Micropropagation of the Indian Birthwort Arsitolochia indica L .

Aristolochia indica L. is a medicinal woody perennial climber plant of immense pharmaceutical value. The species is endangered with possible extinction due to its indiscriminate harvesting as raw material for pharmaceutical industry, to manufacture drugs against cholera, inflammation, biliousness, dry cough and snake bite. A rigorous attempt has been made for development of in vitro propagation procedure for this species, involving four steps, namely: culture establishment, shoot multiplication, rooting and hardening. Aseptic cultures were established by growing nodal segments (1 to 1.5 cm) as explants on Murashige and Skoog (MS) medium containing 5.0 μM N6-Benzyladenine (BA). Five nutrient media, MS, Woody Plant Medium (WPM), Gamborg Medium (B5), Nitsch and Nitsch Medium (NN), and Schenk and Hildebrandt Medium (SH) supplemented with different cytokinins and auxins at a concentration of 10.0 μM were used in this study. Ads at 10.0 μM proved optimum for in vitro shoot multiplication. The treatment resulted in 100% shoot number per explant at 15 days and 61.9% at 30 days on MS medium, 65.2% node number per shoot at 15 days and 196.2% at 30 days on WPM medium and 147.5 and 366.6% node number per explant at 30 days after inoculation on MS medium. The in vitro


INTRODUCTION
Aristolochia indica L. (family-Asclepiadaceae.) is a perennial climber with greenish whitish woody stem growing throughout India especially in the tropical and sub-tropical regions. The active constituent "Aristolic acid" is potent drug used in Ayurvedic, Sidda and Homeopathy systems of medicines. Roots are widely used in joint pains and seeds in inflammation, biliousness, dry cough and dyspepsia. The juice of leaves or roots is said to be a specific antidote for cobra poisoning (Kirtikar and Basu, 1987). The species is rare and endangered with extinction due to its indiscriminate collection and over exploitation from natural resources for commercial purpose by pharmaceutical industries (Rahman, 2001). The conventional propagation is hampered due to low seed viability and poor rooting of vegetative cuttings and emphasizes need for the alternative in vitro propagation method for large scale multiplication, improvement and conservation of the species. The objective of the study was to develop an efficient protocol for its micropropagation.

MATERIALS AND METHODS
The selected (mother) plant from Jabalpur area of Madhya Pradesh, India (Figure 1a) was used to collect twig (s) (Figure 1b), which were washed thoroughly for 15 min under running water for removing the surface debris. The washed twigs were defoliated and cut into nodal explants (approximately 1 to 1.5 cm long and 0.5 to 0.6 cm diameter) (Figure 1c). These explants were washed with 2% Cetrimide ® and kept for 10 min with constant vigorous (shaking 150 rpm) on an orbital shaker incubator followed by rewashing 4 to 5 times with distilled water to remove traces of Cetrimide ® . The washed explants were sterilized for 5 min with HgCl2 (0.1%) and Bavistin ® (1.0%) in the laminar flow cabinet. Finally, the surface sterilized nodal explants were rinsed 4 to 5 times with sterile distilled water and inoculated on MS medium (Murashige and Skoog, 1962) supplemented with 5.0 µM BA for culture establishment ( Figure 1d). The in vitro shoot multiplication (Figure 1e-f) was standardized through a factorial randomized experiment, using single nodal segments from established cultures. In this experiment we screened five nutrient media [MS (Murashige and Skoog, 1962), WPM (Lloyd and McCown, 1980), B5 (Gamborg et al., 1968), NN (Nitsch and Nitsch, 1969) and SH (Schenk and Hildebrandt, 1972  ).

Hardening and transplantation
The in vitro raised plantlets were removed from rooting medium washed with distilled water and the plantlets were subsequently transferred to root trainers containing autoclaved soilrite (Figure 3a) and covered with perforated polythene to maintain humidity which were kept under culture room conditions for about 10 days. Subsequently, they were transferred to perforated polythene bags and kept initially in washing room for 5 days and finally transferred to natural condition (Figure 3b-d).

