Improving Grain Number Per Rice Panicle Using The Genome Editing Technologies

The number of grains per panicle is one of the main composition factors for rice yield. Increasing the grain number could significantly increase the yield of rice. In this paper, we investigated possibility and efficiency of targeting to edit DST gene, a negative regulation of grain number per-panicle in rice, using two genome editing technologies, TALENs and CRISPR/Cas9 system. We hope to create some rice mutants with increase of the number of grains per panicle, which is benefit for the improvement and further utilization of yield related traits in rice breeding. The main results are as follows:Based on the rules of TALENs technology, the operation protocol of TALENs technology was established in rice, a monocot model plant. We designed two TALENs target sites according to the DST gene sequence and constructed two vector plasmids, DST1-TALENs and DST2-TALENs. By the LBA4404 and EHA105, two Agrobacterium strains, the two plasmids were transformed into 2 japonica rice cultivars (Nipponbare and Xiushui 134) and 1 indica rice cultivar (Minghui 86). The statistics and analysis of the regenerated plants found that transformation rate, differentiation rate and the rate of transgenic plants were different with different cultivars, vectors and strains in the transformants of the two TALENs plasmids. Furthermore, the target sites of DST gene were not mutated in all transgenic plants of DST1-TALENs/EHA105 and DST2-TALENs/LBA4404 in Xiushui 134 and Minghui 86. But the target gene mutations were detected in the positive transgenic plants of DST1-TALENs/LBA4404 in Nipponbare, Xiushui 134 and Minghui 86, where target gene mutation rate was 36.8%(7/19),50% (6/12) and 33.3%(1/3).Preliminary analysis of the mutants showed that the grain number per-panicle can increase by an average of 29.8% in Xiushui 134 TO mutants, and those of heterozygous and homozygous T1 mutants in Minghui 86 increase by 21.5% and 39.7%, respectively. The results indicated that the TALENs technology could edit rice DST gene in suitable condition, while the edited mutants can significantly increase the number of grains per panicle. In addition, response of Minghui 86 T2 mutants with DST mutation was further analysized under abiotic stressed, including drought and salt stress. The mutants showed a well root system and normal development comparing with the control. It suggested that DST gene mutation could improve rice tolerance to drought and salt stress.Through the design and screening of the DST gene target site, a gene editing vector was constructed based on CRISPR/Cas9 system. Using Agrobacterium tumefaciens LBA4404, the vector was transformed into Minghui 86, Taijing No.9 and Nipponbare. Statistics and analysis of regeneration plant showed that the transformation rates were 8.6%,26.3% and 30.6%; the positive plant rates 75%,60.8% and 54.5%; the mutated plant rates 90.5%,77.4% and 61.1%, respectively, in the three cultivars. It is interesting that majority of these mutants were homozygous mutants or double allele mutants (80%-94.7%). The results showed that CRISPR/Cas9 system was not only more convenient for the operation process, but also more efficient in gene functional analysis and genetic improvement of rice.