Rapid production of transgenic wheat plants by direct bombardment of cultured immature embryos

Abstract

We have developed an improved protocol for the rapid and efficient production of transgenic wheat. Three plasmids, each containing the selectable bar gene for resistance to the herbicide Basta and the beta-glucuronidase (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 histochemical GUS activity. Twelve independent callus lines showing phosphinothricin acetyltransferase (PAT) activity were recovered from the bombardment of 544 explants (374 immature embryos and 170 one or two month old calli). R0 plants were regenerated from seven of these lines, of which so far five have produced R1 progeny, and two of the latter have produced R2 progeny. PAT activity was detected in each of the plants tested from the seven R0 lines, as well as in a 1:1 or 3:1 ratio in R1 plants following cross or self pollination, respectively. Resistance to topical application of Basta was seen in PAT positive plants and transgenic progeny. Molecular analysis by Southern hybridization showed the presence of the bar gene in all PAT positive R0 and R1 plants analyzed. Hybridization of the bar gene probe with high molecular weight DNA further confirmed integration into nuclear DNA. Both male and female transmission of the bar gene, and its segregation as a dominant Mendelian trait in R1 and R2 plants, were demonstrated. Flowering transgenic R0 plants could be obtained in 7–9 months following excision and culture of immature embryos.