Developing a Method for Making Transgene-Free Gene-Edited Grapevines
Satyanarayana Gouthu and Laurent Deluc*
*Oregon State University, Department of Horticulture, 4017 ALS
Building, Corvallis, OR, 97330 (delucl@oregonstate.edu)
Techniques to obtain transgene-free plants from genome-edited material, either through direct DNA-free genome editing or by eliminating transgenes following conventional transformation methods, are not established in many clonally propagated perennial crop models, including grapevine. We developed a research project in two phases to generate transgene-free, gene-edited grapevine material. In Phase 1, the objective was to create stable transformants in grapevine via Agrobacterium-mediated transformation using an “excisable” genetic cassette containing the gene-editing ingredients targeting gene(s) of interest along with other “foreign” DNA required to select transgenic plants. Phase 2 will deliver a RiboNucleoProtein editing complex (RNP) to the edited plant material from Phase 1 to “excise” the inserted genetic cassette, making the edited plant material GMO-free. To facilitate the entry of the RNP, we are testing a series of nanocarriers called Cell-Penetrating Peptide (CPP), widely used in medical sciences for drug, DNA, and protein delivery to human cells. To the best of our knowledge, no studies report the use of CPP’s for gene editing in plant sciences. As a proof of concept, we have generated a series of edited mutants altering expression of four MLO susceptibility genes whose altered activities are known to confer resistance to mildew in a variety of crops. We will discuss the current progress of the project and the milestones achieved, which include but are not limited to generation of MLO-edited grapevine materials and a set of preliminary experiments showing the ability of CPP to facilitate entry of the RNP into grapevine regenerable tissue (somatic embryos). This will be the first report to demonstrate RNP delivery into intact grapevine cells, if successful. It will offer an alternative to conventional technology (protoplast and viral-mediated transient expression) to generate transgene-free edited grapevines.
Funding Support: AVF, OWB, CDFA, Erath Family Foundation
