Genetic Engineering of Insect Tolerance in Walnut: Introduction of next Generation Constructfor High Expression of a Bt Encodedicp

Replacement trees were planted in the transgenic field plot. A total of 124 plants in the plot now includes 3 Su-2 clones with Bt (~IA(c» and Kan, 3 similar control clones without Bt, 10 Su-2 clones with Kan and GUS, 8 SF-delta clones with Kan, and 2 untransformed control clones (Su-2 and SF-delta). Seven trees (representing 3 clones) flowered in their second season in the field. Polymerase chain reaction technology is being developed to assist in confirming the presence of introduced genes both in the field and in culture. Results from insect feeding trials indicate that the purified insecticidal crystal protein fragments remain active after being homogenized with walnut leaves and applied to the surface of the diet. Significant differences could not be detected in survivalbetweeninsectsfed on livewalnutsembryos containing the£aIA(c) gene and those fed on untransformed or control embryos. A new vector (pMON105l8) which contains a full-length £!:!IA gene, chemically synthesized to match the ~odon usage pattern of dicot plants, was received from Monsanto and modified to contain the GUS marker gene. This vector was used to produce 31 distinct transgenic subclones, 11 of which have been germinated. Ten new embryogenic cultures have been initiated from controlled crosses to provide additional material for gene insertion.