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

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 72 h pre-culturing and 48 h 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 100 µM 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.