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Acta Botanica Hungarica 53(12), pp. 197209, 2011 DOI: 10.1556/ABot.53.2011.12.

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IN VITRO MICROPROPAGATION OF CATHARANTHUS ROSEUS AN ANTICANCER MEDICINAL PLANT


A. A. A. BAKRUDEEN, G. SUBHA SHANTHI T. GOUTHAMAN, M. S. KAVITHA and M. V. RAO
1

Department of Plant Science, School Life Sciences, Bharathidasan University Tiruchirappalli 620 024, Tamil Nadu, India; E-mail: bakru24@yahoo.co.in 2 Institute of Biological Sciences, Faculty of Science, University of Malaya 50603 Kuala Lumpur, Malaysia; E-mail: mvrao_456@yahoo.co.in (Received 24 June, 2008; Accepted 30 September, 2009)

An efficient protocol was standardized using axillary bud and shoot tip explants of Catharanthus roseus an anticancer medicinal plant. The highest number of shoots (19.6 shoots / auxiliary node) was observed after 45 days of culture in the MS medium supplemented with NAA (4.0 mg l1) + BA (4.0 mg l1). Shoots were proliferated and elongated in the same medium. High frequency of rooting (82.5%) was obtained in half strength MS + IBA (4.0) from axillary bud derived shoots. The rooted plantlets were successfully established in soil. Key words: Apocynaceae, auxiliary bud, Catharanthus roseus, micropropagation

INTRODUCTION Catharanthus roseus (L.) G. Don belongs to the family Apocynaceae. It originates from Madagascar, found throughout India. Now it has been spread throughout the tropics and subtropics by human activities through its primary traditional use were for people with diabetes, Catharanthus roseus also has anticancer effect (Pereira et al. 2010). The root is toxic, bitter, acidic, stomachic and used as a tonic. In Hawaii, the plant is boiled to make a poultice to stop bleeding (Van Lersel 1998). In China, it is used as a homemade cold remedy to ease lung congestion, inflammation and sore throats. In the Caribbean, an extract from the flowers was used to make solution to treat eye irritation and infection (Kokil et al. 2007). Catharanthus roseus produces several commercially valuable alkaloids including the anticancer compounds vincristine, vinblastine and the antihyper02366495/$ 20.00 2011 Akadmiai Kiad, Budapest

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tensive compound ajmalicine. Crude extracts of Catharanthus roseus using 50% methanol had significant anticancer activity against numerous cell types in vitro (Ueda et al. 2002). Among the plants analysed in vitro, Catharanthus roseus had the most potent antioxidant properties including Thymus and Salvia (Zheng and Wang 2001). This plant is being exploited on a large scale on commercial basis for its medicinal property. Due to low productivity and high production costs of these alkaloids by cultures of Catharanthus roseus, non-conventional methods have to be employed for better production. It is highly desirable to meet the large scale needs of the various industries and also to conserve the plants in ex situ. Conventionally, it is being propagated through seed, vegetative splits, stem cuttings, where the success is around only 70% and when propagated through seeds, the germination is only 30%. The growing demand for commercial cultivation of this necessitates an alternative faster rate of multiplication. In recent years, tissue culture techniques are being widely used to produce uniform quality, disease free plants at a faster rate within a limited space (Murashige 1974). The present paper describes as an immediate and subsidiary objective of standardisation of a reproducible micropropagation protocol in white varieties of Catharanthus roseus.

MATERIALS AND METHODS Healthy and elite (23 months old) plants of Catharanthus roseus were selected from the medicinal plants garden, Department of Plant Science, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. The young auxiliary node and shoot tip explants were trimmed and washed under running tap water for 5 min followed by a rinse with Teepol solution for 2 min, 70% ethanol (v/v) for 1 min and 0.1% HgCl2 for 5 min, the following explants were washed with sterile distilled water two to four times each, axillary buds were cultured on sterile MS medium (Murashige and Skoog 1962). The nutrient culture medium consisted of MS salts and vitamins gelled with 0.8% (w/v) agar (Hi-media). Various growth regulators, such as 6-benzylaminopurine (BA, 1.05.0 mg l1), 6-furfurylaminopurine (KN, 1.05.0 mg l1), indole3-acetic acid (IAA, 1.08.0 mg l1), indole3-butyric acid (IBA, 1.08.0 mg l1), -naphthalene acetic acid (NAA, 1.05.0 mg l1) and 2,4-dicholorophenoxyacetic acid (2,4-D, 1.05.0 mg l1) were supplemented to MS medium either alone (or) in combination supplemented with 3% (w/v) sucrose (Hi-media) as a carbon source. The pH of the medium was set to 5.6 and than autoclaved at a pressure of 1.06 kg cm2 at 121 C for 15 min. The surface disinfected explants were placed vertically in glass tubes (150 25 mm2) containing

