RAPID PRODUCTION OF TRANSGENIC WHEAT PLANTS BY DIRECT BOMBARDMENT OF CULTURED IMMATURE EMBRYOS

We have developed an improved protocol for the rapid and efficient pro duction of transgenic wheat. Three plasmids, each containing the selec table bar gene for resistance to the herbicide Basta and the beta-gluc uronidase (GUS) reporter gene, were delivered via particle bombardment , directly into immature embryos of two spring and one winter cultivar of wheat four days to two months after culture. Resistant calli were selected on phosphinothricin (PPT) media and screened for histochemica l GUS activity. Twelve independent callus lines showing phosphinothric in acetyltransferase (PAT) activity were recovered from the bombardmen t of 544 explants (374 immature embryos and 170 one or two month old c alli). RO plants were regenerated from seven of these lines, of which so far five have produced R1 progeny, and two of the latter have produ ced R2 progeny. PAT activity was detected in each of the plants tested from the seven RO lines, as well as in a 1:1 or 3:1 ratio in R1 plant s following cross or self pollination, respectively. Resistance to top ical application of Basta was seen in PAT positive plants and transgen ic progeny. Molecular analysis by Southern hybridization showed the pr esence of the bar gene in all PAT positive RO and R1 plants analyzed. Hybridization of the bar gene probe with high molecular weight DNA fur ther confirmed integration into nuclear DNA. Both male and female tran smission of the bar gene, and its segregation as a dominant Mendelian trait in R1 and R2 plants, were demonstrated. Flowering transgenic RO plants could be obtained in 7-9 months following excision and culture of immature embryos.