IN-VITRO PROPAGATION OF TYLOPHORA OVATA (LIND.) HOOK. EX. STEUD – AN IMPORTANT MEDICINAL PLANT

Received on 18 October, 2013; received in revised form, 12 December, 2013; accepted, 16 February, 2014; published 01 March, 2014

IN-VITRO PROPAGATION OF TYLOPHORA OVATA (LIND.) HOOK. EX. STEUD – AN IMPORTANT MEDICINAL PLANT

R. Jeyachandran* and M. Bastin

Department of Botany, St. Joseph’s College (Autonomous), Tiruchirappalli – 620 002, Tamil Nadu, India

ABSTRACT: Efficient in-vitro propagation of Tylophora ovata (Lind.) Hook. Ex. Steud was carried out in this present investigation.  Nodal segments from the healthy grown plants were used as explants for culturing.  Explants were cultured on standard Murashige and Skoog (MS) medium supplemented with different concentration of benzyl amino purine (BAP) and naphthalene acetic acid (NAA) for primary shoot proliferation. Remarkable shoot proliferation was observed in MS medium containing BAP (2.5 mg/L) and NAA (3.5 mg/L). For rooting of the micro shoot, half strength MS medium supplemented with indole acetic acid IAA (2 mg/L) and 3-indole butyric acid IBA (2.5 mg/L) showed the best result. After acclimatization and transplantation, 100% of the in-vitro shoot delivered was found healthy in ex vivo condition.

Growth hormones, In-vitro propagation, Transplantation, Tylophora ovata (Lind.) Hook. Ex. Steud

INTRODUCTION:Tylophora ovata (Lind.) Hook. Ex. Steud (Family Asclepiadaceae) is an important medicinal plant which is used as a folk remedy for the treatment of bronchial asthma, bronchitis, dysentery, inflammation, dermatitis and allergies. The roots are antitussive, carminative, emetic and expectorant. A decoction is used in the treatment of leukaemia, asthma and coughing, traumatic injuries, rheumatoid backaches, pains in the stomach and abdomen, poisonous snakebites _­­­­­­¹.Tylophora ovata is a Chinese traditional toxic medicine. The alkaloid components of T. ovata possess very strong antitumour activities according to literature ². The roots and leaves are used in hydrophobia; dried leaves are more uniform and certain in their action than the roots.

In large doses, the dried leaves may cause fatal poisoning.The dried leaves are used to treat certain forms of allergy ³. Alkaloids mainly present in T. ovata such as tylophoridicine A, O-methyltylo-phorinidine and tylophorinidine has strong antitumor activity ⁴.

S-(+)-Deoxytylo-phorinidine (CAT) is a new phenanthroindolizidine alkaloid, originally extracted from the roots of Tylophora atrofolliculata and Tylophora ovata, potent anticancer activity was observed in this alkaloid ⁵. Plant tissue culture is the process of small pieces of living tissues (explants) isolated from a plant and grown aseptically for indefinite periods on a semi defined or defined nutrient medium ⁶.

Nodal shoot proliferation has much potential as supporting technology for micropropagation of ornamental and medicinal plants. For micropropagation of novel ornamental and medicinal plants direct shoot proliferation using nodal explants is a major biotechnological approaches ⁷.

Hence, the present study is justifiably planned to propagate the valuable medicinal plant Tylophora ovata (Lind.) Hook. Ex. Steud in in-vitro condition with various combinations of plant growth regulators, and transplant the plants from laboratory into field condition. Therefore, this method is a suitable in vitro propagation methods must be evolved to conserve this valuable medicinal species.

MATERIALS AND METHODS: Tylophora ovata (Lind.) Hook. Ex. Stead was collected from agricultural land in Tiruchirappalli District, Tamil Nadu.  The material was identified by Rapinat Herbarium, St. Joseph’s College, Trichy. The collected plant material was established in our department garden for further study. Murashige and Skoog’s medium was used for the cultivation of Tylophora ovata (Lind.) Hook. Ex. Steud at in vitro condition. The MS medium was prepared by adding required amounts of stock solutions and final volume was made up with distilled water, the composition of the MS medium was given in Table 1. The pH of the medium was adjusted to 5.8 using 1 N NaOH/KCl. About 10 ml of the medium was poured into sterile culture tubes. The culture tubes with the MS medium was autoclaved at 121°C for 20 min. at 15 lbs pressure and transferred to the media storage room where they were kept under aseptic condition for further experimental study ⁸.

