In vitro Propagation of Adenia hondala (Gaertn.) de Wilde

Adenia hondala (Gaertn.) de Wilde belonging to the family Passifloraceae is a perennial climbing herb with potential medicinal value. The possibility of in vitro clonal propagation of Adenia hondala was investigated by the use of nodal explants cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of BAP and KN. Optimum treatment was the combination of 1 mg.L-1 BAP and 0.5 mg.L-1 KN that enhanced percent response of explants and the number of multiple shoots per explants. An average of 10.23 shoots per explants was resulted after 32 days of culture. In vitro shoots were elongated in MS medium supplemented with 1 mg.L-1 KN. Half strength MS medium supplemented with 1 mg.L-1 IBA was found to be the best medium for rooting. The rooted plantlets were gradually acclimated ex vitro in mist chamber and successfully established under field conditions with high survival rate.


INTRODUCTION
Adenia hondala belonging to the family Passifloraceae is found in the forests of Western Ghats. It has been red listed as vulnerable in South India. A. hondala is a perennial climber with tuberous roots, simple tendrils, simple and lobed leaves and circular glands between lobes of leaves. The monoecious flowers have oblong petals, 5 stamens, globular ovary and a trifid stigma. The tuber powder is used to treat cough and it increase lactation in nursing mother. The extract of tuber is used to cure intermittent fever, Anonymous, (1999) and the roots are used for the treatment of skin troubles, Anonymous, (2003). Ayurveda is a system of Medicine with historical roots in Indian Subcontinent. 'Vidari ¶ DQ $\XUYHGLF GUXJ LV DQ ingredient of more than 50 Ayurvedic formulations like Chyavanaprash and its annual requirement is about 500± 1000 Metric Tonnes, Sulaiman et al., ( 2014). Ayurveda correlates µvidari ¶ to tubers of Pueraria tuberosa (Roxb. Ex Willd.) DC (Fabaceae) and Kshiravidari to Ipomoea mauritiana Jacq. (Convolvulaceae). However, in Ayurvedic Pharmacopoeia both these species are attributed similar properties and are substituted by each other, Venkatasubramanian et al., (2009). Apart from these, tubers of Adenia hondala (Passifloraceae) and the pith of Cycas circinalis / &\FDGDFHDH DUH DOVR WUDGHG DV µvidari ¶ Ved and Goraya, (2008 ). µVidari ¶ LV XVHG DV DSKURGLVLDF cardiotonic, diuretic and refrigerant, Chopra et al., (1992). In traditional and folklore systems, µvidari ¶ has been used as a tonic, rejuvenator and galactogogue, Mithila et al., (2014). It is reported that use of µYidari ¶ is beneficial in persons suffering from or prone to diabetes and coronary disease problems, Sulaiman et al., ( 2014). Indiscriminate collection, poor seed set and seed germination resulted in the disappearance of this plant from wild habitats. The reason for the diminishing number of A. hondala could be the dwindling forests due to excessive urbanization. Having established its potential as a medicinal plant there is a great necessity for large scale multiplication of this plant which is rapid, simple and genetically stable. In vitro protocols serve as a viable tool for conservation and propagation of germplasm, especially of endangered and threatened plants. There is a single report on in vitro propagation of this plant species (A.hondala) which focuses on in vitro organogenesis and somatic embryogenesis. The present experiment was conducted to develop a successful protocol for rapid clonal propagation of A.hondala through the culture of nodal explants.

Plant sample and experiment design
Stem cuttings with four to six nodes were collected from three months old A. hondala plant. The stem was cut into single node pieces (2 to 3 cm length) and was washed in running tap water for 10 minutes. The nodal explants were then immersed in Bavistin solution (15 g.L -1 ) for 3 minutes with Cefotaxime (200 mg.L -1 ) and Tetracycline (200 mg.L -pressure and 121°C temperature for 15 minutes. All these culture vials were incubated in plant growth room at " ƒ& XQGHU KRXU SKRWRSHULRG ZLWK PRO P-2 s-1 light intensity supplied by cool white fluorescent lamps and 60±65% relative humidity. The nodal explants were then inoculated into MS media (Murashige and Skoog, 1962) supplemented with 3% (w/v) sucrose and 0.8% (w/v) agar. The shoot bud initiation and multiple shoot induction were studied by inoculating the explants on MS media supplemented with 6-Benzylaminopurine -BAP (0.25, 0.5, 1.0, 2.0 mg.L -1 ) either alone or in combination with Kinetin-KN (0.25, 0.5, 1.0, 2.0 mg.L -1 ). Subcultures were carried out at an interval of 14 days. The proliferation rate for each of the treatment was observed. Same media compositions were used to study shoot elongation. During elongation, the length of shoot and the number of nodes were observed and recorded. Half strength and full strength MS media with (0.25, 0.5, 1.0 mg.L -1 ) or without Indole Butyric Acid-IBA were experimented for in vitro rooting. Percentage of root induction, root length and root number were the parameters recorded for each treatment. The rooted shoots were taken out from the culture bottles and washed thoroughly with running tap water to remove traces of medium. These plants were transferred to plastic pots containing a mixture of soil and sand. Almost all rooted plants were acclimated and transferred to field conditions.