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20 ml of culture medium and plugged tightly with non-absorbent cotton. All the cultures were incubated at 252 C under 16 hours photoperiod with light intensity of 60 mol m2 s1 by white fluorescent tubes, and the relative humidity was maintained at 5560%. The experiments were conducted in a completely randomised design. Thirty replicates were used and repeated three times. All the treatments were statistically analysed by Duncans Multiple Range Test (DMRT) (Gomez and Gomez 1976).

RESULTS AND DISCUSSION Maximum frequency of shoots and bud sprouting were formed from explants collected during the month of August and October. Axillary node and shoot tip explant showed direct mode of regeneration and readily developed multiple shoots. Axillary nodes initiation and multiplication failed to develop shoot buds in plant growth regulator free MS medium without growth regulators. Multiple shoot formation from axillary bud explants were observed in MS supplemented with BA and KN alone (or) in combination with IAA, IBA, NAA and 2,4-D. MS media supplemented with BA alone resulted in high bud sprouting frequency and shoot number with maximum percentage of response when compared to KN, after 45 days (Table 1). But a combination of BA and KN treatment showed better overall growth than individual treatments (Table 4). To enhance shoot multiplication, different auxins were combined with the optimised cytokinin concentration. Shoot number was the highest in the media containing NAA (4.0 mg l1) with BA (4.0 mg l1) for axillary node explants of C. roseus, after 45 days (Table 2, Fig. 2ag). KN (3.0 mg l1) with IAA (3.0 mg l1) induced the maximum number of multiple shoots from shoot tip explants, after 45 days (Table 3, Fig. 1ag). Higher concentration of NAA, IAA, IBA induced basal callus, sometimes root formation in IBA at cut ends and prevents multiple shoot induction. The shoots formed from explants of C. roseus showed stunted growth and phenolic exudation, yellowing of leaves. The effective concentration of NAA (4.0 mg l1) requirement for shoot bud induction was active to both the explants. Different media fortified separately with BA (4.0 mg l1) + KN (3.0 mg l1), [BA (4.0 mg l1 + NAA (4.0 mg l1)], [KN (3.0 mg l1) + IAA (3.0 mg l1)], [BA (4.0 mg l1) + KN (3.0 mg l1)] for shoot tip explants and BA (4.0 mg l1) + KN (3.0 mg l1), [BA (4.0 mg l1) + NAA (4.0 mg l1)], [KN (3.0 mg l1) + NAA (4.0 mg l1)], [BA (4.0 mg l1) + KN (3.0 mg l1)] for axillary node explants were examined to determine the optimum salt requirement for shoot initiation and shoot

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multiplication in C. roseus, after 45 days. The shoot buds sprouted slowly with limited development even if they were maintained for longer period in culture. The explants in culture showed phenolic exudation and yellowing of leaves. Culture initiation, multiplication and elongation from shoot tip and axillary node explants were achieved on single optimised culture medium within 50 days, thus suppressing the various stages of micropropagation. Bud sprouting occurred within a week and in about 3040 days multiple shoots developed and the elongation of shoot bud was observed with in 4555 days. Seedling explants did not show significant difference in shoot formation rate to various node positions, whereas the strong influence of node position of shoot tip

Fig. 1. Micropropagation from shoot tip explants of Catharanthus roseus (a = habit, b = shoot initiation, c and d = multiple shoots proliferation, e = rooting, h = rooted plantlets, g = hardening)
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Table 1 Effect of MS medium supplemented with cytokinins on multiple shoots and shoot length induction on Catharanthus roseus, after 45 days Plant growth regulators (mg l1) Percentage of responses (%) Shoot tip Axillary node Multiple shoot numbers MeanS.E. Shoot tip Axillary node Shoot length (cm) MeanS.E. Shoot tip Axillary node

