Department of Life Sciences, Manipur University, Canchipur, Imphal-795003, India (* corresponding author, e-mail: gjs1951@rediffmail.com) Abstract. An efficient micropropagation protocol was developed for Capsicum annuum L. cv. ‘Morok Amuba’, an ornamental chilli cultivar using shoo-tip and axillary shoot-tip explants. Multiple shoot buds were induced from shoot-tip explants on MS medium containing cytokinins alone or in combination with IAA. A maximum number of shoot buds was induced on MS medium containing 10 mg/l Zea followed by 5 mg/l BAP in combination with 1 mg/l IAA. Rooting and elongation of the shoot buds were achieved on MS medium supplemented with 0.5 mg/l IAA or IBA. Axillary shoots were induced on the rooted plantlets by decapitation and the axillary shoot-tips explants were used for further induction of shoot buds by culturing them on a medium containing combinations of BAP with IAA. The shoot buds were rooted on a medium containing 0.5 mg/l IBA. The plantlets showed 80-90% survival during transplantation. Key words: axillary shoot-tip explants, chilli, decapitation, regeneration INTRODUCTION

Chillies are the fruits or berries of plants belonging to the genus Capsicum of the Nightshade family, Solanaceae. The genus Capsicum consists of about 25 wild and 5domesticated species. The five domesticated species are Capsicum annuum L., Capsicum frutescens L., Capsicum chinense Jacq., Capsicum baccatum L., and Capsicum pubescens R & P. (IBPGR, 1983). Of the domesticated species, Capsicum annuum is the most economically important and includes both mild and pungent fruit types. Chillies contain numerous chemicals including steam-volatile oil, fatty oils, capsaicinoids, carotenoids, vitamins, protein, fibre and mineral elements (Bosland and Votava, 2000) and are variously used for different purposes because of their nutritional value, flavour, aroma, texture, pungency and colour in a wide assortment of foods, drugs, and cosmetics, while some are cultivated ornamentally, especially for their brightly glossy fruits with a wide range of colours, shape and sizes. Pepper sprays containing capsicum oleoresin provide ingredients for a non-lethal deterrent or repellent to some human and animal behaviours (De, 2003) and are useful riot control agents and self-defense tools. Chillies also have antifungal property against fungal species belonging to Aspergillus and Fusarium (De Lucca et al., 2006; Ngai and Ng, 2006). Besides the above-mentioned uses, chillies also have medicinal uses. Recently, several studies have also demonstrated anti-cancer or anti-mutagenic effect of the chilli extracts. Carotenoids present in chilli extracts were found to have a synergistic anti-mutagenic and in vitro anti-tumour-promotingactivity (de Mejia et al., 1998; Maoka et al., 2003). Topical capsaicin has been shown to have a safe analgesic effect against many painful conditions such as post-herpetic neuralgia, diabetic neuropathy,osteoarthritis and mouth sores developing after chemotherapy or radiation (Nelson, 1994; Rains and Bryson, 1995). Capsicum annuum L. cv. ‘Morok Amuba’ is cultivated as an ornamental in Manipur (23s47I„- 25s41I„ NL; 93s61I„- 94s47I„ EL; 750-3,600 m above MSL; 1,600-3,430 mm annual rainfall) although its fruits are also edible. The plants have flowers with a purple-coloured corolla, a whitish area near the base and bears mildly pungent fruits, which are dark-purple in colour when young and red when ripe. The conventional method of propagation using seeds is restricted by the short span of viability and low germination rate of seeds. Moreover, chilli plants are also highly susceptible to fungal and viral pathogens (Morrison et al., 1986). Since the plants also lack natural vegetative propagation, tissue culture methods provide a novel way for the asexual multiplication of these chilli pepper plants. Propagation of plants through tissue culture offers a unique advantage over conventional propagation methods for conserving and mass multiplication. In Capsicum, several procedures are available for inducing in vitro plant regeneration (Agrawal et al., 1989; Anilkumar and Nair, 2004; Arroyo and Revilla, 1991; Christopher and Rajam, 1994; Christopherand Rajam, 1996; Gunay and Rao, 1978; Khan et al., 2006; OchoaAlejo and Ireta-Moreno, 1990; Peddaboina et al., 2006; Phillips and Hubstenberger, 1985; Ramirez-Malagon and Ochoa-Alejo, 1996; Szasz et al., 1995; Venkataiah et al., 2003). However, several of these reports suggest a strong influence of genotype on the regeneration process (Christopher and Rajam, 1996; Ochoa-Alejo and Ireta-Moreno, 1990; Ramirez-Malagon and Ochoa-Alejo, 1996; Szasz et al., 1995). Moreover, since in vitro clonal propagation via meristem culture is one of the very few ways for producing large number of true-to-type healthy planting material and the proliferation of multiple shoot buds from shoot-tip explants of Capsicum by the release of axillary buds were reported in limited cases (Anilkumar and Nair 2004; Christopher and Rajam 1994; Khan et al., 2006; Peddaboina et al., 2006). The present research involving culture of shoot-tips and axillary shoots explants of Capsicum annuum L. cv. ‘Morok Amuba’ was therefore, undertaken to develop an efficient in vitro clonal propagation protocol for the cultivar.
MATERIALS AND METHODS

