catharanthus tissue culture protocol

Acta Botanica Hungarica 53(1–2), pp. 197–209, 2011DOI: 10.1556/ABot.53.2011.1–2.20

IN VITRO MICROPROPAGATION OF CATHARANTHUSROSEUS – AN ANTICANCER MEDICINAL PLANT

A. A. A. BAKRUDEEN, G. SUBHA SHANTHIT. GOUTHAMAN, M. S. KAVITHA and M. V. RAO

1Department of Plant Science, School Life Sciences, Bharathidasan UniversityTiruchirappalli 620 024, Tamil Nadu, India; E-mail: [email protected] of Biological Sciences, Faculty of Science, University of Malaya

50603 Kuala Lumpur, Malaysia; E-mail: [email protected]

(Received 24 June, 2008; Accepted 30 September, 2009)

An efficient protocol was standardized using axillary bud and shoot tip explants ofCatharanthus roseus – an anticancer medicinal plant. The highest number of shoots (19.6shoots / auxiliary node) was observed after 45 days of culture in the MS medium supple-mented with NAA (4.0 mg l–1) + BA (4.0 mg l–1). Shoots were proliferated and elongated inthe 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 estab-lished in soil.

Key words: Apocynaceae, auxiliary bud, Catharanthus roseus, micropropagation

INTRODUCTION

Catharanthus roseus (L.) G. Don belongs to the family Apocynaceae. Itoriginates from Madagascar, found throughout India. Now it has been spreadthroughout the tropics and subtropics by human activities through its primarytraditional use were for people with diabetes, Catharanthus roseus also has anti-cancer effect (Pereira et al. 2010). The root is toxic, bitter, acidic, stomachic andused as a tonic. In Hawaii, the plant is boiled to make a poultice to stop bleed-ing (Van Lersel 1998). In China, it is used as a homemade cold remedy to easelung congestion, inflammation and sore throats. In the Caribbean, an extractfrom the flowers was used to make solution to treat eye irritation and infection(Kokil et al. 2007).

Catharanthus roseus produces several commercially valuable alkaloids in-cluding the anticancer compounds vincristine, vinblastine and the antihyper-

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tensive compound ajmalicine. Crude extracts of Catharanthus roseus using 50%methanol had significant anticancer activity against numerous cell types invitro (Ueda et al. 2002). Among the plants analysed in vitro, Catharanthus roseushad the most potent antioxidant properties including Thymus and Salvia (Zhengand Wang 2001).

This plant is being exploited on a large scale on commercial basis for itsmedicinal property. Due to low productivity and high production costs ofthese alkaloids by cultures of Catharanthus roseus, non-conventional methodshave to be employed for better production. It is highly desirable to meet thelarge scale needs of the various industries and also to conserve the plants in exsitu. Conventionally, it is being propagated through seed, vegetative splits,stem cuttings, where the success is around only 70% and when propagatedthrough seeds, the germination is only 30%. The growing demand for com-mercial cultivation of this necessitates an alternative faster rate of multiplica-tion. In recent years, tissue culture techniques are being widely used to pro-duce uniform quality, disease free plants at a faster rate within a limited space(Murashige 1974). The present paper describes as an immediate and subsid-iary objective of standardisation of a reproducible micropropagation protocolin white varieties of Catharanthus roseus.

MATERIALS AND METHODS

Healthy and elite (2–3 months old) plants of Catharanthus roseus were se-lected from the medicinal plants garden, Department of Plant Science, Bharathi-dasan University, Tiruchirappalli, Tamil Nadu, India. The young auxiliarynode and shoot tip explants were trimmed and washed under running tap wa-ter 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 washedwith sterile distilled water two to four times each, axillary buds were culturedon sterile MS medium (Murashige and Skoog 1962).

