In Vitro Rapid Multiplication of Coldenia Procumbens L. from Shoot Tip Explants G

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Journal of Advanced Applied Scientific Research (JOAASR)

Journal of Advanced Applied Scientific Research - ISSN: 2454-3225 G. Jahirhussain et.al JOAASR-RTB-2016- February-2016:18- 28

In vitro rapid multiplication of Coldenia procumbens L. from shoot tip explants G. Jahirhussain*, S. Palanivel , V. Tamilselvan, V. Muniappan, K. Deepa, R. Veerappan P.G. and Research Department of Botany, Government Arts College (Autonomous), Karur -639 005. Tamilnadu, India. Corresponding Author E. mail: [email protected]

Abstract: An efficient protocol for in vitro rapid multiplication of Coldenia procumbens L. has

been developed. The shoot tip explants were cultured on MS basal medium supplemented with

different concentrations of BAP and KIN. The two cytokinins are tested, BAP was found to

develop higher number of shoots from the shoot tip explants when compared to KIN. Higher

number of shoots was produced from all the concentrations of BAP and KIN. The highest

frequency of 100% shoot induction was observed on MS basal medium supplemented with

6 μM BAP and 10 μM KIN. The number of shoots observed on the MS basal medium

supplemented with BAP obtained 14 as well as 11 in KIN. The data's are recorded after 30days

of inoculation.

Key words : Coldenia procumbens L., cytokinins, shoot tip explants.

Journal of Advanced Applied Scientific Research - ISSN: 2454-3225 G. Jahirhussain et.al JOAASR-RTB-2016- February-2016:18- 28

INTRODUCTION grounds, stems reaching 10-50cm long, Coldeniaprocumbens Linn.(Boraginaceae) shaggy with white hairs, branches often is an annual herb, common weed in India. numerous. It is distributed in tropical and It is found widely in south India on waste subtropical zones and found widely in south lands, common in dry rice grounds. The India3-6. Leaves: Crisped, alternate, short [1] [2] genus having 24 species of prostate. and sessaile 2-6×1.2-2cm, obovate to Coldenia procumbens is only species of its oblong, rounded at the apex, warty hair with genus has a place both in the Hortus rosette of basal cells, stomata anomocytic, Bengalensis and Moon’s Catalogue of palisade adaxial, veins 4-6 pairs on each [3] [5],[6] ceylon. This plant is widely used in side. Flowers: Small, 3-4mm long, traditional medicines in india, Africa, white, sessile, inconspicuous, solitary, malaysia. Acetone, water, methanolic axillary, nearly sessile; calyx- 1-1.5cm, extract of dried aerial parts shows weak lobes lanceolate to ovate lanceolate, corolla angiotensin-converting enzyme inhibition a pale yellow and very small.Sepals-5, in vitro. [4] broadly lanceolate, 2.5mm long, petals-5, Taxonomic classification of Coldenia united below into a short tube, stamens-5, procumbens Linn. inserted styles-2.[6],[7] Fruit:3-4mm long, Kingdom: Plantae Composed of four cells wrapped in calyx, Division: Tracheophyta with single seed in each cell. Fruit initially Class: Magnoliopsida splitting into 2 halves, later each half into 2 Order: Boraginales one seeded nutlets with distinct beak.[3] Family: Boraginaceae Fresh leaves of Coldenia Genus: Coldenia procumbens ground and applied to Species: Coldenia procumbens Linn. Rheumatic Swellings, equal parts of dried Synonyms: Creeping Coldenia; Ayurvedic powder mixed with seeds of fenugreek : Tripakshee; causes Suppurations of boils. Fresh leaves Vernacular Names: are used to promote maturation, the Sanskrit - Tripakshee ; decoction of Coldenia procumbens leaves English - Creeping Coldenia when mixed Hindi - tripungkee; with Centella asiatica and Madhuca Telugu -hamsapadu ; longifolia and Ixora coccinia would give Tamil - seruppadai or cherupadai significant result in wound healing, plant It is a prostrate herb usually lying extract also used in treatment of piles, [1] ,[2], [3], [4], [8] ,[9] quite flat on the ground,common on dry rice Leucorrhoea, Menorrhagia. 19

Journal of Advanced Applied Scientific Research - ISSN: 2454-3225 G. Jahirhussain et.al JOAASR-RTB-2016- February-2016:18- 28

