Agrobacterium-mediated transformation of Brassica oleracea var. italica and B. napus with a Trichoderma harzianum endochitinase gene to enhance resistance against fungal pathogens

A Trichoderma harzianum endochitinase gene was introduced into B. oleracea var. italica and B. napus by Agrobacterium tumefaciens-mediated transformation. The cDNA endochitinase, gene was under control of the double CaMV 35-S promoter and the alfalfa mosaic virus enhancer. The nptII gene under control of the nos promoter was used as a selectable marker. Higher transformation efficiency was obtained with the nopaline Agrobacterium strain GV3101 than with the octopine strain LBA4404.; Kanamycin-resistant shoots were analyzed to confirm presence and expression of the endochitinase gene in primary transgenic plants. All kanamycin-resistant shoots showed the endochitinase gene fragment (∼1.4 kb) in the PCR and Southern blot analysis. Levels of endochitinase expression in mature primary transgenic plants in soil were 15 to 37X higher than in non-transgenic plants. T1 plants had higher levels of expression (97 to 208X) than primary transformants.; Inoculation of primary transgenic plants with the fungal pathogens Alternaria brassicicola and Sclerotinia sclerotiorum indicated that disease severity was not significantly different from non-transgenic plants. Broccoli T1 progeny plants inoculated with A. brassicicola showed a significant reduction in disease severity compared to non-transgenic plants; however, no significant reduction was observed in broccoli and rapeseed progeny inoculated with S. sclerotiorum.; The fungicide Bayleton, a demethylation inhibitor, was sprayed on transgenic and non-transgenic broccoli at ED₅₀ dose to determine the level of interaction between endochitinase and fungicide. Sprayed transgenic plants inoculated with A. brassicicola showed important reductions in disease severity compared to non-sprayed transgenic plants; however, these levels of protection were not much higher than the additive expected effect and therefore no synergism was detected. Levels of resistance in non- sprayed transgenic plants were similar to sprayed control plants, suggesting that endochitinase and Bayleton (ED₅₀) provide similar levels of protection against A. brassicicola. Some transgenic lines sprayed with higher doses of fungicide showed similar levels of resistance as the non-sprayed lines, again suggesting no synergistic effect.; Non-inoculated transgenic plants constitutively transcribed the PR-1 gene, unlike non-transgenic plants, suggesting induction of native systemic acquired resistance genes by the heterologous endochitinase gene.