PRODUCTION OF TRANSGENIC MAIZE PLANTS BY DIRECT DNA UPTAKE INTO EMBRYOGENIC PROTOPLASTS

Fertile transgenic maize plants were regenerated after direct transfer of a chimeric gene into maize protoplasts. Plasmid DNA containing mut ant dihydrofolate reductase (DHFR) mouse gene, that confers methotrexa te (MTX) resistance, under the control of the CaMV 35S promoter was in troduced into maize embryogenic protoplasts by polyethylene glycol (PE G) treatment. Transformation was also carried out with a modified plas mid in which the selective marker gene casette was cloned into the Bst BI site of the Ds 1 maize transposable element. Resistant callus tissu es grown in the presence of 10(-6) or 10(-7) M MTX were selected and s hoot or plant regeneration was achieved under hormone-free culture con ditions. The presence of the introduced DHFR gene in DNA isolated from the selected colonies and the primary regenerants (T0) was shown by S outhern hybridization and PCR analysis. PCR primers for the 35S promot er and for two regions of the coding sequence of the DHFR gene were us ed for amplification of the foreign sequence present in maize genomic DNA. The PCR products were hybridized with a mouse DHFR gene specific probe. Synthesis of the mouse DHFR in MTX resistant maize tissues was detected by staining for enzyme activity after native PAGE. The in vit ro regenerated plants could be grown up to maturity in the greenhouse. Cross pollination has resulted in seeds and the F1 progenies were als o analyzed. In addition to the segregation of MTX-resistant and-sensit ive offsprings, molecular evidences based on Southern data and PCR ana lysis have indicated that the introduced gene was transferred into the first sexual generation. This report provides a new example for poten tials in the use of embryogenic cereal protoplasts for production of f ertile transgenic crop plants.