*4.6. Leaf Epidermal Cell Observation*

Epidermal cells in flag leaves were observed following the method used by Yoshikawa et al. [55].

#### *4.7. Trait Measurement*

Plant height, leaf size, and tiller number of WT and mutants were measured in the field at the maturity stage. For tiller number determination, only seed setting tillers were counted. Rice plants were harvested when the grains were fully mature. Grains threshed from each plant were dried, and filled grains were weighed to determine grain yield per plant. Fully filled grains were used for determining grain size and weight. Grain weight was measured based on 100 grains and converted to 1000-grain weight.

#### **5. Conclusions**

Collectively, our findings showed that transgene-free rice plants with targeted mutations can be produced in the T1 generation using the *Agrobacterium*-mediated CRISPR/Cas9 system, and that the *OsFWL4* gene plays a role in the regulation of tillering and plant yield in rice. The specific mutants obtained in this study provide valuable materials for functional analysis of rice *FWL* genes.

#### **Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/3/809/s1.

**Author Contributions:** C.Y. and X.Z. designed the study; Q.G., G.L., H.S., M.X., H.W., J.J., and D.W. performed the experiments; Q.G. and G.L. analyzed the data; and Q.G. and X.Z. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Key Research Program of China (2017YFD0100400), the National Natural Science Foundation of China (31601385), the National Science and Technology Major Project (2016ZX08012-002), and the Natural Science Foundation of Jiangsu Province (BK20160429 and BK20180107).

**Acknowledgments:** We thank Yangwen Qian from the Biogle Genome Editing Center (Changzhou) for assistance with gene transformation. We also thank the anonymous reviewers for their constructive comments and suggestions.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


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