High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize

A high throughput genetic transformation system in maize has been developed with Agrobacterium tumefaciens mediated T-DNA delivery. With optimized conditions, stable callus transformation frequencies for Hi-II immature embryos averaged approximately 40%, with results in some experiments as high as 50%. The optimized conditions include N6 medium system for Agrobacterium inoculation, co-cultivation, resting and selection steps; no AgNo3 in the infection medium and adding AgNo3 in co-cultivation, resting and selection medium; Agrobacterium concentration at 0.5x10(9) c.f.u. ml(-1) for bacterium inoculation; 100 mg l(-1) carbenicillin used in the medium to eliminate Agrobacterium after inoculation; and 3 days for co-cultivation and 4 days for resting. A combination of all of these conditions resulted in establishing a high throughput transformation system. Over 500 T-0 plants were regenerated and these plants were assayed by transgene expression and some of them were also analyzed by Southern hybridization. T-1 plants were analyzed and transmission of transgenes to the T-1 generation was verified. This represents a highly reproducible and reliable system for genetic transformation of maize Hi-II.