Yogananth et al.

ISSN 0976-2272 J. B io s c i. Re s . ,2012.Vol. 3(3):198-202

Effect of different plant hormones on callus induction in Leaf explant of Dre g e a v o lu b ilis Benth. (Asclepiadaceae)
YOGANANTH, N1*, PALANIVEL, S2, PARVATHY, S3, CHANTHURU, A4. AND BHAKYARAJ, R,5 1 Mohamed Sathak Arts and Science College, Chennai - 600034, Tamil Nadu, India. 2 , 3, Dept of Botany, Govt Arts College, Karur, Tamil Nadu, India 4 JJ College of Arts and Science, Pudukkottai, Tamil Nadu, India 5 Indian Institute Crop Processing Technology, Thanjavur, Tamil Nadu, India Abstract Dregea volubilis is a common shrub plant belonging to the family Asclepiadaceae. The leaf juice was anti-inflammatory medicine to treat several diseases including eye ailments, tracheitis and stomachache. The present work is based on developing a protocol for the callus induction in Dregea volubilis from leaf explants. The sterilized explants were inoculated in MS media containing various combination of auxins such as Indole acetic acid (IAA), naphthalene acetic acid (NAA) and 2,4- dichlorophenoxy acetic acid (2, 4-D) and cytokinins such as kinetin and 6 benzyl amino purine (BAP). The highest efficiency of callus formation was observed in the medium containing different concentration of 2, 4-D and kinetin. In vitro generated callus can be used as a source for the isolation of secondary metabolites from D. volubilis. Key words: Dregea volubilis, Perukurunchan, callus induction, auxin, cytokinin For correspondence: bioyogaa@gmail.com Introduction Dregea volubilis which is commonly known as Perukurunchan in Tamil, is an important medicinal woody climber belonging to the family Asclepiadaceae. It is widely used in Indian traditional medicines and the leaf paste to treat rheumatic pain, cough, fever and severe cold (Muthu et al., 2006 and Rajadurai et al., 2009) leaf paste is taken along with pepper to treat dyspepsia (Pandikumar et al., 2007); bark paste, mixed with hot milk is used internally for treating urinary troubles (Silija et al., 2008) and leaf powder is taken orally along with cows milk have antidiabetic activity (Ayyanar et al., 2008). The stems and leaves contain a pigment taraxerol, a triterpenoid, kaempferol, a glycoside of kaempferol and saponins (Sauer et al., 1965). Due to overexploitation and misuse of medicinal plants, we are faced with the problem of losing our precious plant resource in the future. This situation calls for effective and in time conservation measures to enrich our lives with the services of plants. In this regard, Vinod et al., (2003) stressed the need of conservation and sustainable utilization of biodiversity. Different techniques for conservation of plants have been practiced worldwide, the most important being tissue culture (Parabia et al., 2007). Tissue culture is advantageous 198

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in producing multiple copies of a plant species within minimum time and space. Various studies have been carried out on the biochemical (Jeyachandran et al., 2010 and Maruthupandian and Mohan, 2011), Pharmacological (Thomas et al, 1996; Biswas et al., 2010, Maruthupandian et al., 2010) and in vitro propagation (Arulanandam et al., 2011, Vinothkumar et al., 2011, Yogananth et al., 2011) aspects of Dregea volubilis, but no such in vitro callus culture from leaf explants studies have been carried out in this valuable medicinal plant. The objectives of this study was to investigate the influence of plant growth regulators on induction and growth of D. volubilis callus culture as a starting point to produce bioactive compounds in plant cell culture. Materials and methods Young leaves were collected from mature field grown healthy plant of Dregea volubilis maintained in green house at J.J. College of Arts and Science, Pudukkottai, Tamil Nadu, India and washed thoroughly under running tap water and then treated with a few drops of Tween-80 and 1% Savlon for 10 minutes with constant shaking. This is followed by successive three washing with distilled water to make the material free from savlon. Again the explants were washed with 70% ethyl alcohol for few seconds and washed with distilled water for 3-4 times. After that, the explants were transferred to laminar air flow chamber and disinfected with 0.1% HgCl2 for 2 minutes and washed with sterile distilled water for 5-7 times. Then, the explants were placed in sterile Petri plates before inoculation. The sterilized explants were injured all over the surface and used for callus induction. The excised explants were cultured on MS (Murashige and Skoog, 1962) medium augmented with different concentrations of auxins like IAA, NAA