Coldenia procumbens having the Materials and Methods glycosides, phytosterols, proteins, amino Source of Explants acids, fixed oils flavonoids, gums and The field grown Coldenia mucilage as a chief constituents. Alkaloids procumbens Linn. (Boraginaceae) was and tannins are higher in alcoholic extract selected for the source of explants in the than in water extract. Reducing sugars and present Study. Shoot tip segments were phenols are higher in water extract than in used as explants for micropropagation. alcoholic extract. Non-reducing sugars and steroids are equally present in both the Culture Medium extracts. Saponins and fixed oils & fats are The nutrient medium consists of present only in water and alcoholic extracts inorganic salts, carbon source and organic [10] ,[11] respectively. supplements. In addition, vitamins and Micropropagation is one of the growth regulators are also added to the important tool for conservation of medium. In the present study, the basal medicinally important plants. It’s also a medium consists of the mineral salts and viable tool for rapid multiplication within a organic nutrients of Murashige and Skoog short duration. It is a method for production [14] [15] (MS) salts with B5 vitamins are used. of clones. This method conducted under For convenience, throughout this chapter, laboratory condition and it has a chance to MS medium with MS salts plus B5 vitamins produce genetically homogenous plants. is being referred as MS medium. The basal Micro propagation method is highly impact medium is supplemented with various in Plant breeding, Horticulture and concentrations and combinations of Forestry. It is a suitable method for different growth regulators. obtaining a large quantity of genetically homogenous and healthy plant material Growth regulators [12] which can be used for planting. This The prwsent study the hormone technique is an alternative method of concentration was used in µM (micromole). propagation as there is an increase in the These growth regulators were used as supplement to the basal medium propagation rate of plants, availability of individually as well as in different plants throughout the year, protection of combinations. plants against pests and pathogens under Cytokinins: BAP (6benzylaminopurine) controlled conditions and the availability of KIN (6-furfurylaminopurine) uniform clones and uniform production of [13] secondary metabolites. 20

Journal of Advanced Applied Scientific Research - ISSN: 2454-3225 G. Jahirhussain et.al JOAASR-RTB-2016- February-2016:18- 28

Preparation of culture medium Sterilization of explant

The present study was performed in the The explants were taken from the field basal medium with MS salts, B5 vitamins, grown mature plants. The explants 3% sucrose and 0.8% agar.The basal consisting of the shoot tip were surface medium was variously supplemented with sterilized by rinsing in running tap water for factorial combinations of different growth 30 minutes. Then they were washed in an regulators ranging from 2 – 10 µM BAP and agitated solution of liquid detergent KIN for shoot multiplication. After adding (Teepol) for 5 minutes and followed by all the supplements (various concentrations distilled water for 2-3 times for removing of different hormones) to the basal medium, the traces of liquid detergent. After the pH of the medium was adjusted to 5.6. thorough washing, the materials were taken

Sterilization of Culture Medium and in to the Laminar Flow Chamber where they Glassware's were disinfected with 70% alcohol for 30- 60 seconds followed by 0.1% mercuric The culture medium containing high chloride for 3-5 minutes. Finally, the concentration of sucrose supports the materials were thoroughly rinsed with growth of several microorganisms. These sterile distilled water for 4-5 times to microbes generally grow much faster than remove the traces of mercuric chloride. the explants and finally spoil the culture. So it is very essential to maintain a complete Inoculation Procedure aseptic environment inside the culture tube. Before starting inoculation all the Therefore, the culture medium, glassware's, requirements such as culture tubes, forceps and scalpels was sterilized by containing media, spirit lamp, sterile water, autoclaving at 1.06 kg cm-2 and 121°C for glassware and explants, were placed in the 15 min. As well as the same procedure to laminar air flow chamber. The platform follow the sterilization of. During this surface of the chamber was swapped with period much care was taken to avoid 70% alcohol. After swapping the chamber denaturation of growth regulators and with 70% alcohol, the UV light was vitamins that were incorporated into the switched on for 30 minutes. After 30 medium. The culture tubes left free until minutes, the UV light was switched off and agar in the medium become solidified. Then the white fluorescent light was switched on. the tubes were transferred to inoculation Before inoculation, hands were rinsed with chamber for inoculation. absolute alcohol. The instruments were

