Study On The Creation Of Rice Thermo-Sensitive Sterile Lines By CRISPR/Cas9 Gene Editing Technology

Two-line hybrid rice plays an important role in rice production in China,and it has played a major role in ensuring food security.The key to the breeding of hybrid rice is the sterile line with excellent breeding traits.tms5 is the most widely used temperature sensitive sterility gene in China.In this study,the TMS5 was edited by CRISPR/Cas9 technology.The main results are as follows:?1?The expression vector of pCAMBIA1301-Cas9-TMS5-gRNA was constructed.The recombinant construct was then introduced into F197?Oryza sativa L.japonica?via Agrobacterium-mediated transformation method.Fifty-seven tissue culture seedlings were obtained,of which 36 were positive transgenic plants.?2?Analysis the mutation types of TMS5 in T₀ positive transgenic plants.The results showed that 23 plants were detected TMS5 mutation in 36 positive transgenic plants.The rate of TMS5 mutation in transgenic plants was 63.89%.The types of TMS5 mutation included heterozygous mutation and homozygous mutation.There are 15 plants were heterozygous mutations and 8 plants were homozygous mutations.The rate of homozygous mutation was 34.78%.The homozygous mutations were mainly the insertion of single bases A,T,and G between the 3rd and 4th bases before PAM,which accounted for 62.5%,25%,and 12.5%,respectively.However,the single base C insertion was not detected.?3?The fertility transition temperatures and agronomic traits of T₁ homozygous deletion mutants were investigated.The results showed that the fertility transition of tms5 mutants may be 28°C.Both seed setting rates and grain yields in TMS5 mutants were significantly decreased compared with wild-type plants in paddy field condition.It is noteworthy that there are no significant difference of plant height,1000-grain weight and panicle number between tms5 mutant and wild-type plants.