Establish Tomato Male Sterile Lines With Seedling Marker Via Gene Editing And Investigate The Traits Of Hybrids

The utilization of tomato（Solanum lycopersicum）heterosis is an essential approach to increase yield and improve overall tomato quality.However,tomato hybrid seeds are mainly produced by artificial emasculation and pollination at present,which is labor-consuming and time-consuming.In addition,the quality of hybrid seeds cannot be guaranteed.The utilization of male-sterile lines can effectively improve seed production efficiency and reduce seed production cost.CRISPR/Cas9（C lustered regularly interspaced short palindromic repeats（CRISPR）/CRISPR-associated protein 9（Cas9））gene editing system can edit genes related to stamen development simply and efficiently,endow elite inbred lines with male sterility without adverse traits linkage,and quickly create tomato nuclear male-sterile lines which can be used for hybrid seeds production.1.In this study,Ms10³⁵(Male terile 10³⁵),GSTAA（Glutathione S-transferase）and Wo（Woolly）were targeted.Three knockout vectors,PCRI-MS10³⁵,PCRI-GSTAA-MS10³⁵ and p EX-6Xsg R-GSTAA12-Ms10³⁵12-Wo12-Cas9,were constructed,respectively.By Arobacterium-mediated genetic transformation,PCRI-MS10³⁵ was introduced into hairless tomato inbred line 6Q71 to obtain a sterile line CR-ms10³⁵/Wo with no-woolly phenotype,and PCRI-GSTAA-MS10³⁵ was introduced into purple stem tomato inbred line 19A.A green stem labeled nuclear sterile line CR-ms10³⁵/gstaa was obtained.p EX-6Xsg R-GSTAA12-Ms10³⁵12-Wo12-Cas9 was introduced into 19A,but no mutation was detected.2.The morphological traits of CR-ms10³⁵/gstaa were investigated in the field.It was found that compared with 19A,the hypocotyl of male sterile line was green at seedling stage.By observing the flower organs of male sterile lines,it was found that the top of stamens was dehiscent and the stigma extended out of the anther tube.Subsequently,the pollen viability of the sterile line was identified by TTC staining,and no viable pollen grains were found.The male sterile line could not self-cross and bear seeds,but could bear seeds normally by artificial pollination.3.The pollen of maintainer line Ms10³⁵ms10³⁵/GSTAAgstaa was used to pollinate two independent CR-ms10³⁵/gstaa male sterile lines,and the hypocotyl color of their progeny plants was counted,and the fertility was identified at the same time.The results showed that the ratio of purple hypocotyl plants to green hypocotyl plants was close to 1:1.4.CR-ms10³⁵/gstaa and WT were used as female parent and inbred line 5N11 as male parent.Compared with WT,there was no significant difference in the whole fruit growth and development cycle of pollinated sterile lines,especially in the number of seeds per pollinated fruit.The results of molecular marker identification showed that the seed purity of male sterile line was better than that of control.5.The maintenance of male sterile line C R-ms10³⁵/gstaa was achieved by crossing the sterile line with WT to obtain Ms10³⁵ms10³⁵/GSTAAgstaa genotypic plants,and then screening the sterile line from the self-crossing isolated population of Ms10³⁵ms10³⁵/GSTAAgstaa.The main agronomic traits of Ms10³⁵ms10³⁵/GSTAAgstaa were investigated,and it was found that Ms10³⁵ms10³⁵/GSTAAgstaa and 19A had no significant difference in other important agronomic characters except leaves.In addition,the sterile lines isolated by self-cross were evaluated for fertility,and it was found that the sterile characteristics could be maintained.6.The tomato male sterile line C R-ms10³⁵/f3h and inbred line 19A were used as female parents to cross with inbred lines 1#and 8#respectively,resulting in total of 4 hybrid combinations were obtained.The agronomic characters of each F1 hybrids were determined.The results showed that there were no significant differences in plant height,stem diameter,leaf length,leaf width,yield traits,fruit quality traits and seed germination traits between CR-ms10³⁵/f3h green stem marker hybrid combinations and artificial emasculation hybrid combinations.The pollen vigor of each F1 hybrids was observed under electron microscope,and it was found that the pollen grains of male sterile line combination were full,the pollen vigor was strong,and its fertility was well restored.The stamen length,stigma length,petal length and calyx length of each combination were measured.It was found that there was no significant difference in flower organ traits between male sterile line combination and 19A combination.