BA 1.0 40.7e 53.5e 6.10.59d 5.50.34e 2.10.29e 1.80.45e cd bc bc c c 2.0 67.4 6.40.51 6.30.21 2.80.26 2.40.19bc 59.5 3.0 72.5b 6.60.48b 7.10.40b 3.60.17a 3.50.17a 73.4ab a a a a ab 4.0 80.0 8.30.40 7.80.32 3.40.21 2.80.22b 76.2 c d e cd cd 5.0 60.2 5.90.35 6.20.17 2.70.14 2.10.14d 65.0 KN 1.0 43.2e 6.80.39cd 4.10.32e 3.10.16cd 2.00.17cd 62.1c a bc c c ab 2.0 56.0 7.10.43 5.90.18 3.80.28 2.80.32b 72.4 ab a a a a 3.0 70.5 8.40.52 6.50.24 4.10.29 3.10.19a 69.5 d b ab ab c 4.0 62.0 7.30.34 6.20.16 3.40.21 2.20.14c 56.0 5.0 55.3d 6.50.43e 5.40.19cd 2.70.17e 1.70.10e 48.4e Values are mean of 10 replicates per treatment and repeated three times. Values with the same superscript are not significant at 5% probability level according to DMRT

explants were observed. The highest frequency of response and shoot number was observed at node positions 2, 3 and 4 found to be more responsive than from distal and proximal nodes. The maximum root induction was observed in half strength MS medium supplemented with IBA (4.0 mg l1) concentrations after 30 days (Table 5). The full strength MS medium significantly induced the basal callus, reduced root percentage and root length. However, the quarter strength MS medium showed the rooting induction percentage less than half and full strength MS medium (data not shown). After 30 days, rooted plantlets were removed from culture tubes and washed with sterile distilled water to remove media, and they were then transferred to foam cups containing a mixture of autoclaved garden soil, sand and vermiculite (Keltech Energies Ltd., Bangalore, India) (1:1:1), respectively. The cups were covered with polyethylene bags to maintain high humidity. Plantlets were irrigated with MS basal salt solution devoid of sucrose and myo-inositol once in three days for four weeks. The hardened plants were maintained under controlled condition for 30 days and they were acclimatised in garden.

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Table 2 Effect of MS medium supplemented with BA and auxins on multiple shoots and shoot length induction on Catharanthus roseus, after 45 days Percentage Multiple shoot number Shoot length (cm) Plant growth of response (%) MeanS.E. MeanS.E. regulators (mg l1) Shoot tip Axillary Shoot tip Axillary Shoot tip Axillary node node node BA + IAA 4.0 + 1.0 32.5e 46.0d 2.40.42e 3.50.24cd 1.80.26cd 2.40.16e c b ab ab b 2.0 52.5 3.50.19 4.20.18 2.50.19 3.00.22bc 40.0 a a a a a 3.0 65.0 4.40.22 5.00.20 3.40.24 3.40.17b 53.2 b bc c c c 4.0 48.6 3.20.16 3.80.16 1.90.16 4.10.21a 45.6 5.0 40.2de 2.80.20cd 3.20.19e 1.40.10e 2.90.10d 38.0cd BA + IBA 4.0 + 1.0 35.8e 1.80.17de 2.70.14e 1.60.14e 2.20.14d 28.5de d cd b d bc 2.0 44.6 2.90.14 3.40.22 2.10.19 3.50.19a 31.5 3.0 58.2b 3.50.26a 4.20.18a 3.50.22a 3.20.10ab 46.2c a a bc b b 4.0 66.5 2.60.31 3.60.21 2.20.16 2.60.22c 62.0 b c d bc d 5.0 49.0 2.00.18 3.50.24 2.00.10 1.80.12e 55.5 BA + NAA 4.0 + 1.0 58.5e 3.50.26de 2.40.19e 1.60.10e 2.80.14cd 46.2e cd cd bc b cd 2.0 64.2 4.20.18 3.50.28 2.80.24 3.50.19b 58.0 b b a a b 3.0 69.0 5.60.32 4.70.22 3.50.16 4.20.16a 67.5 4.0 75.5a 4.50.21b 3.20.14bc 4.10.19a 3.00.10c 71.2a c c d d c 5.0 66.2 3.70.18 2.90.16 2.90.14 2.60.14e 59.5 BA + 2,4-D 4.0 + 1.0 43.0d 2.70.19de 2.60.14de 1.70.14e 2.20.19cd 32.6d 2.0 51.5b 3.50.24b 3.40.19bc 2.80.22c 2.90.24ab 45.6b a a a a ab 3.0 60.0 3.90.18 4.50.22 2.90.10 3.50.20a 52.5 c bc bc b a 4.0 47.4 3.20.20 3.60.16 3.20.31 2.60.18c 40.0b e de d d cd 5.0 42.5 2.90.14 3.00.10 2.50.18 1.80.10e 36.2 Values are mean of 10 replicates per treatment and repeated three times. Values with the same superscript are not significant at 5% probability level according to DMRT