The explants were first washed with running tap water for 30 min.to remove the soil particles andother extraneous fine particles. The explants parts such as nodal segment were cut from the healthy plant of Tylophora ovata (Lind.) Hook. Ex. Steud and washed with tap water for 5-10 times, and they were soaked in 0.2-0.5% bavistin and 0.03% streptomycin aqueous solution for 10 min. It was gently washed twice with sterile distilled water. The explants were immersed in aqueous solutions of savlon (1.5% v/v chlorohexidine gluconate solution & 3% w/v cetrimide) for 10 min. Then, the explants were washed twice thoroughly with sterile double distilled water. After this treatment, the explants were surface sterilized with 0.01% HgCl₂ aqueous solution for 1 min, and rinsed thoroughly with sterile distilled water ⁹.

The nodal explants were placed in an erect position in the culture tube containing MS medium supplemented with the plant growth regulators like BAP and NAA at different combinations.

The combinations and their concentrations were mentioned in Table 2. The culture bottles were kept in the growth room at 25±2°C, with a photoperiod of 16 h daylight and 8 h night breaks under the cool white fluorescent light. The explants were inoculated at appropriate condition in culture bottles, and the shoot induction rate was observed, triplicates were used for each treatment ⁸. For root induction, Half strength MS basal medium was supplemented with different concentrations (Table 2) of auxin (IAA) 0.25- 2.5 mg/l and IBA 0.25-2.5 mg/l and pH of the medium was adjusted to 5.8. The surface sterilized explants were cut into small pieces implanted on the culture media. All the cultures were maintained in culture room at 25±2°C for 8- 16 h of day and night break under the cool white fluorescent light ⁹ and the root induction rate was observed ¹⁰.

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TABLE 1: EFFECT OF DIFFERENT CONCENTRATION OF BAP AND NAA NODAL ADVENTITIOUS SHOOT REGENERATION FROM NODAL EXPLANTS OF TYLOPHORA OVATA (LIND.) HOOK. EX. STEUD.

Values represent Mean + SD and each experiment was triplicated.

TABLE: 2. EFFECT OF DIFFERENT CONCENTRATIONS OF IBA AND NAA ROOT FORMATION FROM NODAL EXPLANTS OF TYLOPHORA OVATA (LIND.) HOOK. EX. STEUD

RESULTS AND DISCUSSION: Tylophora ovata have been efficiently regenerated from nodal explants when nodal explants of Tylophora ovata were brought from field grown mature plants cultured on MS medium supplemented with different concentrations of BAP, NAA, and IBA. After the inoculation of explants emergence of adventitious shoot buds was observed on two weeks later. Data were noted on different growth parameters from different treatments. The concentration of Cytokinin hormone has been greatly influenced by axillary shoot regeneration from nodal explants. The explants swelled and became develop necrotic tissue three weeks after inoculation. The best result of nodal explants was obtained from the combination of BAP (2.0 – 2.5 mg/L^-1) and NAA (3.5 – 3.5 mg/L^-1) (fig. 1).

The higher frequency (73.2±1.38) of nodal explant regenerated and the production of maximum number of shoots were observed on MS medium supplemented with combination of BAP (2.0, 2.5 mg/L^-1) and NAA (3.0, 3.5 mg/L^-1) (Table 1; fig. 2).  The corresponding explants of longest shoot length of (5.27±0.17 cm) (Table 1, fig. 3).  To develop for rooting, shoot buds from repeated cultures were transferred to half strength MS media supplemented with different concentrations of NAA, IBA and BAP (Table 2, Fig. 2). For best root initiation was achieved (73.3±1.46) on MS medium containing (2.5 mg/L^-1) NAA , BAP (3.0 mg/L^-1 and IBA (2.5 mg/L^-1) proved to be the most effective for rooting of micro shoots (Fig. 3) than that of any other concentration (Table 2). Root achieved plantlets were developed successfully transferred to the paper cup pot containing sterile soil, sand, and organic manure in the ratio of 1:1:1 (Fig. 4).

