Construction and Introduction of CRISPR/Cas9-gRNA-Vinv for Editing A Gene Controlling Cold-Induced Sweetening [CIS] Character on Potato

Abstract

<p>Cold-induced sweetening (CIS) causes the processed potato to have dark (browning) texture and induces high acrylamid formation. This phenomenon involves the activity of the Vacuolar invertase (Vinv) gene in accumulating sugar reducer in tubers under cold conditions. Inhibition of Vinv gene activity plays an important role in developing potato lines tolerance to CIS. The objectives of this study were to (1) construct a CRISPR/Cas9 cassette module carrying the gRNA from the Vinv gene, (2) obtain transgenic Atlantic potato cultivars containing CRISPR/Cas9-gRNA-Vinv cassette through genetic transformation by Agrobacterium tumefaciens, and (3) analyze the transgenic putative potato plant molecularly. The recombinant plasmid containing CRISPR/Cas9-gRNA-Vinv genes was successfully constructed and introduced into the potato genome by A. tumefaciens. Genetic transformation of Atlantic potato with CRISPR/Cas9-gRNA-Vinv cassette was more efficient using leaf explants compared to that using stem segment explants. Transformation of leaf explants resulted in transformation and regeneration efficiency values of 8.8% and 53.6%, respectively, compared to 1.3% and 0% transformation and regeneration efficiencies using stem segment explants. From 12 transformant lines obtained, three lines were found to contain the Cas9 gene. Sequence analysis of the Vinv gene derived from T0 transgenic plant, however, found no mutations occurred in the Vinv gene sequence. Further research is needed to obtain the Vinv gene mutants.</p>