MS medium containing BA was more effective than KN for inducing proliferation of axillary buds as in previous report in medicinal plants (Handique and Bora 1999, Sarker et al. 1997). In Catharanthus roseus, development of shoots with larger internodes was observed on KN supplemented medium, the similar results were observed (Patnaik and Debata 1996, Sabita and Sanghamitra 1997). It shows that KN was necessary for the development of healthy normal shoots (Mondal et al. 1990).
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Table 3 Effect of MS medium supplemented with KN and auxins on multiple shoots and shoot length induction on Catharanthus roseus, after 45 days Plant growth regulators (mg l1) Percentage of response (%) Shoot tip Axillary node Multiple shoot number MeanS.E. Shoot tip Axillary node Shoot length (cm) MeanS.E. Shoot tip Axillary node

KN + IAA 3.0 + 1.0 47.2c 40.5e 1.90.21e 2.10.18e 1.90.12de 2.60.20c 2.0 59.8ab 2.40.10bc 3.10.14c 2.60.20b 3.00.18ab 55.6b a a a a a 3.0 61.5 3.10.18 4.20.21 3.20.19 3.20.14a 64.2 cd c b b bc 4.0 54.2 2.50.14 3.50.14 2.40.14 2.40.22cd 40.0 e d d cd d 5.0 47.0 2.10.10 2.80.20 2.10.19 1.80.16e 32.5 KN + IBA 3.0 + 1.0 32.8de 2.20.16cd 1.80.20e 2.50.12d 1.80.14de 30.2e c d b cd bc 2.0 39.5 3.00.19 2.60.14 2.90.22 2.40.10c 45.0 b c a b a 3.0 52.4 3.50.22 3.50.19 3.20.16 3.20.16a 52.5 4.0 60.0a 2.40.24c 4.10.10a 3.00.10b 2.90.19ab 60.4a cd ab e c de 5.0 55.2 1.80.10 2.90.18 2.40.14 2.00.10d 42.5 KN + NAA 3.0 + 1.0 34.2e 2.60.12e 2.50.14e 1.80.14e 1.90.10e 34.5e 2.0 45.0cd 3.20.10c 3.10.16cd 2.20.20c 2.60.14bc 48.2cd ab ab ab a b 3.0 56.4 4.40.21 4.50.19 2.80.19 3.20.22a 54.0 a a a b a 4.0 62.8 4.80.19 4.0022 3.40.22 2.80.16b 59.8 c c cd c cd 5.0 50.5 3.10.14 3.30.18 2.10.18 2.50.12d 49.3 KN + 2,4-D 3.0 + 1.0 35.4cd 1.90.14e 1.80.21e 2.10.14de 2.00.19e 32.0cd ab b bc d b 2.0 46.8 2.60.10 2.60.14 3.00.22 3.10.20bc 44.2 3.0 49.5a 3.20.14a 3.20.18b 3.60.19a 4.00.22a 48.5a c c b a bc 4.0 38.0 2.80.22 4.00.10 2.80.18 3.20.14b 36.5 e e d bc d 5.0 30.4 2.00.16 3.20.14 2.30.16 2.60.12d 30.0 Values are mean of 10 replicates per treatment and repeated three times. Values with the same superscript are not significant at 5% probability level according to DMRT

In contrast, Reddy et al. (1998) reported that KN did not significantly improve the shoot length and the number of shoots BA concentration varied in seedling and mature explants of Gymnema sylvestre. Combination of BA and KN in the culture medium prompted the multiple shoot induction and shoot sprouting frequency. Superiority of BA, KN combination has been formed for micropropagation of other woody perennials (Das and Mitra 1990, Das et al.

