Development of transgenic broccoli with cryIAa gene for resistance against diamondback moth (Plutella xylostella).

Abstract

Transgenic broccoli (Brassica oleracea L. var. italica) cv. 'Solan Green Head' has been developed by using Agrobacterium tumefaciens strain harboring cryIAa gene for resistance against diamondback moth (Plutella xylostella). By combining the best treatments of 72h pre-culturing and 48h co-cultivation time period, a transformation frequency of 4.92 and 13.83% was obtained from cotyledon and hypocotyl explants, respectively. Supplementing the co-cultivation medium with acetosyringone in a concentration of 100µM enhanced the transformation frequency to 17.92 and 32.11% in cotyledon and hypocotyl explants, respectively. The transgene (cryIAa) integration was confirmed by polymerase chain reaction using gene-specific primers and Southern blot analysis using digoxigenin nonradiolabelled DNA probe. Gene expression in the PCR-positive transgenic events had been confirmed by reverse transcriptase-PCR and quantitative real time-PCR. Insect bioassay proved the effectiveness of the transgene against infestation by diamondback moth (Plutella xylostella) larvae. To the best of our knowledge, this is the first report of optimization of a highly efficient transformation system and transgenic development in broccoli using cry1Aa gene for insect resistance.