The nutrient culture medium consisted of MS salts and vitamins gelledwith 0.8% (w/v) agar (Hi-media). Various growth regulators, such as 6-ben-zylaminopurine (BA, 1.0–5.0 mg l–1), 6-furfurylaminopurine (KN, 1.0–5.0 mgl–1), indole–3-acetic acid (IAA, 1.0–8.0 mg l–1), indole–3-butyric acid (IBA,1.0–8.0 mg l–1), α-naphthalene acetic acid (NAA, 1.0–5.0 mg l–1) and 2,4-dichol-orophenoxyacetic acid (2,4-D, 1.0–5.0 mg l–1) were supplemented to MS me-dium 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 thanautoclaved at a pressure of 1.06 kg cm–2 at 121 °C for 15 min. The surface disin-fected explants were placed vertically in glass tubes (150 × 25 mm2) containing

198 BAKRUDEEN, A. A. A. et al.

Acta Bot. Hung. 53, 2011

20 ml of culture medium and plugged tightly with non-absorbent cotton. Allthe cultures were incubated at 25±2 °C under 16 hours photoperiod with lightintensity of 60 µmol m–2 s–1 by white fluorescent tubes, and the relative humid-ity was maintained at 55–60%.

The experiments were conducted in a completely randomised design.Thirty replicates were used and repeated three times. All the treatments werestatistically analysed by Duncan’s Multiple Range Test (DMRT) (Gomez andGomez 1976).

RESULTS AND DISCUSSION

Maximum frequency of shoots and bud sprouting were formed fromexplants collected during the month of August and October. Axillary nodeand shoot tip explant showed direct mode of regeneration and readily devel-oped multiple shoots. Axillary nodes initiation and multiplication failed to de-velop shoot buds in plant growth regulator free MS medium without growthregulators. Multiple shoot formation from axillary bud explants were ob-served in MS supplemented with BA and KN alone (or) in combination withIAA, IBA, NAA and 2,4-D. MS media supplemented with BA alone resulted inhigh bud sprouting frequency and shoot number with maximum percentageof response when compared to KN, after 45 days (Table 1). But a combination ofBA and KN treatment showed better overall growth than individual treat-ments (Table 4).

To enhance shoot multiplication, different auxins were combined withthe optimised cytokinin concentration. Shoot number was the highest in themedia containing NAA (4.0 mg l–1) with BA (4.0 mg l–1) for axillary nodeexplants of C. roseus, after 45 days (Table 2, Fig. 2a–g). KN (3.0 mg l–1) with IAA(3.0 mg l–1) induced the maximum number of multiple shoots from shoot tipexplants, after 45 days (Table 3, Fig. 1a–g). Higher concentration of NAA, IAA,IBA induced basal callus, sometimes root formation in IBA at cut ends andprevents 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 l–1) requirement for shoot bud in-duction was active to both the explants.

Different media fortified separately with BA (4.0 mg l–1) + KN (3.0 mg l–1),[BA (4.0 mg l–1 + NAA (4.0 mg l–1)], [KN (3.0 mg l–1) + IAA (3.0 mg l–1)], [BA (4.0mg l–1) + KN (3.0 mg l–1)] for shoot tip explants and BA (4.0 mg l–1) + KN (3.0mg l–1), [BA (4.0 mg l–1) + NAA (4.0 mg l–1)], [KN (3.0 mg l–1) + NAA (4.0 mgl–1)], [BA (4.0 mg l–1) + KN (3.0 mg l–1)] for axillary node explants were exam-ined to determine the optimum salt requirement for shoot initiation and shoot

IN VITRO MICROPROPAGATION OF CATHARANTHUS ROSEUS 199


multiplication in C. roseus, after 45 days. The shoot buds sprouted slowly withlimited 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 axillarynode explants were achieved on single optimised culture medium within 50days, thus suppressing the various stages of micropropagation. Bud sproutingoccurred within a week and in about 30–40 days multiple shoots developedand the elongation of shoot bud was observed with in 45–55 days. Seedlingexplants did not show significant difference in shoot formation rate to variousnode positions, whereas the strong influence of node position of shoot tip



Fig. 1. Micropropagation from shoot tip explants of Catharanthus roseus (a = habit, b = shootinitiation, c and d = multiple shoots proliferation, e = rooting, h = rooted plantlets, g = hard-

ening)

explants were observed. The highest frequency of response and shoot numberwas observed at node positions 2, 3 and 4 found to be more responsive thanfrom distal and proximal nodes.

