Using CRISPR/Cas9 Technology To Create Indica Rice Thermo-sensitive Genic Male Sterile Lines

Two-line hybrid rice is the development direction of rice breeding.Indica of thermo-sensitive Genic Male-sterile line is the most widely used type of two-line male sterile.Tms5 is the dominant gene for controlling the TGMS of rice.Hybrid japonica rice developed slowly compared to hybrid indica rice.Lack of practical sterile lines is an important reason.Traditional two-line sterile lines were bred by conventional hybridization,Each generation is concerned with the low temperature propagation of intermediate materials,The process of breeding is slow,and has a chain of complications.So the breeding japonica sterile lines of new technology is of great significance.Genome editing is a new and rapidly growing modern molecular technology..We used CRISPR/Cas9 to editing of the TGMS gene tms5 of rice WJG1 and L11B,After a generation of breeding,Binding molecular identification,Thermonuclear sterility lines with stable heredity and no transgenic gene were obtained,It is proved that CRISPR/Cas9 is an new method for breeding japonica hybrid sterile lines.This research mainly carries on the following work:?1?The CRISPR/Cas9 vector of tms5 gene was constructed;?2?By using agrobacter mediated transformation of two japonica rice materials with good flowering habits of WJG1 and L11B,16 strains of the positive transformation plant of WJG1 and 16 strains of L11B were obtained respectively;?3?tms5 point sequencing was performed for positive transgenic plants of T₀,T₀generation plants have 16 strains in the target locus mutation happened,and cold irrigation was used to deal with the complicated species;?4?T₀ generation tms5 mutant strain was selected,and T₁ generation group was planted.With Hyg marker,the single strain of non-hygromycin was screened,and tms5 target site was amplified and sequenced,and T₁ generation homozygous single strain was preliminarily screened.multiplication of seed in october;?5?The pollen microscopy was carried out for T₁ generation homozygous single plant,and there were 12 sterile lines.