Genetic transformation of grapevine to test significant abiotic stress and pest resistance genes
Abstract
Grapevine transformation is a slow and tedious procedure, but it is an essential technique for establishing the roles of key genes in grapevine, which subsequently leads to better breeding outcomes through marker-assisted selection. In this project we successfully transformed Shiraz to test a range of sodium and chloride exclusion candidate genes, assayed several sets of altered lines for their ability to exclude salt and produced other lines for testing later. Methods for rootstock transformation were also trialled. These techniques were partially successful and, with further refinement, will be useful to support CSIRO’s rootstock breeding projects.
Summary
Rootstocks for winegrapes provide the scion with a number of important protective features, such as resistance to pests like phylloxera and nematodes, exclusion of unwanted salts, control of vigour, and better water use efficiency. Predictions for climate change suggest that stresses such as salt and reduced water quality and quantity will increase over the next few decades. To assist in the protection of the Australian winegrape industry into the future, CSIRO, in partnership with Wine Australia, is developing new rootstocks that can tolerate these stresses to a higher degree than rootstocks currently available. Marker-assisted selection is a large part of the breeding of new rootstocks. To develop perfect markers for a desired trait, the isolation and characterisation of genes that encode these important resistance traits is an essential step.
To assist with the development of markers we are utilising grapevine transformation techniques to test the function of key genes. This project focussed on genes that are candidates for major roles in exclusion of chloride and sodium. Putative chloride exclusion genes were selected at the start of the project, based on previous work conducted with our collaborators, Sam Henderson and Matt Gilliham at the University of Adelaide.
Three putative chloride transporters were chosen because of their patterns of gene expression and their homology to genes of known function in other plants. Transgenic lines were subjected to mixed chlorides in both tissue culture and glasshouse experiments. The results suggest that there could be a role for these genes in the chloride exclusion process.
During the course of a related project (CSP 1302), two new genes for chloride exclusion and six new genes for sodium exclusion were identified using gene mapping techniques. These genes are now being tested in grapevines using transformation techniques. Transgenic lines were produced with reduced HKT1;1 expression and the characterisation of their phenotype supports an active role in sodium exclusion, as reported in the CSP 1302 Final Report, confirming conclusions reached in our publication (Henderson, Dunlevy et al., 2018, New Phytologist). These results assist in decision making regarding marker development for this locus.
While the development of the rootstock transformation methods was not completely successful, considerable progress has been made that will be continued in future projects.
Overall, the project developed capability in scion and rootstock transformation, further improving our world-class grapevine transformation facility, and trained new people in these ever-improving techniques. It has paved the way for the investigation of new gene technologies such as CRISPR, which is designed just to make changes to a single endogenous gene and may speed up breeding of new rootstocks.