Production of transgenic maize plants by direct DNA uptake into embryogenic protoplasts

Abstract

Fertile transgenic maize plants were regenerated after direct transfer of a chimeric gene into maize protoplasts. Plasmid DNA containing mutant dihydrofolate reductase (DHFR) mouse gene, that confers methotrexate (MTX) resistance, under the control of the CaMV 35S promoter was introduced into maize embryogenic protoplasts by polyethylene glycol (PEG) treatment. Transformation was also carried out with a modified plasmid in which the selective marker gene casette was cloned into the BstBI site of the Ds 1 maize transposable element. Resistant callus tissues grown in the presence of 10 −6 or 10 −7 M MTX were selected and shoot or plant regeneration was achieved under hormone-free culture conditions. The presence of the introduced DHFR gene in DNA isolated from the selected colonies and the primary regenerants (T 0) was shown by Southern hybridization and PCR analysis. PCR primers for the 35S promoter and for two regions of the coding sequence of the DHFR gene were used 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 vitro regenerated plants could be grown up to maturity in the greenhouse. Cross pollination has resulted in seeds and the F 1 progenies were also analyzed. In addition to the segregation of MTX-resistant and-sensitive offsprings, molecular evidences based on Southern data and PCR analysis have indicated that the introduced gene was transferred in the first sexual generation. This report provides a new example for potentials in the use of embryogenic cereal protoplasts for production of fertile transgenic crop plants.