Identification of High Linoleic Acid Varieties in Tetraploid Perilla Through Gamma-ray Irradiation and CRISPR/Cas9.

ABSTRACT

Perilla (Perilla frutescens (L.) var frutescens) is a traditional oil crop in Asia, recognized for its seeds abundant in α-linolenic acid (183), a key omega-3 fatty acid known for its health benefits. Despite the known nutritional value, the reason behind the higher 183 content in tetraploid perilla seeds remained unexplored. Gamma irradiation yielded mutants with altered seed fatty acid composition. Among the mutants, DY-46-5 showed a 27% increase in 182 due to the 4 bp deletion of PfrFAD3b and NC-65-12 displayed a 16% increase in 182 due to the loss of function of PfrFAD3a through a large deletion. Simultaneous knockout of two copies of FATTY ACID DESATURASE 3 (PfrFAD3a and PfrFAD3b) using CRISPR/Cas9 resulted in an increase in 182 by up to 75% and a decrease in 183 to as low as 0.3% in seeds, emphasizing the pivotal roles of both genes in 183 synthesis in tetraploid perilla. Furthermore, diploid Perilla citriodora, the progenitor of cultivated tetraploid perilla, harbors only PfrFAD3b, with fatty acid analysis revealing lower 183 levels than tetraploid perilla. In conclusion, the enhanced 183 content in cultivated tetraploid perilla seeds can be attributed to the acquisition of two FAD3 copies through hybridization with wild-type diploid perilla.