Yogananth et al. and 2, 4-D (1.0, 1.5, 2.0, 2.5 and 3.0 mg/l) along with cytokinins like KIN or BAP (0.5 mg/l) , 3% sucrose and 0.8 to 1% agar with pH adjusted to 5.8 before the addition of agar. Culture tubes containing medium were autoclaved at 121C for 15 lbs/inch2 for 15 min. All the inoculated cultures were incubated in growth room in controlled conditions at a temperature of 25 2C, 16 h light/8 h dark photoperiod and continuous illumination was provided by cool white fluorescent tubes at 2000 lux. Each experiment was repeated thrice. Analysis of variance was carried out and the differences between the treatments were determined by DMRT at 5% level of significance using SPSS (SPSS ver. 16.0). Results and Discussion Leaf pieces were used as a primary explants for callus induction. Callus initiation was observed from cut surface of leaves after 3 to 4 week of culture initiation. The leaf explants responded differently based on the concentrations of auxins and cytokinin present in the medium. In general, media containing high auxin and low cytokinin concentrations promote cell proliferation resulting in callus formation (Slater et al., 2003). Plate 1: In v itro callus induction from leaf explant of D. v o lu b ilis Stage wise development in 2, 4-D + KIN concentration A B C

Callus induction in earlier stage 199

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Yogananth et al. was obtained in 1.0 mg/l NAA and 0.5 mg/l KIN combination.
FIG. 2: INFLUENCE OF NAA ON CALLUS INDUCTION, CALLUS GROWTH OF YOUNG STEM EXPLANTS IN COMBINATION WITH KIN AND BAP
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Callus in matured stage Maximum amount of callusing (81.58 1.36 %) was observed on the medium supplemented with combination of 2, 4-D and Kin (2.0 mg/l 2, 4-D, and 0.5 mg/l KIN) after 3 weeks of culture initiation (Fig 3 and Plate E). Minimum response(54.40 + 0.99 %) was obtained in NAA 1.0 mg/l and BAP 0.50 mg/l combination (Fig 2). All the calli derived from leaf explants were pale green and friable in nature (Plate 1, 2, and 3). The results obtained were agreement with the previous reports by other investigators, Gymnema sylvestre (Gopi and Vatsala, 2006; Roy et al., 2008) and Ceropegia juncea (Nikam and Savant, 2009).
FIG. 1: EFFECT OF IAA ON CALLUS INDUCTION, CALLUS GROWTH OF YOUNG STEM EXPLANTS OF Dregea volubilis (L.f.) IN COMBINATION WITH KIN/BAP.
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Among the various concentrations of auxins tested, 2, 4-D with KIN was more effective for callus induction than IAA (Fig 1) and NAA as a source of auxin in leaf explants tested. George, (1996) reported that the 2,4-D shows effect on the RNA metabolism by inducing the transcription of messenger RNA capable of coding proteins required for the growth and hence, promoting a chaotic cell proliferation, i.e., callus formation. In vitro generation of callus can encourage in vitro mass production of bioactive compounds of health benefits from Dregea volubilis plant.
FIG. 6: ROLE OF 2,4-D ON CALLUS INDUCTION, CALLUS GROWTH OF YOUNG LEAF EXPLANTS IN COMBINATION WITH KIN AND BAP
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Conc of IAA with KIN and BAP

The maximum growth rate in terms References of fresh weight (3.56 0.44 g) and dry Arulanandam, L.J.P., Kumar, S.G. and weight (0.33 0.03 g) was observed in the Sovimini, M. 2011. Micropropagation and combination of 2, 4-D 2.5 mg/l and KIN conservation of Medicinal plant Wattakaka 0.5 mg/l. Minimum growth rate 1.19 + 0.34 g fresh weight and 0.10 + 0.02 g dry weight
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