Body of Abstract: Crop genomes acquired innumerable mutations over the years, creating new variations in their genome. Humans have selected a few mutations and generated combinations through breeding to develop today’s improved crop varieties. Although rice as a crop has undergone extensive breeding efforts, many locally grown rice landraces have many undesirable characteristics that hinder their wide adoption. Since conventional breeding is imprecise and suffers from linkage drag issues, removing undesirable traits through breeding remains difficult in those landraces. Genome editing tools empowered us to domesticate those landraces rapidly. We have selected two short-grain aromatic rice landraces, popular in Odisha and West Bengal of India and used as specialty rice. These landraces suffer from frequent lodging due to rain and wind. The green revolution resulted in semi-dwarf high-yielding rice varieties that are lodging resistant; the semi-dwarf nature was bred through the selective breeding of a naturally available mutant sd1 allele with a deletion. We have mimicked the green revolution allele by creating two Cas9-induced cuts in the SD1 gene and produced sd1 alleles with deletion for developing a semi-dwarf version of the two landraces without hampering other quality parameters. Mutants displayed reduced plant height.
Additionally, through a CRISPR-Cas9-multiplex approach, we have targeted two genes to improve the landraces further. We have disrupted the Cytokinin-oxidase-2 (CKX2) gene to increase cytokinin levels in the panicle and subsequently increase grain number. Bacterial blight pathogen secretes TAL effector, which binds to the elicitor binding elements (EBEs) in the promoter of sugar transporter genes to induce their expression and increase disease susceptibility. We successfully edited the EBE in the promoter of the SWEET11 gene to develop bacterial blight disease resistance. Our study shows that rice landraces can be improved rapidly by stacking different beneficial traits through multiplex genome editing.