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We describe a plant protoplast transformation method that provides transformants with a simple pattern of integration of a foreign gene. The approach is to deliver into plant protoplasts by direct gene transfer the Agrobacterium virulence genes virD1 and virD2 with or without virE2, together with a target plasmid containing a gene of interest flanked by Agrobacterium T-DNA border repeat sequences of 25 bp. We present evidence of T-DNA formation in maize protoplasts and its integration into the maize genome. The frequency of VirD1-VirD2-mediated integration events was about 20-35% of the total number of transformants. The addition of virE2 doubled the transformation efficiency. The method described here is of sufficient efficiency and simplicity to be useful for the production of transgenic plants with single-copy well-defined transgenic inserts

Phosphinothricin (PPT) inhibits glutamine synthetase in plant cells, resulting in an accumulation of ammonia that can be toxic. Bar, from Streptomyces hygroscopicus is a gene that codes for phosphinothricin acetyltransferase, an enzyme that detoxifies PPT by acetylation. We used polyethylene-glycol-mediated transformation to introduce bar into protoplasts of maize (Zea mays L.). With a novel assay utilizing the pH indicator chlorophenol red we identified transformants after only two to four weeks of selection on PPT.

A rapid, efficient assay that is nondestructive and semiquantitative for identifying transgenic plants and progeny from Biolistic® and protoplast transformations is described. Leaf sections of maize and wheat plants are placed on an indicator medium containing chlorophenol red and the selection agent. Changes in the color of the medium from red to yellow resulting from altered pH indicate transformed plants within 2-5 d. The method is particularly suited to use with phosphinothricin and could be used with other suitable selectable markers.