Application Of CRISPR/Cas9 Mediated Editing Of TMS5 And LTPL164 In Rice

Two-line hybrid rice breeding with high yield potential is increasingly popular which requires a male sterile parent for producing F₁ seed.Here,thermo-sensitive genic male sterility?TGMS?was induced by applying CRISPR/Cas9 gene editing technology to knockout the gene TMS5.Two TGMS mutants?tms5-1 and tms5-2?,both lacking any residual T-DNA were generated in the background of the indica rice cultivar Zhongjiazao17?cv.YK17?designated as YK17S1 and YK17S2,respectively.Besides,storage lipid is a vital component for maintaining structure of seed storage substances,and valuable for rice quality and food texture.The cr-ltp164 line was also induced by applying CRISPR/Cas9 genome editing technology to modify the putative lipid transfer protein OsLTPL164 in the background of japonica rice cultivar Zhonghua11?cv.ZH11?.The main results obtained from present studies are listed below:1.Both mutants?tms5-1 and tms5-2?produced fully sterile pollens when the plants were grown during the normal season,while their vegetative growth and floral morphology were normal as like cv.YK17.Manual pollination of both mutants generated fully viable grains,showing that the megagametophyte was unaffected by the loss-of-function of TMS5.2.When grown at a sub-optimal temperature?22°C?,both mutants produced viable pollen and successfully produced grain through self-fertilization,but at temperatures 24and 26°C,their pollen was sterile and no grain was set.The pollen sterility behavior as like as a typical male sterile line.3.The new TGMS line has shown good compatibility in two-line hybrid rice breeding system.F₁ hybrids made between plants harboring tms5-1 and three different restorer lines outperformed both parental lines with respect to grain yield and related traits.4.Transcript level of OsLTPL164 was observed abundant in developing seed endosperm and mutant line cr-ltp164 displayed significantly lower than wild type?cv.ZH11?.Histochemical analysis of GUS activity in transgenic plants confirmed that the expression of OsLTPL164 mainly in developing seed endosperm rather than other parts.The cr-ltp164 line displayed less grain size and weight,higher chalky endosperm as compared to cv.ZH11 which suggested that the function of OsLTPL164 is seed specific.5.Lipid and fatty acid content reduced massively in cr-ltp164 line as compared to cv.ZH11.The less accumulation of lipid in cr-ltp164 seed suggested that OsLTPL164 might be functioned as a lipid transporter.Seed germination rate was found lower in cr-ltp164line as compared to cv.ZH11 due to decline of lipid content.