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Keywords:
Amorphous calli, Chilli, Recalcitrant nature, Replicase gene, Plant regeneration
Chilli (Capsicum annuum) is one of the important commercial spice crops in India and well known for its recalcitrant genetic nature. Due to its genetic nature, implicates the limitations in application of genetic engineering. Hence, the present study emphasized with optimization of effective Agrobacterium mediated transformation in major chilli cultivar of Tamil Nadu. The regeneration and transformation potential of chilli cultivar K2 and CO-4 was tested with explants of hypocotyl, leaf and cotyledon for different combinations of plant growth hormones. The maximum per cent calli induction was observed with hypocotyl (61.33%) followed by leaf (57.33%) and cotyledon (21.33%) explants of CO-4 variety. Indeed, K2 variety observed to be not responsive for regeneration as CO-4 and often showed the senescence reaction. The combination of cytokinin 2,4-D (5 mg l−1) with auxin NAA (5 mg l−1) was found to be highly effective in supporting regeneration of chilli. The hypocotyl explant produced amorphous calli at 25–30 days whereas shoot proliferation was observed at 40–43 days. On the other hand, leaf and cotyledon explants yielded calli induction at 30–35 days but failed to give shoot proliferation. There are 4–5 shoot buds were observed from the differentiated calli of inoculated hypocotyl explants. After 50–60 days of complete shoot proliferation, elongated shoots were separated and transferred for the root induction for 10–15 days. The presence of transgene in the putative lines was confirmed through molecular screening with NPTII, 35S promoter and replicase gene specific primers.
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Ahmad N, Siddique I, Anis M (2006) Improved plant regeneration in Capsicum annuum L. from nodal segments. Biol Plant 50(4):701–704
Anonymous (2019) Chilli outlook. Professor Jayashankar Telngana State Agricultural University, Agricultural Market Intelligence Centre
Ashrafuzzaman M, Hossain MM, Ismail MR, Haque MS, Shahidullah SM, Uz-zaman S (2009) Regeneration potential of seedling explants of chilli (Capsicum annuum). Afr J Biotech 8(4):591–596
Bora G, Gogoi HK, Handique PJ (2019) Influence of silver nitrate and glutamine on in vitro organogenesis of Lota Bhot (Capsicum chinense Jacq.), an indigenous pungent pepper variety of Assam. J Appl Biol Biotechnol 7(1):021–028
Christopher T, Rajam MV (1996) Effect of genotype, explant and medium on in vitro regeneration of red pepper. Plant Cell, Tissue Organ Cult 46(3):245–250
Delis M, Garbaczewska GY, Niemirowicz-Szczytt K (2005) Differentiation of adventitious buds from Capsicum annuum L. hypocotyls after co-culture. Acta Biol Cracov Bot 47(1):193–198
Doyle JJ (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15
Gayathri N, Gopalakrishnan M, Sekar T (2015) In vitro micropropagation of Capsicum chinense Jacq. (Naga King Chili). Asian J Plant Sci Res 5(12):13–18
Gunay AL, Rao PS (1978) In vitro plant regeneration from hypocotyl and cotyledon explants of red pepper (Capsicum). Plant Sci Lett 11(3–4):365–372
Iqbal S, Ashfaq M, Shah H (2012) Prevalence and distribution of Cucumber mosaic virus (CMV) in major chilli growing areas of Pakistan. Pak J Bot 44(5):1749–1754
Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Report 5(4):387–405
Joshi A, Kothari SL (2007) High copper levels in the medium improves shoot bud differentiation and elongation from the cultured cotyledons of Capsicum annuum L. Plant Cell, Tissue Organ Cult 88(2):127–133
