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A high throughput genetic transformation system in maize has been developed with Agrobacterium tumefaciensmediated 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 Agrobacteriuminoculation, co-cultivation, resting and selection steps; no AgNo3in the infection medium and adding AgNo3in co-cultivation, resting and selection medium; Agrobacteriumconcentration at 0.5×109c.f.u. ml−1for bacterium inoculation; 100 mg l−1carbenicillin used in the medium to eliminate Agrobacteriumafter 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 T0plants were regenerated and these plants were assayed by transgene expression and some of them were also analyzed by Southern hybridization. T1plants were analyzed and transmission of transgenes to the T1generation was verified. This represents a highly reproducible and reliable system for genetic transformation of maize Hi-II.

For regulatory issues and research purposes it would be desirable to have the ability to segregate transgenes in co-transformed maize. We have developed a highly efficient system to segregate transgenes in maize that was co-transformed using an Agrobacterium tumefaciens2 T-DNA binary system. Three vector treatments were compared in this study; (1) a 2 T-DNA vector, where the selectable marker gene bar(confers resistance to bialaphos) and the β-glucuronidase(GUS) reporter gene are on two separate T-DNA's contained on a single binary vector; (2) a mixed strain treatment, where barand GUS are contained on single T-DNA vectors in two separate Agrobacteriumstrains; (3) and a single T-DNA binary vector containing both barand GUS as control treatment. Bialaphos resistant calli were generated from 52 to 59% of inoculated immature embryos depending on treatment. A total of 93.4% of the bialaphos selected calli from the 2 T-DNA vector treatment exhibited GUS activity compared to 11.7% for the mixed strain treatment and 98.2% for the ciscontrol vector treatment. For the 2 T-DNA vector treatment, 86.7% of the bialaphos resistant/GUS active calli produced R0plants exhibiting both transgenic phenotypes compared to 10% for the mixed strain treatment and 99% for the single T-DNA control vector treatment. A total of 87 Liberty herbicide (contains bialaphos as the active ingredient) resistant/GUS active R0events from the 2 T-DNA binary vector treatment were evaluated for phenotypic segregation of these traits in the R1generation. Of these R0events, 71.4% exhibited segregation of Liberty resistance and GUS activity in the R1generation. A total of 64.4% of the R02 T-DNA vector events produced Liberty sensitive/GUS active (indicating selectable-marker-free) R1progeny. A high frequency of phenotypic segregation was also observed using the mixed strain approach, but a low frequency of calli producing R0plants displaying both transgenic phenotypes makes this method less efficient. Molecular analyses were then used to confirm that the observed segregation of R1phenotypes were highly correlated to genetic segregation of the barand GUS genes. A high efficiency system to segregate transgenes in co-transformed maize plants has now been demonstrated.