Seeds from fresh and healthy ripe fruits collected from local gardens were taken out using forceps and washed with tap water. Seeds were then treated with 0.1% Dhanustin (Carbendazim 50% w/w) for 10-15 min and rinsed three times with distilled water. This was followed by surface sterilization under aseptic conditions with 0.1% HgCl2 solution for5 min, followed by several washes with sterile distilled water. The surface-sterilized seeds were inoculated in 250 ml flasks containing sterile filter paper soaked in sterile distilled water and incubated in the dark for 7-10 days at 25±2 °C. After germination, the seeds were then transferred to culture tubes containing MS (Murashige and Skoog, 1962) basal medium. Shoot-tip leaf explants were derived from four-five week-old in vitro germinated seedlings. Shoot apices (1-1.5 cm) were trimmed from four week-old seedlings and inoculated on a shoot bud induction medium consisting of MS basal medium supplemented with different concentrations of cytokinins, 2-10 mg/l 6-benzyl aminopurine (BAP) or kinetin (Kin) or zeatin (Zea) alone or in combinations of 2-10 mg/l BAP with 1 mg/l indole-3-acetic acid (IAA). The number of shoot buds and the percentage of explants
forming shoot buds were counted after four weeks. The elongated shoot buds (about 2 cm long) obtained from shoot-tip explants were excised and cultured in 250 ml flasks containing 70 ml of rooting media consisting of MS medium supplemented with different concentrations of auxins, 0.5 or 1 mg/l of IAA or indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA) for the rooting of shoot buds. The number of roots (including the main roots and their branches), shoot length and the length of the roots were recorded after six weeks of culture. Axillary shoots were induced on fourweek-old rooted plantlets. These plantlets having 59 leaves were decapitated for inducing axillary shoot development by cutting the tips with a sterile blade. Axillary shoots developing in the axils of leaves of the decapitated plantlets were used for further multiple shoot bud induction by culturing on a medium containing 2-10 mg/l BAP alone or with mg/l IAA and the number of shoot buds were counted after six weeks. The shoot buds proliferated from axillary shoot-tip explants, they were excised and cultured on a rooting medium consisting of MS medium supplemented with different 0.5 mg/l IAA or IBA. The rooted plantlets were gently removed from the flasks and the roots were washed in tap water to remove traces of agar. The plantlets were then transplanted in perforated paper cups containing sand: soil (1:1) and kept covered with clear polythene bags having a few holes on it for the initial 10 days. The plantlets were kept in a 50% shaded net-house and watered daily with tap water to maintain high humidity. After 10 days, humidity was gradually decreased by increasing the size of holes in the polythene bags and the polythene bags were finally removed. Four week-old hardened plants were then transplanted to bigger earthen pots or to the field. All cultures were maintained in a growth chamber at a temperature of 25±2 °C and 16-h photoperiod provided by white fluorescent tubes (30 µmol m-2S-1). All the experiments were repeated thrice and each treatmentfor shoot bud induction from the shoot-tip and axillary shoottip explants and rooting of the shoot buds consisted of ten replicates. Data were analyzed by analysis of variance (ANOVA) followed by Duncan’s multiple range test.
RESULTS