Statistical analysis
All experiments were performed with three replications, having 30 samples each. The effect of various treatments on selected growth parameters was measured quantitatively and statistically tested using analysis of variance (ANOVA) using SPSS (Statistical Package for the Social Sciences) version 11.0. The significance of the mean values of various treatments was DVVHVVHG E\ 'XQFDQ ¶V 1HZ Multiple Range Test (DMRT) at p < 0.05.

Effect of BAP and KN on multiple shoot induction and shoot proliferation from nodal explants
Cultures without any growth regulators were taken as control. No shoot induction was observed in control media. The effect of cytokinis on multiple shoot induction was experimented by culturing the nodal explants on MS medium supplemented with BAP and KN either alone or in different combinations. It was observed that, for the same concentration of BAP and KN tested (0.25, 0.5, 1.0, 2.0 mg.L -1 ), percentage of explants showing shoot induction decreased (Table 1) on KN supplemented (75-78%) media than the media with BAP (81-83%). Among various concentrations and combinations, best results were recorded on medium containing 1 mg.L -1 BAP and 0.5 mg.L -1 KN noticed 87% of explants proliferated within 7 days (Table  1).This particular combination of growth regulators regenerated 10.23±0.40 shoots, (Fig 1) which was found to be the single optimum treatment that promoted highest number of multiple shoots within 32days. MS medium with different combinations of BAP and KN favoured comparatively high multiple shoot regeneration than the treatments with BAP or KN alone. Combinations of BAP (1.0 mg.L -1 ) with KN (0.25, 0.5 mg.L -1 ) significantly increased the shoot number as compared to other treatments with BAP and KN alone ( Table 2). In the present study, increased concentrations of BAP (2 mg.L -1 ) adversely affected the shoot multiplication rate.

Effect of KN on shoot elongation
Same media composition as that of the shoot induction media were experimented for shoot elongation. Shoots obtained from KN supplemented media were significantly longer among all treatments including their combinations ( Table 2). Among all the concentrations and combinations of growth regulators used, MS with 1.0 mg.L -1 KN showed better result for shoot elongation after 12 days of sub culturing with an average length of 4.87± 0.15cm and an optimum number of 4.10 ± 0.40 nodes per explants. In the present study, length of the shoots were shorter in BAP as compared to KN either alone or in their combination ( Table  3). Shoots of KN supplemented media were significantly longer among all cytokinin treatment. The presence of KN in the medium allowed the in vitro shoots to elongate where the morphogenetic response was lower in lower concentrations (0.25, 0. 5mg.L -1 ) as compared to higher concentration (1.0 mg.L -1 ). Moreover higher concentrations of BAP (2 mg.L -1 ) inhibited the shoot length compared to its lower concentrations (0.25, 0.5, 1.0 mg.L -1 ) with or without KN.

Effect of Media and IBA on rooting
To induce rooting, in vitro shoots were transferred to full strength and half strength MS media with (0.25, 0.5, 0.1 mg.L -1 ) or without IBA. The effects of media and IBA treatment on root formation from in vitro shoots were summarized on Table 4 -3, Issue-1, Jan-Feb-2018  http://dx.doi.org/10.22161/ijeab/3.1.22  ISSN: 2456-1878 www.ijeab.com Page | 177 weeks of culture. As compared to half strength MS, full strength MS supplemented with IBA resulted in a significant decrease in the percentage of root induction, number of roots and length of roots.

V.
CONCLUSION In vitro propagation through nodal explants of A.hondala is an easy and economic way for obtaining large number of consistently uniform plants.
Present protocol holds tremendous potential to select, multiply and conserve this genotypes, which are a potential resource of medicinally important constituents and it reduce the dependence on the natural habitat for the supply of raw drugs.