J Cell Physiol. 2020;1–12. wileyonlinelibrary.com/journal/jcp © 2020 Wiley Periodicals LLC | 1 Received: 29 May 2020 | Accepted: 21 July 2020 DOI: 10.1002/jcp.29986 ORIGINAL RESEARCH ARTICLE Targeted mutagenesis of EOD3 gene in Brassica napus L. regulates seed production Muhammad H. U. Khan | Limin Hu | Miaoshan Zhu | Yungu Zhai | Shahid U. Khan | Sunny Ahmar | Olalekan Amoo | Kunpeng Zhang | Chuchuan Fan | Yongming Zhou National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China Correspondence Chuchuan Fan, National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, 430070 Wuhan, China. Email: fanchuchuan@mail.hzau.edu.cn Funding information National Natural Science Foundation of China, Grant/Award Numbers: 31371240, 31671279, 31971976 Abstract Seed size and number are central to the evolutionary fitness of plants and are also crucial for seed production of crops. However, the molecular mechanisms of seed production control are poorly understood in Brassica crops. Here, we report the gene cloning, expression analysis, and functional characterization of the EOD3/CYP78A6 gene in rapeseed. BnaEOD3 has four copies located in two subgenomes, which exhibited a steady higher expression during seed development with differential expression among copies. The targeted mutations of BnaEOD3 gene were efficiently generated by stable transformation of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat) vector. These mutations were stably transmitted to T 1 and T 2 generations and a large collection of homozygous mutants with combined loss‐of‐function alleles across four BnaEOD3 copies were created for phenotyping. All mutant T 1 lines had shorter siliques, smaller seeds, and an increased number of seeds per silique, in which the quadrable mutants showed the most significant changes in these traits. Consequently, the seed weight per plant in the quadrable mutants increased by 13.9% on average compared with that of wild type, indicating that these BnaEOD3 copies have redundant functions in seed development in rapeseed. The phenotypes of the different allelic combinations of BnaEOD3 copies also revealed gene functional differentiation among the two subgenomes. Cytological observations indicated that the BnaEOD3 could act maternally to promote cotyledon cell expansion and proliferation to regulate seed growth in rapeseed. Collectively, our findings reveal the quantitative involvement of the different BnaEOD3 copies function in seed development, but also provided valuable resources for rapeseed breeding programs. KEYWORDS BnaEOD3, Brassica napus, CRISPR/Cas9, seed number, seed size 1 | INTRODUCTION Rapeseed (Brassica napus L., AACC, 2n = 38), is an important crop grown all over the world, providing approximately 16% of the entire global edible vegetable oils for human diets as well as high‐quality animal feed proteins and raw materials for industrial processes (Hu et al., 2018; S. U. Khan et al., 2019). It resulted from a recent allopolyploidy between ancestors of Brassica rapa (2n = 20, AA) and Brassica oleracea (2n = 18, CC). Seed size and seed number are crucial factors for the seed production of crop plants and are also central to the evolutionary fitness of various plants (Orsi & Tanksley, 2009). A previous study