Several regeneration systems reported so far have also shown the critical effect of cytokinin or cytokinin-auxin ratio in regeneration from various explants of Capsicum (Agrawal et al., 1989; Arroyo and Revilla, 1991; Christopher and Rajam, 1994; Ezura et al., 1993; Gunay and Rao, 1978; Phillips and Hubstenberger, 1985; Ramirez-Malagon and Ochoa-Alejo, 1996; Szasz et al., 1995) and other species of Solanaceae (Gleddie et al., 1983; Kowalczyk et al., 1983; Seetharam et al., 2003; Sharma and Rajam, 1995). Therefore, in vitro culture response of shoottip and axillary shoot explants of Capsicum annuum L. cv. ‘Morok Amuba’ on MS medium supplemented with various concentrations of cytokinins (BAP, Kin or Zea) alone or combinations of BAP with IAA have been investigated. After 2-3 weeks of culture on the shoot bud induction medium, about 2-6 buds multiple shoot buds developed from the shoot-tip explants derived from in vitro germinated seedlings. Different concentrations or combinations of growth regulators induced different responses from the shoot-tip explants of the cultivars studied (Table 1). The maximum number of buds was produced on an MS medium containing 10 mg/l Zea followed by 5 mg/l BAP in combination with 1mg/l IAA (Figure 1a). The effectiveness of 1 mg/l Zea alone (Gunay and Rao 1978) or in combination with 0.1 mg/l IAA (Arroyo and Revilla 1991) in pepper tissue cultures has been reported. However, in the present study, the frequency
of shoot buds formed was low (1-2) at 1 mg/l Zea and it increased with increasing concentration of Zea. Some earlier reports of in vitro studies in Capsicum also reported the effectiveness of BAP in combination with IAA in inducing multiple shoot buds in chilli tissue cultures (Arroyo and Revilla, 1991; Franck-Duchenne et al., 1998; Gunay and Rao, 1978; Phillips and Hubstenberger, 1985; Szasz et al., 1995) and our results also suggest the same. MS medium containing Kin alone was found to be the least effective among the three cytokinins (BAP, Zea, and Kin) tested and was not used for further studies. Such ineffectiveness of Kin in shoot bud induction from chilli tissue culture has also been reported earlier (Agrawal et al., 1989; Gunay and Rao, 1978; Phillips and Hubstenberger, 1985).
Table 1 Effect of growth regulators on multiple shoot bud induction from shoot tip explants of Capsicum annuum L. cv. ‘Morok Amuba’ after four weeks of culture BAP 2 2 5 5 10 10 Growth regulators (mg/l) Zea 2 5 10 IAA 1 1 1 Mean number of shoots per explant (mean ± S.E.) 3.0 ± 0.15cd 2.3 ± 0.26de 3.6 ± 0.22bc 4.4 ± 0.27ab 3.4 ± 0.34bc 3.8 ±0.44abc 1.2 ± 0.13f 1.6 ± 0.27ef 4.7 ± 0.30a

Means followed by the same letters arenot significantly different at p=0.01

When the regenerated shoot buds (about 1 cm long) were separated and transferred to MS medium supplemented with IAA, IBA or NAA, rhizogenesis occurred followed by elongation of the shoot buds. IAA and IBA was found superior to NAA with respect to the induction of roots and 40-100% rooting efficiency was recorded (Table 2).
Table 2 Effect of auxins on rooting and elongation of in vitro induced shoot buds from shoot-tip explants of Capsicum annuum L. cv. ‘Morok Amuba’ after six weeks of culture IAA 0.5 1 Auxins (mg/l) IBA 0.5 1 NAA 0.5 1 Rooting (%) 100 60 100 100 100 40 Shoot length (cm) (mean ± S.E.) 2.7 ± 0.35a 1.3 ± 0.29c 2.6 ± 0.26ab 1.9 ± 0.21abc 1.2 ± 0.12c 0.5 ± 0.08c No. of roots (mean ± S.E.) 12.6 ± 0.69cd 7.1 ± 0.78de 38.7 ± 1.61a 28.7 ± 0.32b 17.7 ± 1.52c 1.9 ± 0.78e Root length (cm) (mean ± S.E.) 5.4 ± 0.38a 1.9 ± 0.62bc 3.3 ± 0.23ab 3.8 ± 0.32ab 0.6 ± 0.05c 0.1 ± 0.05c

