Screening and Bioevaluatuion Studies of Stem Bark of Ventilago maderaspatanaGarten. IJRPC. 4(1): 74-82
19. Kuras A, Korbin M, Zurawicz E (2004). Comparison of suitability of RAPD and ISSR techniques for determination of strawberry (Fragaria X Ananassa Duch.) relationship. Plant Cell, Tissue and Organ Culture. 79:189–193.
20. Lavanya M, Venkateshwarlu B, Devi BP (2009). Acclimatization of neem microshoots adaptable to semi-sterile conditions. Indian J. Biotechnol. 8:218–222.
21. Lee SW (2001). Thidiazuron in the improvement of banana micropropagation. The Second International Symposium on Biotechnology of Tropical and Subtropical Species. 5-9 Nov. Taipei, Taiwan, Republic of China. pp. 1-11.
22. Lee SW (2005). Thidiazuron in the Improvement of Banana Micropropagation. Acta Horticulturae. 692: 67–74.
    Lloyd G McCown, B (1981). Commercially feasible micropropagation of mountain laurel, Kalmia latifolia, by the use of shoot tip culture. Proc. Int. Plant Prop. Soc.30: 421-427.
23. Mathur S (2013).  Conservation of Biodiversity through Tissue culture. RRJMB. 2:1-6.
24. McCown BH, Sellmer JC (1987). General media and vessels suitable for woody plant microculture. In: Bonga JM, Durzan DJ (eds.). Cell and tissue culture in forestry, Vol. 1. General principles and biotechnology. Dordrecht, The Netherlands: Martin us Nijhoff Publishers. Pp. 4-14.
25. Metivier PSR, Yeung EC, Patel KR, Thorpe TA (2007). in vitro Rooting of Microshoots of Cotinus coggygria Mill, a Woody Ornamental Plant: in vitro Cell. Dev. Biol.-Plant. 43:119-123.
26. Minocha R, Jain SM (2000). Tissue Culture of Woody Plants and Its Relevance to Molecular Biology, In: S.M. Jain and S. C. Minocha (eds.), Molecular biology of Woody Plants. 1(3):15-339.
27. Mithila J, Hall JC, Victor JMR, Saxena PK (2003). Thidiazuron induces shoot organogenesis at low concentrations and somatic embryogenesis at high concentrations on leaf and petiole explants of African violet (Saintpaulia ionantha Wendl.). Plant Cell Rep. 21:408–414. 
28. Murali TP, Duncan EJ (1995). The effects of in vitro hardening using triazoles on growth and acclimatization of banana. Scientia Horticulturae. 64(4): 243–251.
29. Murashige T, Skoog F (1962). A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol. Plant. 15:473-497.
30. Murthy BNS, Murch SJ, Saxena PK (1998). Thidiazuron: a potent regulator of in vitro plant morphogenesis. in vitro Cell Dev. Biol. 34P:267–275.
31. Murti, Debnath SC, Yeoung YR (2012). Effect of high concentration of thidiazuron (TDZ) combined with 1H-indole-3-butanoic acid (IBA) on Albion strawberry (Fragaria × ananassa) cultivar plantlets induction Rudi Hari.Afr. J. Biotechnol. 11(81): 14696-14702.
32. Phillips RL, Kaepplert SM, Olhoft P (1994). Genetic instability of plant tissue cultures: Breakdown of normal controls. Proc. Nati. Acad. Sci. USA. 91: 5222-5226.
33. Preece JD (2003). A century of progress with vegetative plant propagation. HortScience. 38:1015–1025.
34. Rameshkumar, Ramakrishnan, Periyasamy R, Lakkakula S, Subramani P, Arockiam SR, Manikandan R (2017). in vitroPropagation and Conservation of Useful Endangered Medicinal Plants with Anticancer Activity. J Mol Biol Biotech. 2(3):1-8.
35. Ravindra KN, Thoyajaksha, Narayanappa M. Sharanappa P (2013). Study of Endophytic Fungal Community from Bark of Ventilago maderaspatanaGaertn. Int J Pharm Bio Sci.  4(1):309 – 316.
36. Sasikala C, Sudhakar Y (2015). Effect of compounds isolated from Filicium decipiens and Ventilago maderaspatanaagainst diabetic nephropathy in streptozotocin induced diabetic rats.  Indian J. Pharm. Educ. Res. 49:146-151.
37. Saxena S, Dhawan B (1999). Regeneration and large-scale propagation of bamboo (Dendrocalamus strictus Nees) through somatic embryogenesis. Plant Cell Rep. 18: 438-443.
38. Seed testing manual (2017 1st October) Retrieved from Seed net India Portal https://seednet.gov.in/GraphReport/SeedGraph.aspx.
39. Shirin F, Hossain M, Kabir MF, Sarker SR (2007). Callus induction and plant regeneration from internodal and leaf explants of potato (Solanum tuberosum L.) cultivars. World J. Agric. Sci. 3:1-6.
40. Sharmila S, Ramya EK, Mownika S, Dhivya SM (2020). Micropropagation of an Endangered and Endemic Medicinal Plant Cayratia pedata Var. Glabra, Asian J. Pharm. Clin. Res. 13(6):191-196.
41. Sharma S, Rathi N, Kamal B, Pundir D, Kaur B, Arya S (2010). Conservation of biodiversity of highly important medicinal plants of India through tissue culture technology- a review. Agric. Biol. J. N. Am. 1(5): 827-833.
42. Shirani S, Mahdavi F, Maziah M (2009). Morphological abnormality among regenerated shoots of banana and plantain (Musa spp.) after in vitromultiplication with TDZ and BAP from excised shoot tips; Afr. J. Biotechnol. 8(21):5755-5761.
43. Sindhura S, Eswaraiah MC (2014). Evaluation of Stroke Preventive Effect of Ethanolic Extract of Ventilago maderaspatanaGaertn.against bilateral carotid artery occlusion (Bcao) induced stroke in rats. Int. Res J Pharm. App Sci. 4(2):1-6.
44. Sutter EG (1985). Morphological, physiological and chemical characteristics of epicuticular wax on ornamental plants regenerated in vitro. Ann. Bot., 55: 321-329.
45. Thakur A, Kanwar JS (2008). Micropropagation of ‘Wild Pear’ Pyrus pyrifolia (Burm. F.) Nakai. I. Explant establishment and shoot multiplication. Not. Bot. Hort. Agrobot. Cluj. 35: 103–108.
46. William E. Finch-Savage, Leubner-Metzger G (2006). Seed dormancy and the control of germination. New Phytologist. 171: 501–523. 
47. Wolfe DE, Eck P, Chin CK (1983). Evaluation of seven media for micropropagation of highbush blueberry. HortScience 18:703–705.
48. Yasmin S, Nasiruddin KM, Begum R,Talukdar SK (2003). Regeneration and establishment of potato plantlet through callus formation with BAP and NAA. Asian J. Plant Sci. 2 (12):936-940.
49. Zimmermann RN, Debergh PC (1991). Acclimatization of micropropagated plants to the green house and field. In: Micropropagation - Technology and Applications. Debergh PC, Zimmermann RH (eds.). Kluwer Academic Publishers, Belgium. pp. 71-90.

Copyright: Copyright © 2020 International Research Journals This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.