Highly Efficient CRISPR/Cas9 Mediated Gene Editing in Ocimum basilicum 'FT Italiko' to Induce Resistance to Peronospora belbahrii .

Ocimum basilicum (sweet basil) is an economically important aromatic herb; in Italy, approximately 1000 ha of "Genovese-type" basil are grown annually in greenhouses and open fields and are subjected to Downy Mildew (DM) disease, caused by Peronospora belbahrii , leading to huge crop losses. Mutation of the Susceptibility (S) gene DMR6 (Downy Mildew Resistant 6) has been proven to confer a broad-spectrum resistance to DM. In this work, an effective Genome Editing (GE) approach mediated by CRISPR/Cas9 in O. basilicum 'Italiko', the élite cultivar used to produce "Pesto Genovese D.O.P", was developed. A highly efficient genetic transformation method mediated by A. tumefaciens has been optimized from cotyledonary nodes, obtaining 82.2% of regenerated shoots, 84.6% of which resulted in Cas9+ plants. Eleven T0 lines presented different type of mutations in ObDMR6; 60% were indel frameshift mutations with knock-out of ObDMR6 of 'FT Italiko'. Analysis of six T1 transgene-free seedlings revealed that the mutations of T0 plants were inherited and segregated. Based on infection trials conducted on T0 plants, clone 22B showed a very low percentage of disease incidence after 14 days post infection. The aromatic profile of all in vitro edited plants was also reported; all of them showed oxygenated monoterpenes as the major fraction.