2019 ASHS Annual Conference

Vitamin D3 is important for maintaining bone health and preventing osteoporosis, with possible connections to diabetes, cardiovascular disease, and colon, breast, and prostate cancers. It was recently found that 39.92% of the population is Vitamin D deficient, particularly as age increases. Unlike most major vitamins Vitamin D3 is found in very few plants. However, a precursor of Vitamin D3, 7-dehydrocholesterol (7-Dhc), occurs in tomato (Solanum lycopersicum L.). Under normal conditions 7-Dhc is converted to cholesterol by the 7-dehydrocholesterol reductase-2 (Solyc06g074090.2). We hypothesize that if this gene is inactive, it will cause an increase in 7-Dhc, which will be converted by UV-B to previtamin D3, an isomer of vitamin D3.

The endogenous allele of 7-DR-2 was cloned and sequenced for the purposes of designing the guide RNA. Of the 8160 bp in the cloned fragment, all 5695 sequenced bases match the reference genome. A CRISPR-Cas9 plasmid has been developing, targeting six sites in six different exons. These were selected using CrisprP 2.0. The components of the plasmid include pMOD_A0103 which carries AtCas9 driven by the Cestrum yellow leaf curling virus (CmYLCV) promoter, pMOD_B2103 which carries the sgRNA with Csy4 spacers, and the pMOD_C000 which carries no extra components but is required for the assembly. These were assembled into the transformation backbone pTRANS_201.

The CRISPR construct will be transformed into the new variety MTX-851 that has been developed at the University of Minnesota for short season and dwarf growth habit with good flavor. We have determined that this variety can be transformed and regenerated from tissue callus, presenting an opportunity to use this variety for gene editing and crop improvement.