World Journal of Agricultural Sciences 7 (6): 699-704, 2011 ISSN 1817-3047 IDOSI Publications, 2011

Rapid Micropropagation and Callus Induction of Catharanthusrouses in vitro Using Different Explants
1

Kanta Rani, 3Pushpa Kharb and 2Renu Singh

Department of Biotechnology and Applied Sciences, N C I T College, Israna, Panipat Laboratory of Plant breeding, plant research international, Wageningen UR, The Netherland 3 Department of Biotechnology and Molecular Biology, CCS HAU, Hisar-125004, India

Abstract: The effect of plant growth regulators on callus induction using different explants, to regenerate shoot/root from different explants as well as from calli in cultures of Catharanthus roseus (L.) G. Don was studied. Total of nineteen shoot regeneration media were used, out of these nineteen media three were further used to see the regeneration response and for root regeneration four media were tested supplemented with different concentration of IBA. Based on the results of this study, earliest and maximum (99%) callus induction response was observed on 10th day of inoculation from hypocotyl explants under dark conditions on MS medium supplemented with BAP (1.0 mg/l) + NAA (1.0 mg/l). For shoot proliferation, MS basal medium supplemented with BAP (1.5 mg lG1) + NAA (1.0 mg lG1) was the best followed by MS medium fortified with BAP (3.0 mglG1) + NAA (4.0 mglG1) while half strength MS medium supplemented with IBA (2.5 mglG1) + NAA (0.5 mglG1) gave best rooting response with quality roots. Besides, maximum shoot regeneration, response was observed from hypocotyl calli both under light and dark conditions on media supplemented with BAP (1.5 mglG1) + NAA (1.0 mglG1). Key words:6-Benzyladenine (BAP) % Catharanthus roseus (L.) % Indole butyric acid (IBA) % Naphthalene acetic acid (NAA) % Plant regeneration INTRODUCTION Herbal medicines have always occupied the nuclear place in every traditional system of medicine be it Ayurveda, Unani or Sidtha. More than 21,000 plants have been listed by World Health Organization (WHO) for possessing medicinal value. Catharanthus roseus(L.) G. Don. (commonly known as sadabahar or Periwinkle, family apocynaceae) gained commercial importance because of its alkaloids in different plant pars. The alkaloids like antineoplastindimericvinblastin and vincristine are mainly present in aerial parts, whereas ajmalcine, vinceine, vincamine, raubasin and reserpine are present in roots and basal stem. The dimericindole alkaloids from C.roseus are used for treatment of various human cancers. Selection of suitable experiments depends on various factors such as physiological stage, type of organ tissue, seasonal variations of parent plant from which the explant is selected [1]. Datta and Srivastava [2] initiated callus cultures from different explants in C. roseus of different age and observed detectable quantities of vinblastine in the calli of seedling origin. They also observed that as the callus differentiated into multiple shoots, the vinblastine production increased rapidly equal to that of in vitro seedlings of similar age. There are not many reports available on plantlet regeneration in this plant species. Therefore, an effort is made to study plant regeneration and callus induction study in periwinkle using leaf, shoot tip, hypocotyl, epicotyl and root explants with and without intervening callus phase in both light and dark conditions. MATERIALS AND METHODS Seeds were procured from medicinal aromatic and under-utilized plant section, Department of Plant Breeding, CCS Haryana Agricultural University, Hisar. Seeds were sown in pots and kept in green house. Explants (leaf, hypocotyl, epicotyl and root) of about 3-4mm were excised from 10-15 days old seedlings grown in green house. They were washed with tap water

Corresponding Author: Kanta Rani, Department of Biotechnology and Applied Sciences, N C I T College, Israna, Panipat, Haryana India. Mob: +91-9466948759.