Means followed by the same letters are not significantly different at p=0.01

On NAA containing medium, the roots produced were thick and short with fine root hairs, while on medium containing IAA or IBA, the roots were thin and long with branches and root hairs. The best rooting and elongation of the regenerated shoot buds was achieved on a medium containing 0.5 mg/l IAA or IBA (Figure 1b). Similar effectiveness of IBA and IAA on 60


the rooting of in vitro regenerated chilli plantlets has been reported earlier(Agrawal et al., 1989; Christopher and Rajam, 1994, 1996; Gunay and Rao, 1978; Peddaboina et al., 2006; Shivegowda et al., 2002; Szasz et al., 1995; Venkataiah et al., 2003). Several studies also reported higher effectiveness of NAA in inducing rhizogenesis of the regenerated shoots in Capsicum (Bodhipadma and Leung, 2003; Husain et al., 1999; Siddique and Anis, 2006). However, in the present study, NAA was found to be less effective for root induction and elongation of the shoot buds.

Figure 1. In vitro propagation of Capsicum annuum L. cv. ‘Morok Amuba’: (a) induction of multiple shoots from shoot-tip explant; (b) rooted plantlet; (c) induction of axillary shoots by decapitation; (d) transplanted plantlet

Development of axillary buds following decapitation is a common phenomenon observed in plants. Earlier, we reported a new technique for obtaining axillary shoot explants for chilli tissue culture by inducing axillary shoot proliferation (up to 5) from in vitro raised chilli seedling plantlets by decapitation (Sanatombi and Sharma, 2007). Ramirez-Malagon and Ochoa-Alejo (1996) also reported the formation of buds and elongated shoots from the wounded apical zone of hypocotyl tissues of the chilli pepper seedlings by wounding the seedlings at the curved stage followed by decapitation of the seedlings after 10-14 days of culture on MS medium without growth regulators. However, the buds in the above case were adventitious buds,which do not show clonal fidelity unlike the axillary shoots produced by release of axillary buds from the decapitated plantlets in the present study. In the next set of experiments, the effect of

decapitation on the growth of axillary shoots in the rooted plantlets and the effect of growth regulators on multiple shoot bud induction from the axillary shoot-tip explants were studied. The decapitated plantlets showed the development of axillary shoots within two weeks of culture. About 3-6 young shoots per plantlet were formed within two weeks of culture in all the cultivars (Figure 1c). Further, the effects of growth regulators on multiple shoot bud induction from the axillary shoot-tip explants have been investigated. The response of axillary shoo-tip explants to bud induction media containing different concentrations and combinations of growth regulators were similar to the shoot-tip explants. The axillary shoot-tip explants proliferated to produce the maximum number of shoot buds on a medium containing 5-10 mg/l BAP alone or in combination with 1 mg/l IAA (Table 3). The proliferated shoot buds also showed rooting and elongation on medium containing 0.5 mg/l IBA.
Table 3 Effect of growth regulators on multiple shoot bud induction from axillary shoot explants of Capsicum annuum L. cv. ‘Morok Amuba’ after six weeks of culture Growth regulators (mg/l) BAP IAA 2 2 1 5 5 1 10 Number of shoot buds (mean ± S.E.) 2.6 ± 0.16bc 2.1 ± 0.18c3.7 ± 0.34ab 4.2 ± 0.39a 2.8 ± 0.25bc 3.6 ± 0.40ab

Means followed by the same letters are not significantly different at p=0.01

The regenerated plants showed 80-90% survival during hardening and acclimatization (Figure 1d) and there were no observable variations between the parent plants and in vitro raised plants. The transplanted plantlets established well in pots and in the field.
CONCLUSION