The maximum root induction was observed in half strength MS mediumsupplemented with IBA (4.0 mg l–1) concentrations after 30 days (Table 5). Thefull strength MS medium significantly induced the basal callus, reduced rootpercentage and root length. However, the quarter strength MS medium showedthe rooting induction percentage less than half and full strength MS medium(data not shown). After 30 days, rooted plantlets were removed from culturetubes and washed with sterile distilled water to remove media, and they werethen transferred to foam cups containing a mixture of autoclaved garden soil,sand and vermiculite (Keltech Energies Ltd., Bangalore, India) (1:1:1), respec-tively. The cups were covered with polyethylene bags to maintain high hu-midity. Plantlets were irrigated with MS basal salt solution devoid of sucroseand myo-inositol once in three days for four weeks. The hardened plants weremaintained under controlled condition for 30 days and they were acclimatisedin garden.



Table 1Effect of MS medium supplemented with cytokinins on multiple shoots and shoot length

induction on Catharanthus roseus, after 45 days

Plant growthregulators

(mg l–1)

Percentage of re-sponses (%)

Multiple shootnumbers

Mean±S.E.

Shoot length (cm)Mean±S.E.

Shoot tip Axillarynode



BA1.0 40.7e 53.5e 6.1±0.59d 5.5±0.34e 2.1±0.29e 1.8±0.45e

2.0 59.5cd 67.4bc 6.4±0.51bc 6.3±0.21c 2.8±0.26c 2.4±0.19bc

3.0 73.4ab 72.5b 6.6±0.48b 7.1±0.40b 3.6±0.17a 3.5±0.17a

4.0 76.2a 80.0a 8.3±0.40a 7.8±0.32a 3.4±0.21ab 2.8±0.22b

5.0 65.0c 60.2d 5.9±0.35e 6.2±0.17cd 2.7±0.14cd 2.1±0.14d

KN1.0 62.1c 43.2e 6.8±0.39cd 4.1±0.32e 3.1±0.16cd 2.0±0.17cd

2.0 72.4a 56.0bc 7.1±0.43c 5.9±0.18c 3.8±0.28ab 2.8±0.32b

3.0 69.5ab 70.5a 8.4±0.52a 6.5±0.24a 4.1±0.29a 3.1±0.19a

4.0 56.0d 62.0b 7.3±0.34ab 6.2±0.16ab 3.4±0.21c 2.2±0.14c

5.0 48.4e 55.3d 6.5±0.43e 5.4±0.19cd 2.7±0.17e 1.7±0.10e

Values are mean of 10 replicates per treatment and repeated three times. Values with thesame superscript are not significant at 5% probability level according to DMRT

MS medium containing BA was more effective than KN for inducing pro-liferation of axillary buds as in previous report in medicinal plants (Handiqueand Bora 1999, Sarker et al. 1997). In Catharanthus roseus, development of shootswith larger internodes was observed on KN supplemented medium, the simi-lar results were observed (Patnaik and Debata 1996, Sabita and Sanghamitra1997). It shows that KN was necessary for the development of healthy normalshoots (Mondal et al. 1990).



Table 2Effect of MS medium supplemented with BA and auxins on multiple shoots and shoot

length induction on Catharanthus roseus, after 45 days


(mg l–1)

Percentageof response (%)

Multiple shoot numberMean±S.E.





