CROWN GALL RESISTANT WALNUT ROOTSTOCKS: ANALYSIS OF THE GRAFT UNION FOR TRANSMISSION OF GENETIC AND BIOLOGICAL MATERIALS.

Crown gall disease, caused by the bacterium Agrobacterium tumefaciens, is a source of significant economic losses in California walnut orchards and nurseries. Profits are reduced in the form of, lowered productivity from galled trees, their increased susceptibility to other pathogens and/or adverse environmental conditions and unsalable stock at the level of nurseries. A resistant rootstock was developed as a potential solution to this problem using RNAi technology. These transgenic rootstocks were generated by Agrobaterium-mediated plant transformation and express double stranded RNA corresponding to common and highly conserved Agrobacterium T-DNA encoded genes ipt, iaaM responsible for tumor formation (Escobar et al., 2001; 2002; 2003). Typically when wild type walnut plants are infected with Agrobacterium these genes are introduced into the infected cells leading to their expression that results in the uncontrolled cell proliferation and the crown gall phenotype. The genetically engineered rootstocks are able to block the expression of these genes resulting in the suppression of tumor formation (Escobar et al., 2001; 2002; 2003). Now that the success of this technology has been demonstrated with a transgenic Paradox hybrid genotype, the next step is to obtain detailed analysis of the elite transgenic materials to enable deregulation of these rootstocks that would permit further extensive field testing to establish efficacy under rigorous field conditions at multiple locations and eventual commercial release. A key issue this study is investigating is the stability and movement of the introduced genes and their products across the graft union in bark tissues. The regulations focus on the movement of genetic materials, i.e., seeds and pollen Studies on the long distance movement of genes have been completed and show no movement of genetic materials (genes) and supports the earlier work with tomato (Escobar et al., 2003). The movement of gene products, i.e., RNA, protein and metabolites and to the extent this occurs is the focus of this study. Stegemann & Bock et al.(2009) recently documented short distance movement of genetic materials from rootstock into a few cell layers of the scion within the graft union. In this study we are investigating the graft union site and the tissues immediately above and below as well as pellicle and kernels (nut parts) to detect both short and long distance movement of transgene products from transgenic tissues to wild type scion using the most advanced technologies with highest possible sensitivity and sequence-based accuracy.