Ke J, Khan R, Johnson T, Somers DA, Das A (2001) High-efficiency gene transfer to recalcitrant plants by Agrobacterium tumefaciens. Plant Cell Rep 20(2):150–156
Kehie M, Kumaria S, Tandon P (2012) In vitro plantlet regeneration from nodal segments and shoot tips of Capsicum chinense Jacq. cv. Naga King Chilli. 3 Biotech 2(1):31–35
Ko MK, Soh H, Kim KM, Kim YS, Im K (2007) Stable production of transgenic pepper plants mediated by Agrobacterium tumefaciens. HortScience 42(6):1425–1430
Kothari SL, Joshi A, Kachhwaha S, Ochoa-Alejo N (2010) Chilli peppers—a review on tissue culture and transgenesis. Biotechnol Adv 28(1):35–48
Kumar RV, Sharma VK, Chattopadhyay B, Chakraborty S (2012a) An improved plant regeneration and Agrobacterium-mediated transformation of red pepper (Capsicum annuum L.). Physiol Mol Biol Plants 18(4):357–364
Kumar S, Raj SK, Sharma AK, Varma HN (2012b) Genetic transformation and development of Cucumber mosaic virus resistant transgenic plants of Chrysanthemum morifolium cv. Kundan Scientia Horticulturae 134:40–45
Lee SJ, Paik KH, Kim BD (1993) In vitro plant regeneration and Agrobacterium-mediated transformation from cotyledon explants of hot pepper (Capsicum annuum cv Golden Tower). Korean J Plant Tissue Cult 20(5):289–294
Lee YH, Kim HS, Kim JY, Jung M, Park YS, Lee JS, Choi SH, Her NH, Lee JH, Hyung NI (2004) A new selection method for pepper transformation: callus-mediated shoot formation. Plant Cell Rep 23(1):50–58
Liu W, Parrott WA, Hildebrand DF, Collins GB, Williams EG (1990) Agrobacterium induced gall formation in bell pepper (Capsicum annuum L.) and formation of shoot-like structures expressing introduced genes. Plant Cell Rep 9(7):360–364
Mahto BK, Sharma P, Rajam MV, Reddy PM, Dhar-Ray S (2018) An efficient method for Agrobacterium-mediated genetic transformation of chilli pepper (Capsicum annuum L.). Indian J Plant Physiol 23(3):573–581
Manoharan M, Vidya CSS, Sita GL (1998) A grobacterium-mediated genetic transformation in hot chilli (Capsicum annuum L. var. Pusa jwala). Plant Sci 131(1):77–83
Mok SH, Khalid N (2007) Trouble shooting for recalcitrant bud formation in Capsicum annuum var. Kulai. Asia Pac J Mol Biol Biotechnol 15(1):33–38
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15(3):473–497
Ochoa-Alejo N, Ireta-Moreno L (1990) Cultivar differences in shoot forming capacity of hypocotyl tissue of red pepper pepper (Capsicum annuum) cultured in vitro. Sci Horti 42:21–28
Ochoa-Alejo N, Ramirez-Malagon R (2001) In vitro chili pepper biotechnology. In Vitro Cell Dev Biol Plant 37(6):701–729
Prakash AH, Rao KS, Kumar MU (1997) Plant regeneration from protoplasts of Capsicum annuum L. cv. California Wonder. J Biosci 22(3):339–344
Prakash DP, Deepali BS, Asokan R, Ramachandra YL, Anand L, Hanur VS (2007) Effects of growth regulators and explant-type on Agrobacterium-mediated transformation in brinjal (Solanum melongena L.) cv. Manjarigota. J Hortic Sci 2(2):94–98
Raj RP, Glint VD, Babu KN (2015) In vitro plant regeneration in Capsicum chinense Jacq. (Naga Chili). J Appl Biol Biotechnol 3:030–033
Sanatombi K, Sharma GJ (2008) Capsaicin content and pungency of different Capsicum spp. cultivars. Notulae Botanicae Horti Agrobotanici Cluj-Napoca 36(2):89–90
We are thankful to Department of Biotechnology, NER-BPMC, New Delhi for the financial assistance, Dr. R. Gnanam, Professor and Head, Department of Bioinformatics, CPMB&B for valuable guidance and Professor and Head, Department of Plant Pathology, TNAU, Coimbatore for providing facilities during the course of study.