BA + IAA4.0 + 1.0 32.5e 46.0d 2.4±0.42e 3.5±0.24cd 1.8±0.26cd 2.4±0.16e

2.0 40.0c 52.5b 3.5±0.19ab 4.2±0.18ab 2.5±0.19b 3.0±0.22bc

3.0 53.2a 65.0a 4.4±0.22a 5.0±0.20a 3.4±0.24a 3.4±0.17b

4.0 45.6b 48.6bc 3.2±0.16c 3.8±0.16c 1.9±0.16c 4.1±0.21a

5.0 38.0cd 40.2de 2.8±0.20cd 3.2±0.19e 1.4±0.10e 2.9±0.10d

BA + IBA4.0 + 1.0 28.5de 35.8e 1.8±0.17de 2.7±0.14e 1.6±0.14e 2.2±0.14d

2.0 31.5d 44.6cd 2.9±0.14b 3.4±0.22d 2.1±0.19bc 3.5±0.19a

3.0 46.2c 58.2b 3.5±0.26a 4.2±0.18a 3.5±0.22a 3.2±0.10ab

4.0 62.0a 66.5a 2.6±0.31bc 3.6±0.21b 2.2±0.16b 2.6±0.22c

5.0 55.5b 49.0c 2.0±0.18d 3.5±0.24bc 2.0±0.10d 1.8±0.12e

BA + NAA4.0 + 1.0 46.2e 58.5e 3.5±0.26de 2.4±0.19e 1.6±0.10e 2.8±0.14cd

2.0 58.0cd 64.2cd 4.2±0.18bc 3.5±0.28b 2.8±0.24cd 3.5±0.19b

3.0 67.5b 69.0b 5.6±0.32a 4.7±0.22a 3.5±0.16b 4.2±0.16a

4.0 71.2a 75.5a 4.5±0.21b 3.2±0.14bc 4.1±0.19a 3.0±0.10c

5.0 59.5c 66.2c 3.7±0.18d 2.9±0.16d 2.9±0.14c 2.6±0.14e

BA + 2,4-D4.0 + 1.0 32.6d 43.0d 2.7±0.19de 2.6±0.14de 1.7±0.14e 2.2±0.19cd

2.0 45.6b 51.5b 3.5±0.24b 3.4±0.19bc 2.8±0.22c 2.9±0.24ab

3.0 52.5a 60.0a 3.9±0.18a 4.5±0.22a 2.9±0.10ab 3.5±0.20a

4.0 40.0bc 47.4bc 3.2±0.20bc 3.6±0.16b 3.2±0.31a 2.6±0.18c

5.0 36.2e 42.5de 2.9±0.14d 3.0±0.10d 2.5±0.18cd 1.8±0.10e


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



Table 3Effect of MS medium supplemented with KN and auxins on multiple shoots and shoot

length induction on Catharanthus roseus, after 45 days

Plantgrowth

regulators(mg l–1)