Thus, by inducing multiple shoots (about 5 per explant) from the shoot-tip explants of a seedling followed by in vitro induction of axillary shoots (up to 5 per plantlet) from the regenerated plantlets and further induction of multiple shoot buds from the axillary shoot explants (up to 5) it has been made possible to produce a maximum of about 125 plantlets from a single seedling. This technique, therefore, presents an efficient system of in vitro clonal propagation compared to seed propagation for rapid multiplication, production of disease-free plants, non-seasonal production, germoplasm conservation and facilitating their easy exchange.
REFERENCES
1. Agrawal, S., Chandra, N., and Kothari, S.L., 1988, Shoot-tip culture of pepper for micropropagation. Curr. Sci., 57, 1347-1349. 2. Anilkumar, M., and Nair, A.S., 2004, Multiple shoot induction in Capsicum annuum L. cv. Early California Wonder. Plant Cell Biotech. Mol. Biol., 5(3&4), 95-100. 3. Arroyo, R., and Revilla, M.A., 1991, In vitro plant regeneration from cotyledon and hypocotyl segmentsin two bell pepper cultivars. Plant Cell Rep., 10, 414-416.


Bodhipadma, K., and Leung, D. W. M., 2003, In vitro fruiting and seed set of Capsicum annuum L. cv. Sweet Banana. In Vitro Cell Dev. Biol. Plant, 39, 530-539. 5. Bosland, P. W., and Votava, E.J., 2000, Peppers: Vegetables and Spice Capsicums. Crop Production Science in Horticulture 12. CAB International Publishing, Wallingford, England, UK. p: 204. 6. Christopher, T., and Rajam, M. V., 1994, In vitro clonal propagation of Capsicum spp. Plant Cell Tiss. Org. Cult., 38, 25-29. 7. Christopher, T., and Rajam, M.V., 1996, Effect of genotype, explants and medium on in vitro regeneration of red pepper. Plant Cell Tiss. Org. Cult., 46, 245-250. 8. De Lucca, A. J., Boue, S., Palmgren, M. S., Maskos, K., and Cleveland, T. E. (2006). Fungicidal properties of two saponins from Capsicum frutescens and the relationship of structure. Can. J. Microbiol., 52(4), 336-342. 9. de Mejia G., E., Quintanar-Hernandez, A., and Loarca-Pina, G., 1998, Antimutagenic activity of carotenoids in green peppers against some nitroarenes. Mutat. Res., 416(1-2), 11-19. 10. De, A. K., 2003, Capsicum: The Genus Capsicum. Medicinal and Aromatic Plants- Industrial Profiles Vol. 33. Taylor & Francis, London and New York. p: 275. 11. Ezura, H., Nishimiya, S. and Kasumi, M., 1993, Efficient regeneration of plants independent of exogenous growth regulators in bell pepper (Capsicum annuum L.). Plant Cell Rep., 12,676-680. 12. Frank-Duchenne, M., Wang, Y., Tahar, S.B., and Beachy, R.N., 1998, In vitro stem elongation of sweet pepper in media containing 24-epi-brassinolide. Plant Cell Tiss. Org. Cult., 53, 79-84. 13. Gleddie, S., Keller, W., and Setterfield, G., 1983, Somatic embryogenesis and plant regeneration from leaf explants and cell suspensions of Solanum melongena (eggplant). Can. J. Bot., 61, 656-666. 14. Gunay, A.L., and Rao, P.S., 1978, In vitro plant regeneration from hypocotyl and cotyledon explants of red pepper (Capsicum). Plant Sci. Letts., 11, 365-372. 15. Husain, S., Jain, A., and Kothari, S.L., 1999, Phenylacetic acid improves bud elongation and in vitro plant regeneration efficiency in Capsicum annuum L. Plant Cell Rep., 19, 64-68. 16. Husain, S., Jain, A., and Kothari, S.L., 1999, Phenylacetic acid improves bud elongation and in vitro plant regeneration efficiency in Capsicum annuum L. Plant Cell Rep., 19, 64-68. 17. IBPGR., 1983, Genetic resources of Capsicum. International Board for Plant Genetic Resources, Rome. 18. Khan, H., Siddique, I., Anis, M., 2006, Thidiazuron induced somatic embryogenesis and plant regeneration in Capsicum annuum. Biol. Plant. 50(4), 789-792. 19. Kowalczyk, T. P., Mackenzie, I. A., and Cocking, E. C., 1983, Plant regeneration from organ explants and protoplasts of the medicinal plant Solanum khasianum C. B. Clake var. chatterjeanum Sengupta (Syn. Solanum viarum Dunal). Z Pflanzenphysiol., 111, 55-68. 20. Maoka,T., Mochida, K., Ito, Y., Fujiwara, Y., Hashimoto, K., Enjo, F., Ojata, M., Nobukumi, Y., Tokuda, H., and Nishino, H., 2001, Cancer chemopreventive activity of carotenoids in the fruits of red paprika Capsicum annuum L. Cancer Letts., 172(2), 103-109. 21. Morrison, R.A., Koning, R.E., and Evans, D.A., 1986, Pepper. In: Evans, D.A., Sharp, W.R., and Ammirato, P.V. (eds). Handbook of Plant Cell Culture. vol 4. McGraw Hill, New York. pp. 554-573. 22. Murashige, T., and Skoog, F., 1962, A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15, 473-497. 23. Nelson, C., 1994, Heal the burn: Pepper and lasers in cancer pain therapy. J. Natl. Canc. Inst., 86, 1381. 24. Ngai, P. H., and Ng, T. B., 2006, A lectin with antifungal and mitogenic activities from red cluster pepper (Capsicum frutescens) seeds. Appl. Microbiol. Biotech., 74(2), 366-371. 25. Ochoa-Alejo, N., Ireta-Moreno, L., 1990, Cultivar differences in shoot-forming capacity of hypocotyl tissues of chilli pepper (Capsicum annuum L.) cultured in vitro. Scientia Hort., 42, 21-28. 26. Peddaboina, V., Christopher, T., Subhash, K., 2006, In vitro shoot multiplication and plant regeneration in four Capsicum species using thidiazuron. Scientia Hort., 107(2), 117-122. 27. Phillips, G.C., and Hubstenberger, J.F., 1985, Organogenesis in pepper tissue cultures. Plant Cell Tiss. Org. Cult., 4, 261-269. 28. Rains, C., and Bryson, H. M., 1995, Topical capsaicin. Areview of its pharmacological properties and therapeutic potential in post-herpetic neuralgia, diabetic neuropathy and osteoarthritis. Drugs Aging, 7(4), 317-328. 29. Ramirez-Malagon, R., and Ochoa-Alejo, N., 1996, An improved and reliable chilli pepper (Capsicum annuum L.) plant regeneration method. Plant Cell Rep., 16, 226-231.