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Table 4 Effect of MS medium supplemented with cytokinins on multiple shoots and shoot length induction on Catharanthus roseus, after 45 days Percentage Multiple shoot number Shoot length (cm) Plant growth of response (%) MeanS.E. MeanS.E. regulators (mg l1) Shoot tip Axillary Shoot tip Axillary Shoot tip Axillary node node node BA + KN 1.0 + 1.0 42.4e 47.5cd 9.10.19d 11.40.12d 2.40.26d 2.40.16d 2.0 51.2c 11.20.18b 14.60.20ab 3.10.19b 2.90.22c 48.5bc a a a a a 3.0 60.5 14.70.14 15.50.16 4.40.24 3.40.17b 58.6 b ab bc c bc 4.0 56.8 10.50.22 13.20.20 2.60.16 4.10.21a 52.5 d e e de e 5.0 43.0 8.00.16 11.00.18 1.40.10 2.90.10de 44.0 BA + KN 2.0 + 1.0 58.2cd 11.40.18d 11.80.28de 1.60.14de 2.20.14cd 56.2de b b c b bc 2.0 70.4 13.60.20 14.50.42 2.10.19 3.50.19a 69.8 a a a a a 3.0 79.2 14.20.16 16.70.54 3.50.22 3.20.10ab 78.0 4.0 63.1c 13.80.42ab 14.20.40bc 2.20.16b 2.60.22c 65.6bc d e e d d 5.0 56.0 9.50.10 11.80.36 2.00.10 1.80.12e 59.4 BA + KN 3.0 + 1.0 58.4e 12.90.26c 12.20.30e 1.60.10e 2.80.14d 62.4d 2.0 75.2b 14.30.18b 14.80.38b 2.80.24cd 3.50.19b 69.5b a a a a b 3.0 81.5 15.80.32 16.40.42 3.50.16 4.20.16a 79.5 bc c cd c a 4.0 67.5 12.20.21 13.00.26 4.10.19 3.00.10bc 68.2 de cd e cd c 5.0 60.2 10.50.18 12.40.35 2.90.14 2.60.14de 61.2 BA + KN 4.0 + 1.0 65.8de 13.40.10d 14.20.34d 1.70.14e 3.10.10cd 64.2cd b b bc bc bc 2.0 78.8 15.10.22 15.30.22 2.50.22 3.30.14c 75.0 3.0 85.2a 17.50.16a 19.60.28a 2.90.10b 3.50.20ab 84.6a c bc b b a 4.0 72.0 15.60.24 16.00.42 3.20.31 3.90.14a 69.4 e d e de d 5.0 66.5 12.10.19 13.00.36 2.00.18 2.00.10e 63.8 BA + KN 5.0 + 1.0 52.4de 10.20.12cd 12.20.24cd 2.40.26d 2.40.19cd 56.5cd b b ab ab b 2.0 65.0 13.60.10 14.20.42 3.10.19 3.10.10b 62.1 a a a a a 3.0 72.5 14.40.19 15.00.36 4.40.24 3.80.16a 67.4 c bc c c bc 4.0 64.6 11.80.22 13.80.28 2.60.16 2.60.14c 59.0 5.0 60.2d 9.30.10e 11.50.30e 1.40.10e 1.90.12e 51.5e Values are mean of 10 replicates per treatment and repeated three times. Values with the same superscript are not significant at 5% probability level according to DMRT