KN + IAA3.0 + 1.0 47.2c 40.5e 1.9±0.21e 2.1±0.18e 1.9±0.12de 2.6±0.20c

2.0 55.6b 59.8ab 2.4±0.10bc 3.1±0.14c 2.6±0.20b 3.0±0.18ab

3.0 64.2a 61.5a 3.1±0.18a 4.2±0.21a 3.2±0.19a 3.2±0.14a

4.0 40.0cd 54.2c 2.5±0.14b 3.5±0.14b 2.4±0.14bc 2.4±0.22cd

5.0 32.5e 47.0d 2.1±0.10d 2.8±0.20cd 2.1±0.19d 1.8±0.16e

KN + IBA3.0 + 1.0 30.2e 32.8de 2.2±0.16cd 1.8±0.20e 2.5±0.12d 1.8±0.14de

2.0 45.0c 39.5d 3.0±0.19b 2.6±0.14cd 2.9±0.22bc 2.4±0.10c

3.0 52.5b 52.4c 3.5±0.22a 3.5±0.19b 3.2±0.16a 3.2±0.16a

4.0 60.4a 60.0a 2.4±0.24c 4.1±0.10a 3.0±0.10b 2.9±0.19ab

5.0 42.5cd 55.2ab 1.8±0.10e 2.9±0.18c 2.4±0.14de 2.0±0.10d

KN + NAA3.0 + 1.0 34.5e 34.2e 2.6±0.12e 2.5±0.14e 1.8±0.14e 1.9±0.10e

2.0 48.2cd 45.0cd 3.2±0.10c 3.1±0.16cd 2.2±0.20c 2.6±0.14bc

3.0 54.0ab 56.4ab 4.4±0.21ab 4.5±0.19a 2.8±0.19b 3.2±0.22a

4.0 59.8a 62.8a 4.8±0.19a 4.0±022b 3.4±0.22a 2.8±0.16b

5.0 49.3c 50.5c 3.1±0.14cd 3.3±0.18c 2.1±0.18cd 2.5±0.12d

KN + 2,4-D3.0 + 1.0 32.0cd 35.4cd 1.9±0.14e 1.8±0.21e 2.1±0.14de 2.0±0.19e

2.0 44.2ab 46.8b 2.6±0.10bc 2.6±0.14d 3.0±0.22b 3.1±0.20bc

3.0 48.5a 49.5a 3.2±0.14a 3.2±0.18b 3.6±0.19a 4.0±0.22a

4.0 36.5c 38.0c 2.8±0.22b 4.0±0.10a 2.8±0.18bc 3.2±0.14b

5.0 30.0e 30.4e 2.0±0.16d 3.2±0.14bc 2.3±0.16d 2.6±0.12d




Table 4Effect of MS medium supplemented with cytokinins on multiple shoots and shoot length

induction on Catharanthus roseus, after 45 days


(mg l–1)







BA + KN1.0 + 1.0 42.4e 47.5cd 9.1±0.19d 11.4±0.12d 2.4±0.26d 2.4±0.16d

2.0 48.5bc 51.2c 11.2±0.18b 14.6±0.20ab 3.1±0.19b 2.9±0.22c

3.0 58.6a 60.5a 14.7±0.14a 15.5±0.16a 4.4±0.24a 3.4±0.17b

4.0 52.5b 56.8ab 10.5±0.22bc 13.2±0.20c 2.6±0.16bc 4.1±0.21a

5.0 44.0d 43.0e 8.0±0.16e 11.0±0.18de 1.4±0.10e 2.9±0.10de

BA + KN2.0 + 1.0 56.2de 58.2cd 11.4±0.18d 11.8±0.28de 1.6±0.14de 2.2±0.14cd

2.0 69.8b 70.4b 13.6±0.20c 14.5±0.42b 2.1±0.19bc 3.5±0.19a

3.0 78.0a 79.2a 14.2±0.16a 16.7±0.54a 3.5±0.22a 3.2±0.10ab

4.0 65.6bc 63.1c 13.8±0.42ab 14.2±0.40bc 2.2±0.16b 2.6±0.22c

5.0 59.4d 56.0e 9.5±0.10e 11.8±0.36d 2.0±0.10d 1.8±0.12e

BA + KN3.0 + 1.0 62.4d 58.4e 12.9±0.26c 12.2±0.30e 1.6±0.10e 2.8±0.14d

2.0 69.5b 75.2b 14.3±0.18b 14.8±0.38b 2.8±0.24cd 3.5±0.19b

3.0 79.5a 81.5a 15.8±0.32a 16.4±0.42a 3.5±0.16b 4.2±0.16a

4.0 68.2bc 67.5c 12.2±0.21cd 13.0±0.26c 4.1±0.19a 3.0±0.10bc

5.0 61.2de 60.2cd 10.5±0.18e 12.4±0.35cd 2.9±0.14c 2.6±0.14de

BA + KN4.0 + 1.0 64.2cd 65.8de 13.4±0.10d 14.2±0.34d 1.7±0.14e 3.1±0.10cd

2.0 75.0b 78.8b 15.1±0.22bc 15.3±0.22bc 2.5±0.22bc 3.3±0.14c

3.0 84.6a 85.2a 17.5±0.16a 19.6±0.28a 2.9±0.10b 3.5±0.20ab

4.0 69.4c 72.0bc 15.6±0.24b 16.0±0.42b 3.2±0.31a 3.9±0.14a

5.0 63.8e 66.5d 12.1±0.19e 13.0±0.36de 2.0±0.18d 2.0±0.10e

BA + KN5.0 + 1.0 56.5cd 52.4de 10.2±0.12cd 12.2±0.24cd 2.4±0.26d 2.4±0.19cd

2.0 62.1b 65.0b 13.6±0.10ab 14.2±0.42ab 3.1±0.19b 3.1±0.10b

3.0 67.4a 72.5a 14.4±0.19a 15.0±0.36a 4.4±0.24a 3.8±0.16a

4.0 59.0c 64.6bc 11.8±0.22c 13.8±0.28c 2.6±0.16bc 2.6±0.14c

5.0 51.5e 60.2d 9.3±0.10e 11.5±0.30e 1.4±0.10e 1.9±0.12e




Table 5Effect of full MS and 1/2 MS medium supplemented with auxins on root induction in

