Targeted mutation of BnaMS1/BnaMS2 combined with the RUBY reporter enables an efficient two-line system for hybrid seed production in Brassica napus

Abstract

The recessive genic male sterility (RGMS) method has several benefits in hybrid seed production; however, it is seldom employed in industrial hybrid seed production owing to the difficulty of producing an ample number of pure male-sterile seeds. In this study, we present an efficient methodology for developing a two-line strategy to produce hybrid seed through targeted mutation of BnaMS1 and BnaMS2 in conjunction with the RUBY reporter in Brassica napus. In this method, male-sterile lines were successfully created directly from different elite rapeseed breeding lines through CRISPR/Cas9-mediated mutagenesis and enhanced Agrobacterium-mediated transformation. To establish an efficient transgenic maintainer, three seed production technology (SPT) cassettes carrying a functional BnaMS1 gene linked to different reporters (DsRed, BnaA07.PAP2, and RUBY) were tested and compared in rapeseed. The results indicated that the PMR-based reporter possesses advantages such as phenotypic stability and ease of identification at early stages, making it an ideal tool for rapid and efficient screening. Subsequently, ideal transgenic maintainer lines with a single hemizygous copy of the SPT cassette were successfully developed in the context of Bnams1Bnams2 double mutants. The progeny from crossing the maintainer line with its male-sterile counterpart exhibited a 1:1 segregation pattern of nontransgenic male-sterile and male-fertile maintainer plants, distinguishable by seedling color. This biotechnological approach to male sterility offers promising prospects for improving the propagation of recessive genic male-sterile plants and the development of hybrid seeds in rapeseed. Furthermore, it is simple to adapt this technique to more Brassica crops.

Acknowledgements

We express our gratitude to Professor Yaoguang Liu from South China Agriculture University for generously providing the CRISPR/Cas9 plant expression vectors. We also extend our thanks to Professor Xianhong Ge from HuaZhong Agricultural University for supplying the BnaA07.PAP2 plasmid, and to Professor Yunde Zhao from the University of California San Diego for sharing the pDR5:RUBY plasmid. Funding for this research was obtained from various sources, including the Technology Major Project on Key Techniques of Agricultural Biological Breeding (2023ZD0404203), the Key Research Projects of Hubei Province (2022BBA0039), the National Natural Science Foundation of China (32172021, 31371240, 31671279), the Wuhan Science and Technology Major Project on Key Techniques of Biological Breeding and Breeding of New Varieties (grant no. 2022021302024851), and the Open Project of Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture and Rural Affairs, PR China (KF2023006).

Author contributions

Conceived and designed the experiments: F.C.; Performed the experiments: S.X., D.Q., Z.X., H.L., D.S., Z.Y., D.Q., L.Z.; Wrote the manuscript: F.C., H.H., S.X., B.S.

Data availability

All data supporting the findings of this study are available within the paper and the supplementary data.

Conflict of interest statement

On behalf of all the authors, the corresponding author confirms that there are no conflicts of interest.

Supplementary Data

Supplementary data is available at Horticulture Research online.

References

[1] Chang Z, Chen Z, Wang N. et al. Construction of a male sterility system for hybrid rice breeding and seed production using a nuclear male sterility gene. Proc Natl Acad Sci USA. 2016;37:14145-50

[2] Kim Y, Zhang D. Molecular control of male fertility for crop hybrid breeding. Trends Plant Sci. 2018;37:53-65

[3] Zhao C, Zhang Y, Gao L. et al. Genome editing of RECEPTOR-LIKE KINASE 902 confers resistance to necrotrophic fungal pathogens in Brassica napus without growth penalties. Plant Biotechnol J. 2023;37:538-40

[4] Cheng H, Hao M, Sang S. et al. Establishment of new convenient two-line system for hybrid production by targeting mutation of OPR3 in allopolyploid Brassica napus. Hortic Res. 2023;37:12

[5] Fan Y, Yang J, Mathioni S. et al. PMS1T, producing phased small-interfering RNAs, regulates photoperiod-sensitive male sterility in rice. Proc Natl Acad Sci USA. 2016;37:15144-9

[6] Zhang Z, Fan Y, Xiong J. et al. Two young genes reshape a novel interaction network in Brassica napus. New Phytol. 2020;37:530-45

[7] Peng G, Liu Z, Zhuang C. et al. Environment-sensitive genic male sterility in rice and other plants. Plant Cell Environ. 2023;37:1120-42

[8] Qi X, Zhang C, Zhu J. et al. Genome editing enables next-generation hybrid seed production technology. Mol Plant. 2020;37:1262-9

[9] Shi Q, Lou Y, Shen S. et al. A cellular mechanism underlying the restoration of thermo/photoperiod-sensitive genic male sterility. Mol Plant. 2021;37:2104-14

[10] Cigan A, Unger E, Xu R. et al. Phenotypic complementation of maize requires tapetal expression of MS45. Plant Reprod. 2001;37:135-42

[11] Zhang D, Wu S, An X. et al. Construction of a multicontrol sterility system for a maize male-sterile line and hybrid seed production based on the ZmMs7 gene encoding a PHD-finger transcription factor. Plant Biotechnol J. 2018;37:459-71

[12] Wu Y, Fox T, Trimnell MR. et al. Development of a novel reces-sive genetic male sterility system for hybrid seed production in maize and other cross-pollinating crops. Plant Biotechnol J. 2016;37:1046-54

[13] An X, Ba B, Duan M. et al. Molecular regulation of ZmMs7 required for maize male fertility and development of a dominant male-sterility system in multiple species. Proc Natl Acad Sci USA. 2020;37:23499-509