Journal of Advanced Applied Scientific Research - ISSN: 2454-3225 G. Jahirhussain et.al JOAASR-RTB-2016- February-2016:18- 28 sterilized by dipping in absolute alcohol combinations or whichever is the best for followed by flaming and cooling. further multiplication. To facilitate higher number of shoot formation, the explants The inoculation was carried out in were also subcultured on conical flasks the vicinity of flame. The surface sterilized and/or culture bottles which can provide explants were aseptically transferred to the more space and more medium for growth respective culture media in the Laminar and multiplication. Flow Chamber. The explants were taken out from beaker and at the same time the cotton Experimental Design, Data Collection plug of the culture tube was slightly opened and Statistical Analysis in front of the spirit lamp flame, the explant was put it the medium and immediately The design of all the experiments covered with cotton plug. The explants with was a complete randomized block and each nodal regions were inserted in the medium experiment consisted of five explants per vertically. Cultures were transferred to flask and five replicate culture flasks per fresh media with the same hormone concentration at 4 week intervals. plant growth regulator treatment. The parameters recorded were frequency Culture Conditions (number of cultures responding in terms of The cultures were maintained in a multiple shoot proliferation and root culture room at 25±2°C under 16 hr development), number of shoots per photoperiod with a light intensity of 30-40 explant, shoot length, number of roots per µM m-2 s-1 supplied by cool white fluorescent tubes. These growth conditions shoot, root length and survival rate (%). All were referred to as standard culture of the experiments were repeated five times. conditions for in vitro studies. The analysis of variance (ANOVA)

Culture Maintenance appropriate for the design was carried out to detect the significance of differences among The shoot tip explants regions, were initially cultured on MS solid medium the treatment means were compared using in test tubes. After 4 weeks, the initiated Duncan’s Multiple Range Test (DMRT) at shoot multiples were subcultured on MS a 5% level of significance. [16] basal medium fortified with the same growth regulator concentrations and

Journal of Advanced Applied Scientific Research - ISSN: 2454-3225 G. Jahirhussain et.al JOAASR-RTB-2016- February-2016:18- 28

Results and Discussion induce more number of shoots when compared to KIN. Among different Shoot Multiplication concentrations of BAP, the MS basal Shoot tip explants of Coldenia procumbens medium supplemented with 6 µM BAP Linn. were cultured on MS basal medium showed the highest number of 10.6 shoots supplemented with different concentrations per explant and mean shoot length of 5.42 of BAP and KIN of both these cytokinins cm. ranging from 2 - 10 µM for shoot The basal medium fortified with different multiplication. Multiple shoots were concentrations of KIN induced less number initiated within 15 days of inoculation. of shoots when compared to BAP. Maximum number of shoots was observed Maximum number of 9 shoots per explant in 25-30 days. The data in respect of shoot was induced on MS basal medium induction frequency, number of shoots and containing 10 µM KIN and mean shoot length of shoots on different concentrations length 5.34 cm. These results showed that of each hormone on shoot tip explants were both the cytokinins tested were found to presented in Tables 1 and Figs.1. initiate and proliferate shoots from the

Shoot multiplication from the shoot tip nodal explants. However, BAP was found explants: to be more suitable than KIN for shoot

The shoot tip explants were initially grown multiplication. on MS basal medium supplemented with In micro propagation technique, shoots are directly induced from the nodal explant BAP or KIN alone in different with axillary buds where meristematic concentrations ranging from 2 - 10 µM. tissue is present. This technique is primarily These two cytokinins BAP was found to used to produce pathogen free plantlets.

Journal of Advanced Applied Scientific Research - ISSN: 2454-3225 G. Jahirhussain et.al JOAASR-RTB-2016- February-2016:18- 28

Fig. 1. Shoot induction from the shoot tip explant of Coldenia procumbens L. Nowadays, it is widely used to get a mass In many plants, multiple shoots were propagation within a short period. Since obtained from the shoot tips or axillary the meristematic region is the very active buds by administering BAP or KIN. site, the axillary buds are readily [19],[20],[21],[22],[23], [24] In the present study proliferated. The efficiency of shoot nodal explants with axillary bud were multiplication depends on plant growth taken as explants source. The nodal regulators and types of explants. [17], [18] explants showed active site of positive morphogenetic response and readily developed multiple shoots.

