, Volume 8, Issue 4, pp 323-333

High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize

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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 0.5×109 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 T0 plants were regenerated and these plants were assayed by transgene expression and some of them were also analyzed by Southern hybridization. T1 plants were analyzed and transmission of transgenes to the T1 generation was verified. This represents a highly reproducible and reliable system for genetic transformation of maize Hi-II.