Agrobacterium-mediated transformation of bell pepper (Capsicum annuum L.)using a binary vector system
DOI:
https://doi.org/10.24154/jhs.v20i2.4310Keywords:
Agrobacterium, California Wonder, Capsicum, regeneration, transformationAbstract
This study established an efficient Agrobacterium tumefaciens-mediated transformation system for Capsicum annuum L. cv. California Wonder. Cotyledon and hypocotyl explants were co-cultivated with Agrobacterium strain LBA 4404, harboring the binary vector pCAMBIA2301, carrying the npt-II (neomycin phosphotransferase II) and gus (â-glucuronidase) genes. The effects of bacterial cell density and co-cultivation time on transformation efficiency were evaluated. Optimal transformation efficiencies of 43.3% for cotyledon and 33.3% for hypocotyl explants were achieved using an Agrobacterium concentration of OD600 = 0.6 and a 48-h co-cultivation period. As a pre-requisite, in vitro regeneration media were optimized: cotyledon explants exhibited shoot organogenesis on Murashige & Skoog (MS) medium supplemented with 8 mg/L 6-benzylaminopurine (BAP) and 6 mg/L indole-3-acetic acid (IAA), while hypocotyls produced shoots on 6 mg/L meta-topolin (mT) and 4 mg/L IAA. Shoot elongation was maximized on MS medium containing 2 mg/L mT, 1 mg/L gibberellic acid (GA3), and 0.5 mg/L IAA. Root induction on MS medium with 1 mg/L IBA. Bacterial overgrowth was controlled with 500 mg/L cefotaxime, and transformants were selected on 60 mg/L kanamycin. PCR analysis verified stable gus gene integration in putative transgenic plantlets. This optimized protocol offers a valuable tool for introducing agronomically important genes to enhance biotic and abiotic stress tolerance in C. annuum, facilitating genetic improvement of this challenging crop
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