DIRECT ORGANOGENESIS OF A CRITICALLY ENDANGERED MEDICINAL LIANA, COSCINIUM FENESTRATUM (GAERTN.) COLEBR. (MENISPERMACEAE)

An efficient and reproducible regeneration protocol via direct organogenesis has been established using nodal explants of Coscinium fenestratum. Nodal explants were inoculated on MS medium supplemented with BAP and TDZ at 2.0 and 1.0mg/L respectively were produced high frequency of shoots (91.01%). The elongated shoots subcultured on half strength MS medium supplemented with IBA alone at 0.6mg/L produced high frequency of roots (97.42%). Rooted plantlets were acclimatized and established in garden soil, sand and vermicompost in the ratio 1:1:1 with good survival rate under greenhouse conditions. This protocol can be efficiently used for mass multiplication and conservation of this critically endangered medicinal liana in its native habitat.


INTRODUCTION
Plants with medicinal properties have been known for thousands of years and have been used as traditional medicines by the people to treat disease. Traditional medicine has remained as the most affordable and easily accessible source of treatment in the primary healthcare system of developing countries including India. Medicinal plants have a great importance due to their potential uses in pharmaceutical industries. Plant tissue culture is well known biotechnological tool for the rapid propagation of medicinal plants for the purpose of commercialization and conservation (1). The species which are failed in successful propagation through seeds or lacking vegetative reproduction can be multiplied through in vitro regeneration by employing this technique. Greater demand for these plants especially for the purpose of food and medicine is one of the causes of their rapid depletion from primary habitats. Micropropagation offers a great potential for large scale multiplication of such useful species and subsequent exploitation and also the application of this technology provides materials required for the isolation of drugs by the pharmaceutical industries without depleting natural plant resources.
The plant, Coscinium fenestratum (Gaertn.) Colebr. (Menispermaceae) is widely used as a medicinal plant in many Southeast Asian countries, mainly Oorali tribes of Idukki district and Kaadar tribes of Thrissur district for fever, muscle pain, abdominal pain, inflammation, ulcers, wounds, jaundice, burns, skin diseases, snake bite poisoning and diabetic food supplements (2). It is a main source of berberine in southern part of India (3,4).
It is estimated that about 114 tons of woody stem bark of this species is extracted annually from the Western Ghats. Due to these wide medicinal importances, this species is severely exploited from its wilds in the western part of Tamil Nadu, India (5). In light of this fact, the present study was aimed to carry out the in vitro regeneration studies in the endangered medicinal liana, C. fenestratum.

Plant material and surface sterilization
Healthy and immature nodal segments of the study species, C. fenestratum were collected from Velliangiri hills, Coimbatore district, Tamil Nadu, India is used as explant (Fig. 1). These segments were washed under running tap water followed by treatment with a surfactant, tween 20 (5% w/v) for 5 min. To eliminate the fungal contamination, the explants were treated with carbendazim (5% w/v) fungicide also for 5 min followed by 2 or 3 rinses in sterile double distilled *Correspondence: Karthika, K., Department of Botany, Kongunadu Arts and Science College, Coimbatore -641 029, Tamil Nadu, water. To eliminate bacterial contamination, Red soil + sand 1:1 explants were also treated with antibiotics (5% w/v) (Ampicillin and Rifampicin) for 5 min Decomposed coir waste + perlite + vermicompost 1:1:1 followed by three rinses in sterile double distilled water. Furthermore, surface sterilization was carried out by dipping the explants in 0.1% mercuric chloride (HgCl2) for 3 minutes followed by 3-4 rinses in sterilized double distilled water to remove traces of HgCl2.

Establishment of culture and shoot regeneration
For the shoot induction, nodal segments were inoculated vertically on MS medium (6) containing different concentrations and combinations of growth regulators such as BAP (ranging from 0.5 -3.0 mg/L), TDZ (0.5 mg/L and 1mg/L), 2,4-D (0.5mg/L) and Kn (0.5mg/L). These cultures were incubated at 25±2°C temperature under light. The percentage of explants responding, number of shoots per explant and length of the shoots can be recorded after 40 days of culture.

Rooting of in vitro derived shoots
The proliferated shoots of 2-3cm length will be excised from the culture and transferred to half strength MS medium supplemented with various concentrations of auxin (IBA and IAA ranging from 0.2 to 1.6 mg/L and NAA ranging from 0.5 to 3.0 mg/L) for root formation. After two weeks, the rooting attributes viz., the per cent shoots responding for rooting, number of roots per shoot and root length will be measured.

Percentage of response
The percentage of response of explants can be calculated as per the following formula:

Number of explants responded
Twenty five plantlets per potting mixture will be tested in the green house and growth rate can be calculated after 30 days of hardening. After acclimatization, the plantlets will be exposed to natural environmental conditions.

Observations and data analysis
The cultures were regularly subcultured on fresh medium after 4-5 week interval. The experiments were repeated thrice with twenty five replicates per treatment. All the values were expressed as mean ± standard deviation (SD) of two determinations and subjected to one-way analysis of variance (ANOVA) followed by post hoc Duncan's multiple range test using SPSS software (version 9, SPSS Inc., Chicago, USA). p < 0.05 was chosen as the criterion for statistical significance.