Acclimatized field survival rate of this plant was 75%. This protocol was suitable for conservation medicinal.

                   FIG. 1                                 FIG. 2                                             FIG. 3                                              FIG. 4

FIGURE 1:  SHOWING THE IN VITRO REGENERATED PLANTS OF TYLOPHORA OVATA

FIGURE 4: PROCESS OF HARDENING

CONCLUSION: The in vitro-propagated medicinal plants furnish a ready source of uniform, sterile, and compatible plant material for biochemical characterization and identification of active constituents ^{11, 12}. In addition, compounds from tissue cultures may be more easily purified because of simple extraction procedures and the absence of significant amounts of pigments, thus possibly reducing the production and processing costs ¹³. In the present study, a fruitful protocol was set up for Tylophora ovata (Lind.) Hook. Ex. Steud through shoot induction. This protocol can be exploited for the conservation of this valuable germplasm resource.

ACKNOWLDEGMENT: We would like to thank the management of St. Joseph’s College (Auto-nomous), Tiruchirappalli-620002 for providing laboratory facilities to carry out this research work.

REFERENCES:

1. S. R. Singh, R. Singh, A. K. Dhawan. Biochemical Changes related to shoot differentiation in callus cultures of Tylophora indica WIGHI and ARN. J Indian Bot. Soc. 2009. 88(3-4): 49-53.
2. Institute of medical and pharmaceutical science of Zhuang autonomous region of Guangxi. Experimental studies on antitumor agents of Tylophora ovata (Lind.) Hook. Ex. Steud.  Acta Biochim Biophys sin, 1977; 9(2): a 131-138, b. 139-145.
3. Available from: http://clinicalsciences.wordpress.com/ article/ayurveda-traditional-indian-system-of-3fy5eowy8suq3-130/.(Ayurveda-traditional Indian system of  medicine Achievements and contributions to medicine. April 3, 2012).
4. Zhen Yue-Ying et. al., Antitumor alkaloids isolated from Tylophora ovata. Acta Botanica Sinica 2002; 44(3): 349-353.
5. Liu ZJ et. Al. Anticancer effect and neurotoxicity of S-(+)-deoxytylophorinidine, a new phenanthroindolizidine alkaloid that interacts with nucleic acids. J Asian Nat Prod Res, 2011; 13(5): 400-408.
6. Ignacimuthu S. Plant Biotechnology, Oxford and IBH publishing Co. Pvt. Ltd, 1997; 180.
7. Ashok K. Jain and Mudasir Bashir. In-vitro Propagation of a Medicinal Plant Portulaca grandiflora. Hook World Journal of Agricultural Sciences 2010; 6(3): 327-330.
8. Murashige T and Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 1962; 15: 473-497.
9. Tiwari V, Singh BD and Tiwari KN. Shoot regeneration and somatic embryogenesis from different explants of Brahmi (Bacopa monniera (L.) Wettst.). Plant Cell Rep. 1998; 17: 538-543.
10. Anilkumar M and Sajeevan RS. Micropropagation of Musa acuminata colla. Plant Cell Biotechnol. Mol. Biol. 2005; 6: 159-162.
11. Mahendran TS and Sampath P (2005). In vitro propagation of Coleus forskohli – A threatened medicinal plant. Recent advances in medicinal plant research: vision 21st century.
12. Thorat SP, Swanth RB, Garande VK and Patgaonkar DR. Studies on the effect of IBA and NAA on rooting of cutting in nerium. J. Asian Horticul. 2006; 2: 312-313.
13. Wakhlu AK, Bajwa PS. Regeneration of uniform plants from somatic embryos of Papaver somniferum (opium poppy). Phytomorphology, 1986; 36: 101–105.
    

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    How to cite this article:

    Jeyachandran R and Bastin M: In-vitro propagation of Tylophora ovata (Lind.) Hook. Ex. Steud – an important medicinal plant. Int J Pharm Sci Res 2014; 5(3): 1083-86.doi: 10.13040/IJPSR.0975-8232.5(3).1083-86

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