Development of Novel Rice Germplasm for Salt-Tolerance at Seedling Stage Using CRISPR-Cas9

Round 1

Reviewer 1 Report

Salinity is one of the most important abiotic stresses affecting the world rice production. The cultivation of salinity-tolerant cultivars is the most cost-effective and environmentally friendly approach for salinity control. In recent years, the CRISPR/Cas9 system has been widely used for target-site genome editing; however, its application for the improvement of elite rice cultivars has rarely been reported. In this study, an elite three-line indica restorer in breeding, R192, was taken as the receptor, and the CRISPR/Cas9 technology was used to perform the directional editing of OsRR22, the main effect gene controlling salt tolerance in rice. With the recent introduction of China's agricultural gene editing plant management policy, this study has shown good theoretical value and potential application value of breeding salt-tolerant hybrid rice varieties for application and promotion. However, before being accepted in the journal, the manuscript should be revised at a minor point. In Figure 4, the comparison analyses among wild type and mutants should be performed and the significant differences or not should be shown in the figure.

Author Response

Salinity is one of the most important abiotic stresses affecting the world rice production. The cultivation of salinity-tolerant cultivars is the most cost-effective and environmentally friendly approach for salinity control. In recent years, the CRISPR/Cas9 system has been widely used for target-site genome editing; however, its application for the improvement of elite rice cultivars has rarely been reported. In this study, an elite three-line indica restorer in breeding, R192, was taken as the receptor, and the CRISPR/Cas9 technology was used to perform the directional editing of OsRR22, the main effect gene controlling salt tolerance in rice. With the recent introduction of China's agricultural gene editing plant management policy, this study has shown good theoretical value and potential application value of breeding salt-tolerant hybrid rice varieties for application and promotion. However, before being accepted in the journal, the manuscript should be revised at a minor point. In Figure 4, the comparison analyses among wild type and mutants should be performed and the significant differences or not should be shown in the figure.

Response: According to the Reviewer' advice, we have added the signs of significant difference in Figure 4.

Author Response File: Author Response.docx

Reviewer 2 Report

Currently, among the cloned salt-tolerant  genes in rice, OsRR22 is one of the most important negative regulatory genes for salt tolerance in rice.  The authors knocked out the OsRR22 gene in rice by CRISPR /cas9 technology and obtained the knockout mutants. Through the experiment of NaCl treatment at seedling stage, it was found that knocking out OsRR22  significantly increased the salt tolerance of rice. OsRR22  is a known salt tolerant gene, and I don't think this work is worth publishing.
1. The authors speculate that OsRR22 gene may be related to photosynthesis and water retention capacity under salt stress, but there is no experimental evidence to support this conclusion.
2. Table 2, only a small number of transgenic seedlings, this statistic is meaningless.
3. Figure 4, no significant difference analysis.
4. The language needs to be modified. The Abstract is not brief enough.

Author Response

1. The authors speculate that OsRR22 gene may be related to photosynthesis and water retention capacity under salt stress, but there is no experimental evidence to support this conclusion.

Response: In this study, we would like to development of salt-tolerant rice lines by CRISPR/Cas9 technology to show the theoretical and potential application value of breeding salt-tolerant rice varieties. After comparison of plant height, the number of green leaves, total fresh weight, and total dry weight among WT, M16, and M18 after salt treatment at seedling stage, we speculate that OsRR22 gene may be related to photosynthesis and water retention capacity under salt stress. Because the physiological function of the OsRR22 gene was studied after obtaining the hst1 mutant of the OsRR22 gene (Niwagaba 2018), which is consistent with our speculation, we didn't do more experiments. According to the Reviewer' advice, the speculative content in the abstract was deleted for without experimental evidence .

2. Table 2, only a small number of transgenic seedlings, this statistic is meaningless.

Response: Table 2 shows the general situation of gene-edited seedlings in order to show them more clearly. Because this study is not for the study of gene-editing methods, there are not many seedlings, and we did not interpret the statistical results in the results. According to the Reviewer' opinion, we agree to delete the proportion numbers in Table 2 or delete Table 2.

3. Figure 4, no significant difference analysis.

Response: According to the Reviewer' advice, we have added the signs of significant difference in Figure 4.

4. The language needs to be modified. The Abstract is not brief enough.

Response: According to the Reviewer' advice, we revised the abstract and . part of our manuscript.

Author Response File: Author Response.docx

Reviewer 3 Report

this is the dose of salt, or the application of the salt element twice

Comments for author File: Comments.pdf

Author Response

this is the dose of salt, or the application of the salt element twice

Response: According to the Reviewer' advice, we revise the statement of salt tolerance treatment.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The author answered my question. Although I still feel that this manuscript is too simple, it is now a complete story with no obvious mistakes.