Statisticaly analysis
Each experiment had three replicates for in vitro shoot multiplication and rooting. Each replicate had 10 propagules. The data were subjected to two way (factor) analysis of variance for both the experiments with "F" test for ascertaining level of significance. If the data were found significant at p ≤ 0.05, LSD0.05 was computed for comparison of treatment means.

In vitro shoot multiplication
The effect of cultured media, cytokinin sources and their     436% at 30 days in comparison to TDZ, which produced the lowest value for the parameter. As for interaction, SH medium with 10.0 µM Ads registered the highest value for the parameter at 30 days after sampling.

In vitro adventitious rooting
Auxin sources and their combinations with different media induced significant rooting and root number per explant at both the stages of sampling.

Percent rooting
SH medium produced significantly high percent of rooting. The enhancement of rooting in SH medium was 327.8% at 21 days and 655% at 28 days in comparison to MS medium. MS, B 5 , NN and WPM produced minimum effect on rooting. NAA produced significantly maximum rooting (%), which was 228 at 21 days and 443.7% at 28 days after inoculation in compared to IAA producing minimum value for rooting. SH medium with10.0 µM NAA maximum rooting at both stages of sampling (Table 4).

Root number per explants
SH medium produced maximum root number per explant at both the stages of sampling. The enhancement of root number per explant was 4300% at 21 days and 394% at 28 days after inoculation in comparison with WPM, MS and NN medium. NAA was found to have significant effect on root number per explant at both stages of sampling and resulted in 800% at 21 days and 2900% at 28 days more than that obtained in IAA. SH medium  (Table 5).

DISCUSSION
The micro-propagation of A. indica comprises four steps, namely: establishment of culture from nodal explants, shoot multiplication, root induction and hardening and acclimatization. The present investigation was intended for the standardization of culture medium and plant growth regulators at second and third steps followed by hardening procedure. For shoot multiplications, the best in vitro combination was SH medium supplemented with 10.0 µM Ads. There is no published report on the in vitro shoot multiplication of A. indica using SH medium. The suitability of SH medium in the present study contrasts with earlier reports of micropropagation for this species wherein MS medium was found to be the most effective (Siddique et al., 2006a;2006b;Pattar and Jayraj, 2012).
The results indicate that the species requires low amount of nitrogen for growth and differentiation of new shoots. Adenine sulphate was found as the most suitable cytokinin for shoot multiplication. Similar results have been reported in the medicinal plant Cichorium intybus also, where multiple shoots proliferation was observed on medium supplemented with BA, IAA and adenine sulphate (Nadagopal and Ranjitha Kumari, 2006). For in vitro rooting also, better performance was obtained on SH medium. High concentration of thiamine (Vitamin B 1 ) included in SH medium seems to be synergistic with auxins for facilitation of rhizogenesis as reported in teak by Ansari et al. (2002). Of the various auxin treatments, NAA was found to be the best auxin for A. indica. Superiority of NAA for in vitro rooting may be attributed to its synthetic nature and stability. Further, NAA also eludes the auxin oxidizing/ degrading enzyme systems of the plants (Jacobs, 1972). IAA was found to be inferior to both NAA and IBA. In literature also there are reports of IBA and NAA being more effective than IAA, because of the instability of the latter (Gaspar and Coumans, 1987).

Conclusion
The study demonstrates successful development of in vitro propagation procedure for A. indica. The procedure offers a potential system for conservation and mass propagation using explants derived from mature plants. SH (medium supplemented with 10.0 µM Ads has been found the best for efficient and rapid multiplication of in vitro shoots, while SH medium supplemented with 10.0 µM NAA for optimum induction of in vitro adventitious roots. Further, the hardening procedure reported here ensures 70 to 80% field survival of micropropagated plants of A. indica.