Generation Of A Thermo-sensitive Genic Male Sterile Line By Using CRISPR/Cas9 Gene Editing System

Two-line hybrid rice breeding is an efficient breeding method,and the key lies in the acquisition of temperature-sensitive male sterile lines.In order to quickly obtain excellent temperature-sensitive male sterile lines in rice,two targets on tms5 genes of four excellent rice lines AB9808Q,BAS P12,AB9738LG and AB9300 were edited using the CRISPR/Cas9 gene editing system,and they were obtained separately.The corresponding tms5 edited TGMS series are GXU41,GXU47,GXU40 and GXU36.The editing plants were tested for target mutations,the editing TGMS lines without foreign DNA were screened,and fertility conversion,agronomic traits and rice quality were analyzed and evaluated.The main findings are as follows:（1）Tms5 genome editing.The obtained T₀generation regenerated plants were subjected to target PCR amplification and sequencing analysis,and the mutations of all transformed plants were all analyzed.The results showed that:the transformation efficiency of the GXU41 line reached 75%,and the double homozygous mutation rate reached 25%.The transformation efficiency of the GXU47 line reached 75%,and the double homozygous mutation rate reached13.3%.The transformation efficiency of the GXU40 line reached 78.6%,and the double homozygous mutation rate reached 18.2%.The transformation efficiency of GXU36 reached 83.3%,and the double homozygous mutation rate reached15%.A certain number of T-DNA-free homozygous TGMS plants was screened in each T₁generation double homozygous mutant line.（2）The fertility conversion characteristics of TGMS line were edited by tms5.The anther morphologies of T₁double homozygous mutant plants treated at 21℃,22℃,23℃and 24℃were observed under a stereo microscope with super depth of field,and 1%I₂-KI solution was used to identify pollen fertility,then spikelet fertility were calculated at maturity.The results showed that the pollen fertility of T₁generation tms5 editing TGMS lines double homozygous GXU41 at 21℃,22℃,23℃and 24℃was 63.5%,43.2%,12.8%,0%,respectively,and the spikelet fertility was 41.6%,24.5%,5.5%,0%,respectively;the pollen fertility of GXU47 was 74.3%,45.9%,14.4%,0%,respectively,and the spikelet fertility was 52.7%,26.8.%,7.3%,0%,respectively;the pollen fertility of GXU40 was 51.9%,42.5%,6.3%,0%,respectively,and the spikelet fertility was 32.8%,21.4%,2.3%,0%,respectively;the pollen fertility of GXU36 was 46.3%,27.1%,2.5%,0%,respectively,and the spikelet fertility was23.7%,12.6%,0%,0%,respectively.（3）The agronomic traits of Tms5 edited TGMS line.At the maturity stage of T₁generation,3 plants were randomly selected to examine the main agronomic traits under each temperature treatment.The results showed that all tms5 edited TGMS lines and wild type（WT）had no difference in effective ear length,flag leaf width,flag leaf length and thousand-grain weight;while all tms5 edited TGMS lines had significant differences in average plant height（p<0.05）compared with WT.There is a very significant difference in the average number of effective panicles（p<0.01）between them.（4）The quality traits of Tms5 edited TGMS line,GXU41-5S.The seeds of GXU41-5S under the condition of 21℃were harvested and sent to the Rice and Product Quality Supervision,Inspection and Testing Center of the Ministry of Agriculture and Rural Affairs for rice quality analysis.The results show that various rice quality indicators meet the requirements of the first-class standard for high-quality edible rice,and GXU41-5S is a high-quality TGMS system.The above research results show that the use of the CRISPR/Cas9 editing system to edit the temperature-sensitive male sterility gene tms5 can efficiently obtain rice TGMS lines without foreign DNA.The critical temperature for fertility conversion of these lines is 24℃,which meets the breeding requirements of temperature-sensitive male sterility.In particular,GXU41-5S has excellent quality and has important application value in the breeding of two-line hybrid rice varieties.