Cytokinin vectors mediate marker-free and backbone-free plant transformation.

Conventional Agrobacterium-mediated transformation methods rely on complex and genotype-specific tissue culture media for selection, proliferation, and regeneration of genetically modified cells. Resulting transgenic plants may not only contain selectable marker genes but also carry fragments of the vector backbone. Here, we describe a new method for the production of transgenic plants that lack such foreign DNA. This method employs vectors containing the bacterial isopentenyltransferase (ipt) gene as backbone integration marker. Agrobacterium strains carrying the resulting ipt gene-containing "cytokinin" vectors were used to infect explants of various Solanaceous plant species as well as canola (Brassica napus). Upon transfer to hormone-free media, 1.8% to 9.9% of the infected explants produced shoots that contained a marker-free T-DNA while lacking the backbone integration marker. These frequencies often equal or exceed those for backbone-free conventional transformation.

Reduction of protease inhibitor activity by expression of a mutant Bowman-Birk gene in soybean seed.

A mutant Bowman-Birk gene was created that encoded an inactive high-sulfur product. It was used to transform soybean line Asgrow 3237. Transformants bearing the mutant gene were identified by GUS expression, PCR analysis, and Southern analysis. The amount of steady state mRNA from the mutant gene in the transformed plants showed that the gene was highly expressed, but the amount of message from the unmodified Bowman-Birk gene did not change detectably. Proteins synthesized at the direction of the mutant Bowman-Birk gene accumulated in seeds of the transformed plants, and there was a marked decrease in the ability of extracts prepared from these seeds to inhibit trypsin and chymotrypsin despite the presence of Kunitz trypsin inhibitor. The more prevalent mRNA from the mutant gene was considered to out-compete message from the native genes to decrease the amount of active Bowman-Birk inhibitor.