Journal of Advanced Applied Scientific Research - ISSN: 2454-3225 G. Jahirhussain et.al JOAASR-RTB-2016- February-2016:18- 28

BAP KIN Percentage of Number of Shoots length response (%) shoots 2 µM -- 80 8.8±2.16 3.88±0.63 4 µM -- 90 10±1.22 4.7±0.54 6 µM -- 100 14±0.70 5.42±0.36 8 µM -- 95 9.8±2.58 4.72±0.14 10 µM -- 85 11.2±1.48 4.7±0.37 -- 2 µM 95 5.4±1.14 3.86±0.65 -- 4 µM 90 6.2±1.30 4.24±0.15 -- 6 µM 95 6.8±1.09 4.56±0.27 -- 8 µM 90 8.8±0.83 4.66±0.23 -- 10 µM 100 11±1.58 5.34±0.23 Table 1. Effect of different concentrations of cytokinins on shoot induction from the shoot tip explant of Coldenia procumbens L.

The propagation rate and morphogenetic used. In the present study both these response significantly varied to a greater cytokinins are used independently to find extent according to the explant type. Shoot their efficiency in shoot multiplication. Of tips have always been preferred for in vitro the two cytokinins used, BAP was found to studies because they can be handled easily be more effective in shoot induction and and restore their regeneration potential proliferation than KIN. In several studies over other explants. Some earlier findings BAP was most effective in inducing bud showed that more number of shoots were break resulting in the sprouting of a large produced from the nodal explants. [23],[25], number of shoots. [23],[27],[28],[29] [26] The cytokinins, BAP and KIN, were The cytokinin, BAP has been independently used to induce shoot commonly used for the induction of multiplication. The induction of multiple organogenesis in many plants. A shoots varied with the cytokinin types and comparison of the relative effect of concentrations. Though several growth different cytokinins for multiple shoot regulators are available for shoot formation revealed in order of multiplication, BAP and KIN are widely

Journal of Advanced Applied Scientific Research - ISSN: 2454-3225 G. Jahirhussain et.al JOAASR-RTB-2016- February-2016:18- 28

effectiveness was BAP >KIN > Zeatin > Centella asiatica [38] and Plumbago

[30] Adenine. It was stated that BAP is zeylanica L. [39] effective for meristem, shoot tip and bud Conclusion cultures.[31] reported that higher concentrations of cytokinins reduced the Micro propagation was carried out from number of micro propagated shoots. A the shoot tip explants of Coldenia similar phenomenon was observed earlier procumbens Linn. Shoot tip explants were [32] and also in the present study. grown on MS basal medium supplemented with different concentrations BAP or KIN. The potential for shoot multiplication Of the two cytokinins BAP was found to appeared to be strong in the presence of induce more number of shoots from nodal BAP alone in the culture medium. The explants when compared to KIN. The MS stimulatory effect of multiple shoot basal medium supplemented with 6 µM formation was similar to that reported in BAP showed the maximum number of other plant species.[17],[23] The number of 10.6 shoots per shoot tip explant and 10 shoots produced per explant in the medium µM KIN produced the maximum number supplemented with BAP was significantly of 9 shoots per shoot tip explant. higher than other cytokinins. In general, References KIN was less effective than BAP and 2iP 1. Krishnarao Mangeshrao Nadkarni, A. K. Nadkarni, (2-isopentenyl adenine) in shoot induction. Indian materia medica, 3, Vol 1, Popular Book Depot, Bombay,1955, 371. The MS medium containing BAP 2. CSIR, The Wealth of India: A Dictionary of Indian Raw Materials and Industrial Products, Vol I, was more effective than KIN for induction A-B, Council of Scientific & Industrial Research and proliferation of axillary buds as in (CSIR), New Delhi , India,1950, 307. 3. White Law Anisile, Materia Indica, Vol II, Rees, [33],[34],[35],[36],[37],[38] previous reports. The Longman, Orme, Brown, and Green, London, 1826, 435-436. superior effect of BAP for shoot bud 4. G.H.Schmelzer, A.Gurib-Fakim, R.Arroo, induction on High frequency shoot tip C.H.Bosch, Plant Resources of Tropical Africa 11(1) Medicinal Palnts 1, Backhuys Publisher, multiplication and exvitro rooting of Netherlands, 2008, 186-187. 5. David f cutler, Ted Botha, Christiaan Edward Johannes Botha and Dennis Wm Stevenson, Plant

