An efficient protocol for Agrobacterium-mediated transformation of Australian commercial canola cultivars using seedling explants is described. Seedling explants provide flexibility and reduction in labour and maintenance costs of explant sources. Five commercial genotypes of canola were successfully transformed using the developed protocol. A transformation efficiency of 67% was obtained for genotypes Oscar and RK7 from cotyledon explants, which was higher than the rate for the most commonly used cultivar Westar (33%). Comparison of different seedling explants showed that although transgenic plants could be regenerated from all explant types (cotyledons, hypocotyls, and roots) used, the number of plants regenerated per explant type varied among the cultivars. Cotyledons produced the maximum number of transgenic shoots (RK7, RI25, Oscar, and Westar cultivars), whereas root explants produced the lowest numbers of shoots. Therefore, cotyledons and hypocotyls can be considered as ideal explants for the Agrobacterium-mediated transformation of these Australian canola cultivars. Integration and expression of the introduced transgene were analysed by DNA gel blot, leaf disc test, and GUS expression assays. Analysis of progeny showed that the transgene was stably inherited. The possibility of producing male sterile lines using an antisense approach was also explored. For this, Bcp1, a gene shown to be vital for viable pollen development, was targetted. Pollen ablation and lack of seed set were observed in the transgenic plants. Histochemical tests showed an intact tapetum layer and well developed pollen in control plants, whereas degraded tapetum and ablated pollen were noted in the transgenic plants. These results indicate that it is possible to generate stable transgenic male-sterile lines of canola using this strategy.
Production of male sterile transgenic plants