Shoot regeneration
The response of nodal explants for direct shooting was varied according to the combinations and concentrations of the growth regulators, BAP, TDZ, 2,4-D and Kn in the MS medium. The MS medium containing the growth regulators, BAP and TDZ at 2.0 and 1.0mg/L respectively exhibiting high degree of shooting characters like per cent explants response to shoot initiation (91.01%) and number of shoots per explant (3.87 shoots/explant) and shoot length (5.92cm) ( Table 1, Fig. 2(b-g)). Due to better response of node for direct shooting, the node derived shoots were taken for further studies on rooting and hardening to derive the elite Percent response = Number of explants cultured × 100 plantlets.

Rooting of in vitro derived shoots 2.5. Hardening of plantlets in the greenhouse
Rooted shoots will be thoroughly washed to remove the adhering gel followed by dipping in 1% (w/v) fungicide, Bavistin solution for 10-15min to remove the fungal contamination. The plantlets will be transplanted to various types of sterilized potting media as detailed below: The rooting attributes such as percentage of shoot produced roots, number of roots produced per shoot and root length have been significantly varied depending on the concentration of individual supplementation of auxins like IBA, IAA and NAA in half strength MS medium for the study species, C. fenestratum are exhibited in Tables Fig. 2h). However, the rooting percentage was Vermicompost + red soil 1:1 significantly reduced at the lower concentrations of IAA and NAA.

Acclimatization
Hardening experiments were conducted for the study species by using various hardening media to determine the survivability rate of plantlets. For the node derived (88%) in vitro regenerated plantlets, the survivability rate was significantly higher in the hardening medium composed by garden soil, sand and vermicompost in the ratio of 1:1:1 by volume followed by the hardening medium consisting of red soil, sand and vermicompost in the ratio of 1:1:1 by volume for node derived (78%) in vitro regenerated plantlets (Table 3, Fig. 2i). Table 3. Effect of different composition of hardening medium on survivability rate of node derived plantlets of the species, Coscinium fenestratum.

DISCUSSION
Generally, in vitro regeneration techniques have been found to be effective in circumventing cross ability barriers encounted in conventional methods. A wide range of Menispermaceae plants have now been successfully propagated using in vitro techniques (Tinospora cordifolia (7,8), Tinospora formanii (9) and Cissampelos pareira (10). This technique facilitates the introduction of successfully produced in vitro plantlets into the suitable micro-sites in natural communities to enhance the population of valuable plant species which have failed/less efficient in natural reproduction processes. The supplementation of growth hormones like auxins and cytokinins individually or in combinations with MS medium at different concentrations is having varied response with respect to callus formation and organogenesis in many plant species (11). In the present study, the in vitro regeneration responses of the study species, Coscinium fenestratum are discussed below.
The subculturing of node for shoot formation of the species, Coscinium fenestratum onto the MS medium containing BAP (2.0mg/L) + TDZ (1.0mg/L) was found to be most effective. Cytokinins are one of the most important hormones for shoot proliferation in many plant species (12). Further, a wider survey of literature suggests that BAP is the most reliable and effective cytokinin (13). Kyozuka (14) reported that cytokinins can induce activation of meristems and cause shoot proliferation. Thomas and Gangaprasad (15) already reported the requirement of more quantity of BAP for effective shoot formation in the species, Enicotema axillare. Amin et al. (16) explained that in many cases, the growth regulators like many kinds of cytokinins served as better available source of nitrogen for organogenesis particularly shoot formation. Similarly, in Vitex negundo, Sahoo and Chand (17) reported BAP as the most effective growth hormone for shoot bud induction. Multiple shoots were formed from epicotyl explants of Coscinium ferestratum on MS medium supplemented with cytokinins (18,19). Huetteman and Preece (20) reported that Thidiazuron is a potent cytokinin hormone used for the growth of woody plants in tissue culture.
The root induction during the subculturing of node derived shoots was significantly higher in the half strength MS medium with IBA at 0.6mg/L. It indicates that the growth regulator, auxin is most essential for rooting attribute. Similar trend of results have been reported in other Menispermaceae member, Tinospora cordifolia (8). Tanimoto (21) have already reported the importance of auxins in the root formation during the subculturing of secondary explants. Similar kind of observation on the low level requirement of auxins for better rooting was reported in many species (22)(23)(24).
Hardening is a crucial step prior to transplantation of plant to soil. The well developed plantlets of the study species were acclimatized in various potting media. The node derived plantlets were responded well in the medium containing garden soil, sand and vermicompost. It may be due to the presence of suitable physical and chemical conditions of respective potting media for the survivability of these study species. The same hardening medium compositions were used by Jamuna and Paulsamy (25,26) for the growth of the medicinal plant, Hypochaeris radicata. These specific hardening media was also recommended for the micropropagation of epicotyl explants of the study species, Coscinium fenestratum for better survival in the field and they were morphologically similar to the mother plants (19).
From the present study, it can be concluded that the auxin and cytokinin interact synergistically to control the balance of cell division and differentiation. The protocol developed can be used to regenerate the species massively and hence for commercial purpose.