J. Steven Tebbets

High levels of expression of full-length cryIA(c) gene from Bacillus thuringiensis in transgenic somatic walnut embryos

Abstract A full-length synthetic version of the cry IA(c) gene, expressing a Bacillus thuringiensis (Bt) insecticidal crystal protein (ICP), was transferred into walnut somatic embryos. Sixty one transgenic embryo lines or clones were obtained and bioassayed with first instar codling moth larvae. In 34% of these lines, designated as ‘class A’, expression was high enough to obtain 80–100% mortality. A total of 20% were designated ‘class B’, which produced a mortality between 25 and 70% and also caused a decreased rate of larval development. Insect mortality and development from the remaining 46% of the lines were indistinguishable from that of the control. Expression of a chimeric gene encoding β -glucuronidase (GUS) was evaluated to serve as a linked but unselected marker gene. About 62% of the class A embryo lines showed correspondingly high activity for the GUS gene. However, 38% of the class A would have been missed if the level of GUS activity was used as the sole indicator. Detectable levels of protein corresponding to cry IA(c) could be found only in class A, but not in class B or C clones. Southern analysis of border regions revealed single inserts for class A clones and multiple inserts for classes B and C.

Low levels of expression of wild type Bacillus thuringiensis var. kurstaki cryIA (c) sequences in transgenic walnut somatic embryos

Abstract Insecticidal crystal protein fragments (ICPFs) of Bacillus thuringiensis encoded by cry IA (c) and cry IA (b) were previously shown to be lethal to key target walnut insect pests (codling moth, navel orangeworm and Indianmeal moth). One of these genes, cry IA (c), was used to transform walnut somatic embryos using a binary vector (pWB139) in which this gene was expressed as a protein fusion with the kanamycin resistance gene from bacteria. Transgenic embryos representing individual transformation events were germinated to produce shoots that were maintained as micropropagated shoot lines in the laboratory. Transgenic shoots were then grafted onto seedling rootstocks and transplanted into the field. DNA analysis (Southern blotting) demonstrated that these shoots were transformed by pWB139 or by the control construct pWB149 (which does not contain the cry IA (c) gene). Insect feeding trials of walnut embryos grown in vitro and infested with neonatal Indianmeal moth, codling moth or navel orangeworm larvae showed non-significant mortality; confirming insufficient or incomplete expression of ICPs in the transgenic embryos. Therefore, transformation of somatic walnut embryos with the vector pWB139 was ineffective in protecting the embryos from damage by lepidopteran insect larvae.