Improving in vitro culture and rigenaration conditions for Agrobacterium-mediated maize transformation

Experimental conditions for improving the tissue Culture regenerative potential and embryogenic capability of maize cultures after Agrobacterium-mediated transformation have been investigated. The parameters were investigated in five elite inbreds per se and in their crosses with A188. Embryogenic calli obtained from the inbreds spanned in a range of values from hardly responsive (11.5%) to highly responsive (79.1%) cultures. Immature embryos explanted from crosses of each inbred with A188 were all enhanced in their embryogenic and regenerative capability, with values ranging from 72.7 to 79.6%. The same genotypes, were subjected to Agrobacterium-mediated transformation, considering as target tissue either freshly explanted or five days-precultured immature embryos. Tissues were transformed with the Agrobacterium strain EHA105 with a standard binary vector based on pCAMBIA3301. The addition of 400mg/I L-cysteine in the co-cultivation step, as antioxidant to reduce callus necrosis, was also tested. Results indicated that addition of L-cys in the infection step with non pre-cultured embryos in general reduces regenerative capability, inhibiting the embryogenic development of the infected embryos. Conversely, the adoption of a pre-culture period of five days on callus induction medium, largely overcomes the inhibition. Calli treated and non-treated with L-cys exhibited comparable embryogenic potential; in addition, the regenerative capability was significantly enhanced. The protocol described is discussed as an improvement in Agrobacterium-mediated maize transformation.