Sanatombi, K., and Sharma, G.J., 2007, Micropropagation of Capsicum frutescens L. using axillary shoot explants, Scientia Hort., 113, 96-99. 31. Seetharam, Y. N., Jyothisaran, G., Sujeeth, H., Barad, A., Sharanabasappa, G., and Panchal, T. S., 2003, In vitro plant regeneration from nodal and leaf explants of Solanum surattense Burm. F.: A medicinal plant. Plant Cell Biotech. Mol. Biol., 4(1&2), 17-22. 32. Sharma, P., and Rajam, M.V., Genotype, explant, and position effects on organogenesis and embryogenesis in eggplant (Solanum melongena). J. Exptl. Bot., 46, 135-141. 33. Shivegowda, S. T., Mythili, J. B., Anand, L., Saiprasad, G. V. S., Gowda, R., and Gowda, T. K. S., 2002, In vitro regeneration and transformation in chilli pepper (Capsicum annuum L.). J. Hort. Sci. Biotech., 77(5), 629-634. 34. Siddique, I., and Anis, M., 2006, Thidiazuron induced high frequency shoot bud formation and plant regeneration from cotyledonary node explants of Capsicum annuum L. Indian J. Biotech., 5, 303-308. 35. Szasz, A., Nervo, G., and Fari, M., 1995, Screening for in vitro shoot forming capacity of seedlingexplants in bell pepper (Capsicum annuum L.) genotypes and efficient plant regeneration using thidiazuron. Plant Cell Rep., 14, 666-669. 36. Venkataiah, P., Christopher, T., and Subhash, K., 2003, Thidiazuron induced high frequency adventitious shoot formation and plant regeneration in Capsicum annuum L. J. Plant Biotech., 5(4), 245-250.