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containing few drops of teepol, treated with fungicide solution (Bavistin 0.4% w/v) for 30 min followed by 4 to 5 times rinsing with distilled water. Different growth regulators namely indole-3-acetic acid (IAA), indolebutyric acid (IBA), naphthalene acetic acid (NAA), 2,4-dicholophenoxyacetic acid (2,4-D), kinetin, 6benzylaminopurine (BAP), were tested with full and half strength Murashige and Skoog (MS) basic medium [3]. Effectiveness of MS basal medium was also reported earlier in other crops [4]. Standard procedure was followed for the preparation of media [5]. The pH of the media was adjusted to 5.7 prior to sterilization done at 15 lbs/in2 for 15 min. All media were solidified with 8g/l agar. Cultures were maintained at 26C with 16 h light (light intensity 502gmsG1)/ 8 h darkness or completely dark conditions. The data of all the experiments conducted under present investigations were presented as mean of the three repeats. Data were analysed statistically by completely randomized design using one-way analysis of variance (ANOVA) using GenStat software 12 ed. RESULTS AND DISCUSSION In the in vitro studies, the application of growth regulators in the medium disturbs the internal polarity and causes dedifferentiation which results into callus formation. In a study by Rohtas [6] found that MS medium supplemented with BAP alone or along with NAA was the best for callus induction.

Callus Induction Days to Callus Induction on Media Supplemented with Different Growth Regulators: Range of days to callus induction for all the explants inoculated on all the media under light and dark conditions was 10-22 days (Fig. 1A). With regard to individual explants, days to callus induction from leaf (12-22 days), hypocotyl (10-19 days), epicotyl (12-19 days) and root (11-20 days) were almost in the same range under both light and dark conditions. Out of various explants used, callus induction was found to be the earliest (10-11 days) in hypocotyl explants (Fig. 1B) on media supplemented with MS + BAP (1.5 mg/l) + NAA (1.0 mg/l) under light and dark conditions. Similar results were observed by Ramavat et al. [7] in C. roseus. Callus induction took maximum (22 days) in leaf explants on media MS + BAP (0.5 mg/l) + 2, 4-D (2.0 mg/l). Highest percentage of callus induction was obtained from hypocotyl (94.9% and 92.2%) followed by leaf (92.2 and 90.2%) under light and dark conditions, respectively. Epicotyl Explant: Superiority of media with MS + BAP (0.1 mg/l) + 2,4-D (2.0 mg/l) medium for epicotyl-derived callus (87.9 and 90.8%) under both light and dark conditions was observed (Table 1). Callus induction response from epicotyl explants ranged between 64.1 to 90.8 per cent under light and dark conditions, respectively. No callus induction response was observed on MS medium without any growth regulators.

Table 1: Days to callus induction from different explants under light and dark conditions in C. roseus (L.) G. Don Leaf Medium Basal Salt MS MS BAP (2.0) BAP (0.1)+ 2, 4-D (2.0) BAP (0.5)+ 2, 4-D (2.0) BAP (1.0)+ NAA (1.0) BAP (1.5)+ NAA (1.0) BAP (1.5)+ NAA (2.0) BAP (2.0)+ NAA (1.0) BAP (3.0)+ NAA (2.0) BAP (3.0)+ NAA (4.0) BAP (1.5)+ IAA (0.2) BAP (1.5)+ IAA (0.5) BAP (1.5)+ IAA (1.0) 2, 4-D (2.0) Kinetin (0.5)+ 2, 4-D (2.0) Kinetin (1.0)+ NAA (1.0) -------------------------Light 19 22 18 18 19 20 21 13 21 19 17 20 17 15 14 Dark 19 19 18 17 18 19 20 12 19 19 17 18 15 15 14 Hypocotyl ---------------------------Light 18 19 17 11 17 19 17 16 18 17 15 18 14 13 Light 20 18 14 15 12 17 15 16 17 15 16 13 14 Epicotyl -------------------------Dark 20 15 14 15 11 15 14 15 17 14 16 13 13 Dark 18 16 17 10 17 19 17 15 17 16 14 18 14 13 Root ------------------------Light 19 12 15 16 17 18 17 14 17 16 15 17 15 14 Dark 19 12 14 17 16 17 15 14 17 16 16 16 14 14

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Fig. 1: A: B: C: D: E: F: G: H:

Initiation of Callus formation in C. roseus Shoot regeneration from hypocotyl derived calli in C. roseus Initiation of multiple shoot formation on shooting medium in C. roseus Maximum multiple shoot formation from hypocotyl derived calli in C. roseus Maximum root induction on rooting medium supplemented with IBA (2.5mg/l) + NAA (0.5 mg/l) in C. roseus Root formed were hard, thick and hairy in C. roseus Transfer of regenerated plantlets into pot containing FYM (1:1:1) Survived plantlets with morphologically normal healthy stem and leaves in C. roseus