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Table 5 Effect of full MS and 1/2 MS medium supplemented with auxins on root induction in Catharanthus roseus, after 30 days Rooting response Root length Plant growth regulators (%) (cm) (mg l1) MS + IAA 1.150.19h 1.0 32.5ef 2.0 44.6d 1.840.27cd bc 3.0 52.8 1.900.10c a 4.0 65.8 2.100.15a b 5.0 54.3 2.000.18b 6.0 37.8e 1.650.19e g 7.0 29.6 1.500.21f h 8.0 23.5 1.240.24g g 1/2 MS + IAA 1.600.17ef 1.0 44.6 2.0 49.3ef 2.000.27d d 3.0 57.5 2.180.10b a 4.0 72.3 3.330.19a b 5.0 65.0 2.100.21bc 6.0 62.0bc 1.800.24e e 7.0 50.4 1.420.18g h 8.0 35.0 1.000.15h ef MS + IBA 1.100.14f 1.0 34.6 2.0 42.5d 1.180.27de a 3.0 67.8 1.500.10c b 4.0 52.4 2.000.25a bc 5.0 47.0 1.650.17b 6.0 35.3e 1.280.20d g 7.0 23.4 1.000.16fg h 8.0 18.0 0.750.19h 1/2 MS + IBA 1.860.94ef 1.0 57.0f de 2.0 60.3 2.300.36d bc 3.0 68.2 2.350.27bc a 4.0 82.5 3.650.47a 5.0 69.1b 2.500.28b d 6.0 61.0 2.000.39e g 7.0 46.8 1.700.27g h 8.0 29.7 1.300.35h Values are mean of 10 replicates per treatment and repeated three times. Values with the same superscript are not significant at 5% probability level according to DMRT

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1996, Komalavalli and Rao 1997, Ravishankar and Jagadish Chandra 1989, Reuveni et al. 1990, Roy et al. 1998). Generally to induce the shoot regeneration, higher concentration of cytokinins and lower concentration of auxins are added to the MS basal media. Multiple shoot induction from shoot tip and axillary node explants has been suggested as a potential tool for mass multiplication of plants through in vitro. In our study among the various concentrations of BA involved (4.0 mg l1) BA

Fig. 2. Micropropagation from axillary node explant of Catharanthus roseus (a = axillary bud initiation, b and c = multiple shoot formation, d = multiple shoot proliferation, e = rooting, f = hardened plants, g = field developed plants)
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with KN (3.0 mg l1) gave good survival percentage. Similar result was observed in C. roseus, MS medium supplemented with KN and BA each at 0.2 mg/l + IAA (0.1 mg l1) (Adinpunya et al. 1997). The presence of auxins did not significantly enhance the response. Combination of BA and KN in the culture medium prompted the multiple shoot induction and shoot sprouting frequency. Our results indicated that NAA (4.0 mg l1) concentration could modify positively the shoot induction response. When with cytokinins (BA and KN) as observed in the propagation of Asclepias (Chi Won and John 1985), Gymnema (Reddy et al. 1998) and in Hemidesmus (Patnaik and Debata 1996) species, whereas the presence of NAA suppressed shoot formation and callus production in the propagation of latex producing plants (Tideman and Hawker 1982). IAA and IBA were used individually with full, half and quarter strength MS basal medium for rooting. In our study IBA was found to be more potent auxin for highest percentage of rooting. The highest root induction (82.5%) was observed on half-strength MS basal medium supplemented with auxins. The root lengths were varied in all MS basal strength with IAA or IBA concentrations. Similar results were observed in Madhuca longifolia (Rout and Das 1993), Gymnema sylvestre (Komalavalli and Rao 2000) and Eclipta alba (Baskaran and Jayabalan 2005). Junaid et al. (2004) observed that matured green embryos of C. roseus were cultured on MS medium supplemented with optimised BAP (0.5 mg l1) produced shoots and roots. The protocol established in this study can be used for the efficient multiplication of Catharanthus roseus. MS basal medium in combination of BA (4.0 mg l1) and KN (3.0 mg l1) be influenced the better shoot induction and shoot multiplication. Half strength of MS basal medium with IBA (4.0 mg l1) effectively induced the number of roots per plant. Plantlets produced from this protocol will contribute to the rehabilitation of Catharanthus roseus and help to a greater extend to reduce the presence on the natural populations. Such plants could also be used as a source for characterisation of secondary metabolites that are medicinally active compounds and will increase its probability in both the traditional and modern health care systems.
* Acknowledgements We sincerely thank Dr V. Kumaresan and Dr A. R. Lavanya, Department of Plant Science, Bharathidasan University, Tiruchirappalli, 620 024, Tamil Nadu, India.

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