Journal of Advanced Applied Scientific Research - ISSN: 2454-3225 G. Jahirhussain et.al JOAASR-RTB-2016- February-2016:18- 28

Anatomy An Applied Approach, Wiley-Blackwell, 17. Jahirhussain, G., Kala, K., Alagumanian, S., UK, 2008, 198. Muniappan, V., Tamilselvan1, V. and Rajkumar, 6. Wu Zheng-yi, Peter H. Raven , Flora of China, 16, P. (2015). In vitro micropropagation of Coldenia Missouri Botanical Garden,1995,337-338. procumbens L. From nodal explants. World 7. Ram Nath Chopra, Indigenous drugs of India:their Journal of Pharmaceutical Research, Volume 4, medical and economic aspects, The Art Press, Issue 12, 1324-1336. Calcutta, 1933, 477. 18. Mohamed, S.V., Jawahar, M., Thiruvengadam, M., 8. Chopra RN, Nayar SL, Chopra IC, Glossary of Indian Jeyakumar, M. and Jayabalan, N. (1999). Effect of Medicinal Plants, Council of scientific and cytokinins on the proliferation of multiple shoots in industrial Research, New Delhi,1958,74. horse gram (Macrotyloma uniflorum (L.) Verdc.). 9. Lather A, Gupta V, Garg S, Singh A, Sachdeva Jour. Plant Biotech.1: 79-83. K,Journal of natura conscientia, vol2(1), 2011, 19. Kackar, N.L., Solanki, K.R., Singh,M. and Vyas,S.C. 255-265. (1991). Micropropagation of Prosopis cineraria 10. Beena.P, Purnima,S,Kokilavani.R, Identification & Ind. J. Exp Biol. 29,65-67. Estimation of Wedelolactone in Coldenia 20. Varghese,S.K., Inamdar,J.A., Kalia, K., Procumbens, Scholars Research Library, Der Subramanina,R.B. and Nataraj, M. (1993). Pharmacia Lettre: 3(4),2011,320-324. Micropropagation of Aegle marmelos (L.) Corr. 11. Venkata S.S.N.kantamreddi, Y.Nagendra lakshmi and Phytomorphology 43(1&2),87-92. V.V.V.Satyanarayana Kasapu, Preliminary 21. Bennet, I.J., McComb,J.A., Tonkin, C.M. and Phytochemical Analysis Of Some Important McDavid, D.A.J. (1994). Alternating Cytokinins in Indian Plant Species, International Journal of Multiplication Media Stimulates In vitro Shoot Pharma And Bio Sciences, vol 1, issue 4, 2010, B- Growth and Rooting of Eucalyptus globulus Labill. 351-358. Ann. Bot., 74,53-58. 12. Pierik, R.L.M. (1987). In vitro Culture of Higher 22. Kumar, S., Chander, S., Gupta, H. and Sharma, D.R. Plants. Martinus Nijhoff Publishers. The Hague. (1998). Micropropagation of Actinidia deliciosa Plant Tissue Culture. 14 (1): 9-16. from axillary buds. Phytomorphology 48(3): 303- 13. Bajaj,Y.P.S., Furmanowa, M. and Olszowsks, O., 307. (1988). Biotechnology of the micropropagation of 23. Subbaiya, S., Alagumanian, S., Jahirhussain, G. and medicinal and aromatic plants. In: Biotechnology Nagarajan, T.(2015). In vitro rapid in Agriculture and Forestry, Medicinal and multiplication of Solanum trilobatum L. From shoot aromatic Plants I. (ed.) Bajaj, Y.P.S.). Springer - tip explants. World Journal of Pharmaceutical Berlin,Heidelberg, New York, Tokyo, 4 : 60-103. Research, Volume 4, Issue 12, 1954-1969. 14. Murashige, T. and Skoog, F. (1962). A revised 24. Baskaran, P. Jayabalan, N. (2005) An efficient medium for rapid growth and bioassay with micropropagation system for Eclipta alba - a tobacco tissue culture. Physiologia Plant., 15, 473- valuable medicinal herb. In Vitro Cell. Dev. Biol. 497. Plant. 41 : 532-539. 15. Gamborg O.L., Miller R.A., Ojima K. (1968): 25. Jain, N. and Babbar, S.B. (2003). Regeneration of Nutrient requirements of suspension cultures of 'juvenile' plants of black plum, Syzygium cuminii soybean root cells. Experimental Cell Research, Skeels, from nodal explants of mature trees. Plant 50: 151–158. Cell, Tissue and Organ Culture 73: 257-263. 16. Gomez, K. A. and Gomez, K. A. (1976). Statistical 26. Tefera W, Wannakrairoj S (2004). Micropropagation procedures for agricultural research with emphasis of krawan (Amomum krervanh Pierre ex Gangnep). on Rice. Philippines International Rice Research Sci. Asia 29:9-15. Institute, Los Bans.Grieve, A Modern Herbal. 27. Kadota, M. and Niimi, Y. (2003). Effects of Penguin. cytokinin types and their concentrations on shoot