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Table 1: Mean percent response of callus induction from different explants under light and dark conditions in C. roseus (L.) G. Don Leaf* --------------------------------------Medium Basal Salt MS MS BAP (2.0) BAP (0.1)+ 2, 4-D (2.0) BAP (0.5)+ 2, 4-D (2.0) BAP (1.0)+ NAA (1.0) BAP (1.5)+ NAA (1.0) BAP (1.5)+ NAA (2.0) BAP (2.0)+ NAA (1.0) BAP (3.0)+ NAA (2.0) BAP (3.0)+ NAA (4.0) BAP (1.5)+ IAA (0.2) BAP (1.5)+ IAA (0.5) BAP (1.5)+ IAA (1.0) 2, 4-D (2.0) Kinetin (0.5)+ 2, 4-D (2.0) Kinetin (1.0)+ NAA (1.0) (Mean S.E.)* Light 0(4.050.01) 68.4(56.080.01) 70.2(57.250.17) 64.9(53.950.06) 80.0(63.780.66) 74.6(60.050.60) 72.5(58.700.63) 90.2(72.250.81) 67.7(55.670.32) 71.4(57.970.29) 76.2(61.120.44) 51.3(46.050.78) 68.9(56.420.33) 57.0(49.100.39) 54.0(47.600.44) (Mean S.E.)* Dark 0(4.050.01) 69.4(56.750.33) 73.6(59.360.09) 68.4(56.080.01) 80.9(64.440.66) 78.9(62.980.01) 77.8(62.220.48) 92.2(73.901.17) 68.9(56.440.48) 74.0(59.690.31) 78.3(62.590.40) 53.0(47.510.06) 71.2(57.870.79) 57.1(49.370.44) 55.0(48.140.35) Hypocotyl** ------------------------------------(Mean S.E.)* Light 0(4.050.01) 78.0(62.400.19 84.4(67.160.21) 88.9(70.890.56) 92.2(74.410.88) 89.2(71.320.35) 88.7(70.770.62) 89.9(71.971.04) 88.0(70.150.01) 71.6(58.120.66) 75.5(60.650.60) 77.5(62.010.51) 55.6(48.480.01) 59.4(50.690.50) 83.2(66.220.57) 82.3(65.480.38) (Mean S.E.)* Dark 0(4.050.01) 81.8(65.090.01) 85.4(67.970.19) 90.0(72.020.01) 94.9(77.640.40) 89.6(71.670.35) 89.5(71.621.09) 91.2(73.361.34) 89.3(71.400.62) 73.7(59.450.01) 76.7(61.430.18) 78.3(62.590.40) 60.6(51.400.53) 61.1(51.690.01) 85.2(67.760.41) 83.1(66.120.51) Epicotyl*** --------------------------------------(Mean S.E.)* Light 0(4.050.01) 64.1(53.510.19 87.9(70.100.58) 76.4(61.260.55) 80.6(64.240.58) 82.0(65.280.51) 84.1(66.880.64) 73.9(59.560.37) 78.1(62.450.43) 70.0(57.080.38) 72.5(58.700.63) 75.0(60.360.69) 65.9(54.540.50) 0(4.050.01) 69.0(56.500.30) 67.8(55.730.35) (Mean S.E.)* Dark 0(4.050.01) 64.8(53.930.10) 90.8(72.900.50) 78.4(62.630.42) 84.4(67.120.56) 82.4(65.600.34) 84.9(67.520.64) 75.9(60.940.31) 79.1(63.170.30) 71.9(58.290.60) 75.9(60.940.63) 77.9(62.330.37) 68.9(56.250.22) 0(4.050.01) 70.9(57.650.57) 69.9(57.040.25) Root**** ------------------------------------(Mean S.E.)* Light 0(4.050.01) 46.9(43.470.29 51.2(46.100.73) 51.8(46.340.56) 61.8(52.120.61) 71.4(57.970.74) 63.4(53.070.30) 64.2(53.570.35) 67.4(55.470.43) 54.2(47.720.29) 54.4(47.830.38) 58.3(50.050.01) 54.3(47.740.40) 0(4.050.01) 47.8(44.010.01) 44.5(42.130.27) (Mean S.E.)* Dark 0(4.050.01) 47.3(43.720.10) 53.9(47.490.20) 54.2(47.720.29) 65.6(54.350.34) 74.6(60.020.29) 63.6(53.170.01) 66.1(54.700.34) 68.1(55.900.43) 54.8(48.040.10) 55.6(48.480.01) 59.5(50.570.70) 53.0(46.990.23) 0(4.050.01) 50.0(45.270.01) 46.6(43.320.15)

