GENETIC ENGINEERING OF INSECT TOLERANCE IN VAUruT: IMPROVED TRANSFORMATION EFFICIENCY

The genes encoding the insecticidal crystal proteins (ICPs) of BacIllus thuringiensis are good candidates for protecting walnut plants against insect pests. Ve have estimated the dose response of three important walnut pests (codling moth, navel orangeworm and Indianmeal moth) with two highly purified insecticidal proteins of Bacillus thurJ.nglensis and show that these three insects are highly sensitive to these proteins. Sublethal effects of the insecticidal proteins on development and reproduction are also being determined. In order that the genes encoding these proteins be transferred to walnut we have developed a gene transfer system. The Agrobacterium-mediated gene transfer systemthat relieson repetitive embryogenesis to regenerate transgenic walnut plants was made more efficient by using a combination of a selectable (kanamycin resistance) and a scorable (beta-glucuronidase, GUS) marker gene. The transformation with DNA containing these two genes greatly facilitated early screening and selection. Introduction of the GUS marker gene allows positive identification of transformed secondary embryos as early as 5-6 weeks after inoculation. DNA analysis of a representative subset of lines (n-13) derived from secondary embryos confirmed transformation and provided evidence for multiple insertion events in single inoculated primary embryos. Ve have now used this gene transfer system to introduce a gene [cryIA (c) gene of BacIllus thurlnglensIs] conferring tolerance to insect pests into walnut embryos. These embryos have been regenerated to form shoots which are being grafted onto seedling rootstock in the greenhouse. Ve have applied for a permit to grow these transgenic plants in a field test plot.