Brassica oleracea cv. Pride of India is one of the most promising vegetable cultivars commercially grown as cash crop in Himachal Pradesh, India. However, its overall production is severely hampered by diamondback moth (Plutella xylostella), a notorious pest. To avoid yield losses caused by this pest, cryIAa gene was introduced into cabbage cv. Pride of India using Agrobacterium-mediated transformation method. In an attempt to maximize the transformation frequency, critical determinant factors such as explant type, pre-incubation and co-cultivation period, and acetosyringone effect were successfully optimized. The highest transformation frequency (4.67% and 14.50%) in cotyledon and hypocotyl explant was achieved with a pre-incubation period of 72 h and co-cultivation period of 48 h. Furthermore, transformation frequency was enhanced in cotyledon (18.66%) and hypocotyl (32.00%) explants, when selective regeneration medium was fortified with 100 µM acetosyringone, respectively. The transgene (cryIAa) integration and copy number were confirmed using PCR and Southern blotting. Reverse transcriptase PCR and quantitative real-time PCR analyses were performed that proved transcriptional expression of cryIAa gene in PCR-positive transgenic events. Transgenic cabbage-fed diamondback moth larvae showed significantly higher mortality, thereby proving transgene effectiveness against insect pest control.
Keywords: Cotyledon; Genetic transformation; Hypocotyl; Insect bioassay; Insect resistance; cry1Aa gene.
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