70.6(57.460.001) 72.3(58.590.60)

*CD = 1.318 (Light);CD = 1.434 (Dark); **CD = 1.544 (Light);CD = 1.548 (Dark); ***CD = 1.351 (Light);CD = 1.212 (Dark), ****CD = 1.192 (light); CD = 0.789 (Dark) *CV = 1.472CV = 1.561**CV = 1.517 CV = 1.490 ***CV = 1.515 CV = 1.328 ****CV=1.641(Light); CV= 1.080 (Dark)

Table 2: Effect of different growth regulators on shoot regeneration in C roseus (L.) G. Don Basal Salt MS MS BAP (0.1) BAP (1.0) BAP (2.0) BAP (1.0)+ NAA (1.0) BAP (1.5)+ NAA (1.0) BAP (1.5)+ NAA (2.0) BAP (2.0)+ NAA (1.0) BAP (3.0)+ NAA (2.0) BAP (1.5)+ IAA (0.2) BAP (1.5)+ IAA (0.5) BAP (1.5)+ IAA (1.0) Kinetin (1.0)+ NAA (1.0) Kinetin (2.5)+ NAA (0.05) No. of explants cultured 86 94 90 80 94 107 111 114 102 120 108 110 115 97 No. of explants responding 22 44 58 57 64 95 97 78 71 85 79 86 66 59 Mean percent response (meanS.E)* 25.6 (30.690.37) 46.8 (43.460.06) 64.3 (53.630.40) 71.2 (57.860.32) 68.0 (55.900.61) 88.9 (71.000.85) 87.4 (69.620.43) 68.4 (56.100.34) 67.6 (55.590.36) 70.8 (57.610.64) 73.1 (59.080.31) 78.2 (62.520.42) 57.4 (49.530.19) 59.8 (50.900.14) No. of shoots /explant 1-2 2-3 1-3 3-4 15-17 10-12 4-5 4-5 4-8 4-10 5-12 1-2 1-2

CD = 1.269 (at 5% level of significance) *Transformed Value CV = 1.366 Table 3: Indirect shoot regeneration response from calli obtained from different explants in C. roseus (L.) G. Don. on media supplemented with BAP and NAA No. of explants NAA (mg/l) 1.0 2.0 4.0 BAP (mg/l) 1.5 3.0 3.0 Explants Hypocotyl Hypocotyl Leaf cultured 47 62 56 No. of explants responding 34 34 22 Mean percent response (MeanS.E.)* 72.4 (38.980.58) 55.2 (58.650.36) 39.1(48.270.21) No of shoots regenerated per calli 10-15 10-12 5-7

CD = 1.452(at 5% level of significance) *Transformed value CV = 1.466 Table 4: Root induction response on different media in C. roseus (L.) G. Don MS Strength MS MS MS MS + MS IBA (mg/l) 1.0 1.0 2.5 2.5 NAA (mg/l) 0.5 Quality of roots + + ++ +++ Mean (%) response (Mean S.E.)* No. of shoots cultured 50.0 (45.270.01) 63.9 (53.360.84) 75.9 (60.940.63) 90.0 (72.020.01) 22 18 25 30 No. of shoots responding 11 14 19 27

CD = 1.745 (at 5% level of significance), + = Low moderate; ++ = Moderate; +++ = Good *Transformed value CV = 1.576