Catharanthus roseus, after 30 days

Plant growth regulators(mg l–1)

Rooting response(%)

Root length(cm)

MS + IAA 1.0 32.5ef 1.15±0.19h

2.0 44.6d 1.84±0.27cd

3.0 52.8bc 1.90±0.10c

4.0 65.8a 2.10±0.15a

5.0 54.3b 2.00±0.18b

6.0 37.8e 1.65±0.19e

7.0 29.6g 1.50±0.21f

8.0 23.5h 1.24±0.24g

1/2 MS + IAA 1.0 44.6g 1.60±0.17ef

2.0 49.3ef 2.00±0.27d

3.0 57.5d 2.18±0.10b

4.0 72.3a 3.33±0.19a

5.0 65.0b 2.10±0.21bc

6.0 62.0bc 1.80±0.24e

7.0 50.4e 1.42±0.18g

8.0 35.0h 1.00±0.15h

MS + IBA 1.0 34.6ef 1.10±0.14f

2.0 42.5d 1.18±0.27de

3.0 67.8a 1.50±0.10c

4.0 52.4b 2.00±0.25a

5.0 47.0bc 1.65±0.17b

6.0 35.3e 1.28±0.20d

7.0 23.4g 1.00±0.16fg

8.0 18.0h 0.75±0.19h

1/2 MS + IBA 1.0 57.0f 1.86±0.94ef

2.0 60.3de 2.30±0.36d

3.0 68.2bc 2.35±0.27bc

4.0 82.5a 3.65±0.47a

5.0 69.1b 2.50±0.28b

6.0 61.0d 2.00±0.39e

7.0 46.8g 1.70±0.27g

8.0 29.7h 1.30±0.35h


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 ofcytokinins and lower concentration of auxins are added to the MS basal media.Multiple shoot induction from shoot tip and axillary node explants has beensuggested 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 l–1) BA



Fig. 2. Micropropagation from axillary node explant of Catharanthus roseus (a = axillary budinitiation, b and c = multiple shoot formation, d = multiple shoot proliferation, e = rooting,

f = hardened plants, g = field developed plants)

with KN (3.0 mg l–1) gave good survival percentage. Similar result was observedin C. roseus, MS medium supplemented with KN and BA each at 0.2 mg/l +IAA (0.1 mg l–1) (Adinpunya et al. 1997). The presence of auxins did not signifi-cantly enhance the response. Combination of BA and KN in the culture me-dium prompted the multiple shoot induction and shoot sprouting frequency.

Our results indicated that NAA (4.0 mg l–1) concentration could modifypositively 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 produc-tion in the propagation of latex producing plants (Tideman and Hawker 1982).

IAA and IBA were used individually with full, half and quarter strengthMS basal medium for rooting. In our study IBA was found to be more potentauxin 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 concen-trations. Similar results were observed in Madhuca longifolia (Rout and Das1993), Gymnema sylvestre (Komalavalli and Rao 2000) and Eclipta alba (Baska-ran and Jayabalan 2005). Junaid et al. (2004) observed that matured green em-bryos of C. roseus were cultured on MS medium supplemented with optimisedBAP (0.5 mg l–1) produced shoots and roots.

The protocol established in this study can be used for the efficient multi-plication of Catharanthus roseus. MS basal medium in combination of BA (4.0mg l–1) and KN (3.0 mg l–1) be influenced the better shoot induction and shootmultiplication. Half strength of MS basal medium with IBA (4.0 mg l–1) effec-tively induced the number of roots per plant.

Plantlets produced from this protocol will contribute to the rehabilitation ofCatharanthus roseus and help to a greater extend to reduce the presence on thenatural populations. Such plants could also be used as a source for characteri-sation of secondary metabolites that are medicinally active compounds and willincrease its probability in both the traditional and modern health care systems.

*

Acknowledgements – We sincerely thank Dr V. Kumaresan and Dr A. R. Lavanya, Depart-ment of Plant Science, Bharathidasan University, Tiruchirappalli, 620 024, Tamil Nadu, India.



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