Grapevine genetic improvement through conventional breeding is difficult due to the extreme heterozygosity of the Vitis genome. As a result, new cultivars produced by crossing two elite parent cultivars are of intermediate fruit and wine quality. Genome editing involves the modification of genetic sequences found solely in the Vitis genome and accelerates the outcomes of conventional breeding for trait improvement. In the current study, a grapevine phytoene desaturase 1 (PDS 1) gene was utilized as a target sequence to optimize CRISPR/Cas-9-mediated genome editing.
Embryogenic cultures from Vitis vinifera ‘Thompson Seedless’ were initiated from micropropagation cultures. Following co-cultivation with Agrobacterium harboring a CRISPR/Cas9 construct that targeted the PDS1 gene, and subsequent culture, edited plant lines were generated and identified based on their bleached appearance, and malformed leaves and stems. PCR and DNA sequencing was carried out to calculate the editing efficiency, which was expressed as the total number of lines generated versus the number exhibiting mutations at the PDS1 gene. Among the various lines recovered and tested, greater than 80% of edited lines exhibited large scale deletions and insertions at the PDS1 alleles. This included random nucleotide substitutions and deletions, which could be positively correlated with the plant phenotype. The observations indicate that CRISPR/Cas9-mediated editing could precisely generate biallelic mutants and thus be used to target traits of commercial importance for rapid genetic improvement. We are currently using genome editing to target genes involved in disease resistance and quality improvement such as novel berry colors and decreased browning. Genome editing can be successfully utilized for rapid improvement of commercial grapevine cultivars while bypassing limitations imposed by conventional breeding techniques.