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Hypocotyl Explants: Highest percentage of callus induction (92.2 and 94.9%) was obtained from hypocotyl explants on medium having MS + BAP (1.0 mg/l) + NAA (1.0 mg/l) medium under light and dark conditions, respectively (Table1). Minimum callus induction response (55.6 and 60.6%) was obtained on media with BAP (1.5 mg/l) + IAA(1.0 mg/l) under both light and dark conditions. Leaf Explants: Maximum callus induction response (90.2 and 92.2%) was obtained on medium supplemented with BAP (3.0 mg/l) + and NAA (2.0 mg/l) under light and dark conditions, respectively (Table1). Minimum callus induction response (51.3% and 53.0%) was observed on medium supplemented with MS + BAP (1.5 mg/l) + IAA(1.0 mg/l) under light and dark conditions, respectively. No callus formation was observed on plane medium i.e. MS with no growth regulators.Media supplemented with NAA (1.0 mg/l) + BAP (1.5) was observed to be more effective for callus induction as per cent callus induction response was decreased from 74.6 to 51.3 under light and from 78.9 to 53.0 under dark when it was replaced by IAA (1.0 mg/l) + BAP (1.5 mg/l). Root Explant: Callus induction obtained from root explant under both light and dark conditions has been described in Table 1. Range in per cent callus induction response under light and dark conditions (71.4. and 74.6%) were observed. Media supplemented with MS + BAP 1.5 mg/l + NAA 1.0 mg/l) medium was the best for callus induction under both light and dark conditions. Root explants failed to form callus when inoculated on media without any growth regulators. Media supplemented with BAP 3.0 mg/l + NAA 2.0 mg/l and on kinetin 0.5 mg/l + 2, 4-D 2.0 mg/l show no callus formation. Shoot Regeneration Direct Shoot Regeneration: Direct shoot regeneration from shoot tip explants was monitored till 25th day after inoculation on MS media supplemented with various concentrations and combinations of Cytokinins and Auxins (Table2; Fig. 1C). shoot regeneration was observed as percent response and number of shoots per explant (88.9 and 15-17) on media containing BAP (1.5 mg/l) + IAA(1.0 mg/l) followed by ( 87.4 and 10-12) at BAP (1.g mg/l) + NAA(2.0 mg/l) then (78.2 and 5-12) at BAP (1.5 mg/l) + IAA(1.0 mg/l).

Indirect Shoot Regeneration: Shoot regeneration from calli was observed on different media i.e. MS + BAP (1.5 mg/l) + NAA (1.0 mg/l), MS + BAP (3.0 mg/l) + NAA (2.0 mg/l) and MS + BAP (3.0 mg/l), 4.0 mg/l NAA) (Table 3). Maximum (72.4%) shoot regeneration from hypocotyl derived calli was observed on BAP (1.5) + NAA (1.0) medium (Fig. 1D) and minimum (39.1%) shoot regeneration from leaf derived callus was observed on BAP (3.0) + NAA (4.0) medium. Root Induction and Transfer of Regenerated Plantlets to Soil: The shoots obtained from callus were surgically excised under aseptic conditions and inoculated on rooting media (Table 4). Rooting was best observed (90%) when excised shoots were pretreated for two hours with liquid 1/2 MS medium containing IBA (2.5 mg/l) and then transferred on solid MS media containing IBA(2.5 mg/l) + NAA (0.5 mg/l) (Fig 1E). This result confirmed by the previous study by Mujib et al. [8]. They also reported root induction on MS basal medium containing 0.5 mg/l NAA. Root induction response was observed to be 63.9 and 50.0 per cent on full strength MS+ IBA (1.0 mg/l) and MS+ IBA (1.0 mg/l) media, respectively. Regenerated roots were varies in quality from hard, thick and hairy to thin, feeble with less hair (Fig. 1F). Survival rate of regenerated plantlets on MS media supplemented with various synthetic growth regulators was observed to be 50 per cent. The regenerated plantlets were later on transferred to pots containing F: Y: M in 1:1:1 ration (Fig 1G). Plantlets grew vigorously in the net house (Fig. 1H). REFRENCES 1. Murashige, T., 1977. Clonal crops through tissue culture. In: Barz, W., E. Reinhard and M.H. Zenk, (Eds.). Proc. Life Sciences: Plant Tissue Culture and its Biotechnological Applications.Springer-Verlag, Berlin, pp: 392-403. Datta, A. and P.S. Srivastava, 1997. Variation in vinblastine production of Catharanthus roseus during in vivo and in vitro differentiation. Phytochemistry, 46(1): 135-137. Murashige, T. and F. Skoog, 1962. A revised medium for rapid growth and bioassays withtobacco tissue cultures. Physiol. Plant, 15: 473-497.

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Rohtas, A., 2001. Micropropagation of SalvandoraoleoidesDecne (Jhal) through tissue culture. M.Sc. Thesis, Department of Botany, CCS Haryana Agricultural University, Hisar, India. Mujib, A., S. Das, S. Dey and B. Bhattacharya, 1995. Influence of agitation in vitro cultivation of C. roseus (L.) G. Don. multiple shoot. Phytomorphol., 45(3and4): 239-245.

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