The production of marker-free genetically engineered broccoli with sense and antisense ACC synthase 1 and ACC oxidases 1 and 2 to extend shelf-life

The production of transgenic broccoli (Brassica oleracea) with increased shelf-life using an Agrobacterium rhizogenes-mediated co-transformation protocol is reported. An Agrobacterium rhizogenes Ri vector, pRi1855:GFP was constructed to allow expression of the green fluorescent protein to identify insertion of Ri T-L-DNA into plant cells. The Brassica oleracea ACC synthase 1 and ACC oxidase 1 and 2 cDNAs in sense and antisense orientations were co-transformed into GDDH33, a doubled haploid calabrese-broccoli cultivar. Transformation efficiency was 3.26%, producing 150 transgenic root lines, of which 18 were regenerated into mature plants. The floral buds from T-0 broccoli heads were assayed for post-harvest production of ethylene and chlorophyll levels. Buds from T-0 lines transformed with ACC oxidase 1 and 2 constructs produced significantly less post-harvest ethylene at 20 degrees C than the untransformed plants and chlorophyll loss was significantly reduced over a 96 h post-harvest period. The T-0 plants transformed with sense and antisense ACC synthase 1 had a significantly reduced 24 h post-harvest ethylene peak and delayed chlorophyll loss. A positive correlation between post-harvest bud ethylene production and chlorophyll loss was described by a regression. This demonstrates that the shelf-life of a very perishable vegetable may be increased up to 2 days at 20 degrees C by reducing post-harvest ethylene production.