Journal of Advanced Applied Scientific Research - ISSN: 2454-3225 G. Jahirhussain et.al JOAASR-RTB-2016- February-2016:18- 28

proliferation and hyperhydricity in in vitro pear 36. Velayutham, P. , Karthi, C., Nalini, P. and cultivar shoots. Plant Cell, Tissue and Organ Jahirhussain, G. (2012). In vitro Regeneration and Culture 72: 261-265. Mass Propagation of Hybanthus enneaspermus 28. Vasudevan, A., Selvaraj, N., Ganapathi, S., (L.) F. Muell. from the stem explants through Kasthurirengan, S., Ramesh Angbazhagan and callus culture.Journal of Agricultural Technology, Manickavasagam, M. (2004). Glutamine: a suitable 8(3):1119-1128. 2. nitrogen source for enhanced shoot 37. Komalavalli, N. and Rao, M.V., (2000). In vitro multiplication in Cucumis sativus L. Biologia micropropagation of Gymnema sylvestre - A Plantarum 48(1): 125-128. multipurpose medicinal plant. Plant Cell Tiss. Org. 29. Martinussen, I., Nilsen, G., Svenson, L., Junttila, O. Cult. 61:97-105. and Rapp, K.(2004). In vitro propagation of 38. Alagumanian, S., Jahirhussain, G. and Rao, M. V. cloudberry (Rubus chamaemorus). Plant Cell, (2015). High frequency shoot tip multiplication Tissue and Organ Culture 78: 43-49.Merck Index. and exvitro rooting of Centella asiatica (L.) Urb. - 1989 Ed. S. Budavari, 11th Ed., NJ, USA. a industrial medicinal herb. World Journal of pp:1371. Pharmaceutical Research, Volume 4, Issue 12, 30. Jahir Hussain, G., Malaimuthu, C., Velayutham, P. 1983-1998. (2010). In vitro regeneration from the shoot tip 39. Velayutham,P., Jahir Hussain, G. and Baskaran, P. of Melia composita willd. (syn. Melia dubia (2005). In vitro plantlet formation from nodal Heirn, not of cav.) an important multipurpose explants of Plumbago zeylanica L. – an important tree. Online journal of Biotechnology Research. medicinal plant. J. Swamy Bot. Cl. 22, 117-120. Vol.1 : 170-176.. 31. Hu CY, Wang PJ (1983) Handbook of plant cell culture. In: Evans DA, Sharp WR, Ammirato PV, Yamada Y (eds.) Meristem shoot tip and bud culture. New York, Macmillan, pp177–227. 32. Nagarajan, T., Alagumanian, S., Jahirhussain, G. and Subbaiya, S. (2015). In vitro mass propagation of Bacopa monnieri (Linn.) Wettst from nodal explant - a multipurpose medicinal plant. World Journal of Pharmaceutical Research, Volume 4,Issue 12,1970-1982.

33. Krishnan,P.N., Sudha,C.G. and Seeni,S.(1995). Rapid propagation through shoot tip cultures of Trichopus zeylanicus Gaertn., a rare ethnomedicinal plant. Plant cell reports.14:708- 711.

34. Padmapriya H., A.V.P. Karthikeyan, G. Jahir Hussain, C. Karthi (2011). An efficient protocol for in vitro propagation of Solanum nigrum L. from explant. J. of Agri. Tech. Vol.7(4): 1063-1073. 35. Reddy SP., Rama Gopal G., Laxshmi Sita G. (1998) In vitro multiplication of Gymnema sylvestre R. Br.—An important medicinal plant. Curr Sci 75:843–845.