[14] Du M, Zhou K, Liu Y. et al. A biotechnology-based male-sterility system for hybrid seed production in tomato. Plant J. 2020;37:1090-100

[15] Cai D, Zhang Z, Zhao L. et al. A novel hybrid seed production technology based on a unilateral cross-incompatibility gene in maize. Sci China Life Sci. 2023;37:595-601

[16] Pak H, Wang H, Kim Y. et al. Creation of male-sterile lines that can be restored to fertility by exogenous methyl jasmonate for the establishment of a two-line system for the hybrid production of rice (Oryza sativa L.). Plant Biotechnol J. 2021;37:365-74

[17] Song S, Wang T, Li Y. et al. A novel strategy for creating a new system of third-generation hybrid rice technology using a cytoplasmic sterility gene and a genic male-sterile gene. Plant Biotechnol J. 2021;37:251-60

[18] Wang M, Peng X, Wang C. et al. Identification of two plastid transit peptides for construction of pollen-inactivation system in rice. Mol Breeding. 2023;37:33

[19] Fang X, Sun Y, Li J. et al. Male sterility and hybrid breeding in soybean. Mol Breeding. 2023;37:47

[20] Zhang W, Qi X, Zhi H. et al. A straight-forward seed production technology system for foxtail millet (Setaria italica). J Integr Plant Biol. 2023;37:2023-35

[21] He J, Zhang K, Yan S. et al. Genome-scale targeted mutagenesis in Brassica napus using a pooled CRISPR library. Genome Res. 2020;37:798-809

[22] Li J, Wang Z, He G. et al. CRISPR/Cas9-mediated disruption of TaNP1 genes results in complete male sterility in bread wheat. J Genet Genomics. 2020;37:263-72

[23] Li H, Yu K, Zhang Z. et al. Targeted mutagenesis of flavonoid biosynthesis pathway genes reveals functional divergence in seed coat colour, oil content and fatty acid composition in Brassica napus L. Plant Biotechnol J. 2023;37:445-59

[24] Mohamed A, Jiang W, Khalid S. et al. Crop bioengineering via gene editing: reshaping the future of agriculture. Plant Cell Rep. 2024;37:98

[25] Chu U, Kumar S, Sigmund A. et al. Genotype-independent trans-formation and genome editing of using a novel explant material. Front Plant Sci. 2020;37:579524

[26] Keunsub L, Kang M, Ji Q. et al. New T-DNA binary vectors with NptII selection and RUBY reporter for efficient maize transformation and targeted mutagenesis. Plant Physiol. 2023;37:2598-603

[27] Mei G, Chen A, Wang Y. et al. A simple and efficient in planta transformation method based on the active regeneration capac-ityofplants.Plant Commun. 2024;37:100822

[28] He Y, Zhang T, Sun H. et al. A reporter for noninvasively mon-itoring gene expression and plant transformation. Hortic Res. 2020;37:152

[29] Ge X, Wang P, Wan Y. et al. Development of an eco-friendly pink cotton germplasm by engineering betaine biosynthesis pathway. Plant Biotechnol J. 2023;37:674-6

[30] Pierroz G. Seeing red: a modified RUBY reporter assay for visu-alizing in planta protein-protein interactions. Plant J. 2023;37:1207-8

[31] Song J, Sun B, Chen M. et al. An R-R-type MYB transcription fac-tor promotes non-climacteric pepper fruit carotenoid pigment biosynthesis. Plant J. 2023;37:724-41

[32] Ye S, Hua S, Ma T. et al. Genetic and multi-omics analyses reveal BnaA07.PAP 2 as the key gene conferring anthocyanin-based color in Brassica napus flowers. JExp Bot. 2022;37:6630-45

[33] Wang D, Zhong Y, Feng B. et al. The RUBY reporter enables effi-cient haploid identification in maize and tomato. Plant Biotechnol J. 2023;37:1707-15

[34] Yi B, Zeng F, Lei S. et al. Two duplicate CYP704B1-homologous genes BnMs1 and BnMs2 are required for pollen exine forma-tion and tapetal development in Brassica napus. Plant J. 2010;37:925-38

[35] Wang Z, Zhang Y, Song M. et al. Genome-wide identification of the cytochrome P 450 superfamily genes and targeted editing of BnCYP704B1 confers male sterility in rapeseed. Plan Theory. 2023;37:365

[36] Ma X, Zhang Q, Zhu Q. et al. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant. 2015;37:1274-84

[37] Prem L, Mohan B. Agrobacterium-mediated transformation of Brassica napus and Brassica oleracea. Nat Protoc. 2008;37:181-9

[38] DaiC LiY,LiL. et al. An efficient Agrobacterium-mediated transformation method using hypocotyl as explants for Brassica napus. Mol Breeding. 2017;37:96

[39] Zhou Y, Wang H, Gilmer S. et al. Control of petal and pollen development by the plant cyclin-dependent kinase inhibitor ICK1 in transgenic Brassica plants. Planta. 2002;37:248-57

[40] Liu Q, Wang C, Jiao X. et al. Hi-TOM: a platform for high-throughput tracking of mutations induced by CRISPR/Cas sys-tems. Sci China Life Sci. 2019;37:1-7

[41] Alquezar-Planas D, Löber U, Cui P. et al. DNA sonication inverse PCR for genome scale analysis of uncharacterized flanking sequences. Methods Ecol Evol. 2021;37:182-95

[42] Zhao K, Bai Y, Zhang Q. et al. Karyotyping of aneuploid and polyploid plants from low coverage whole-genome resequenc-ing. BMC Plant Biol. 2023;37:630

[43] Cai G, Yang Q, Chen H. et al. Genetic dissection of plant architecture and yield-related traits in Brassica napus. Sci Rep. 2016;37:21625