Abstract
Flowering plant sexual reproduction requires double fertilization, yielding embryo and endosperm seed compartments: the latter supports embryo growth and seed germination. In an experiment to generate haploid embryos through inhibition of pollen phospholipase activity in sunflower (Helianthus annus), we serendipitously discovered that emasculated sunflowers spontaneously form parthenogenic haploid seed. Exploration of genetic, chemical and environmental factors demonstrated that a specific genotype background enabled high parthenogenesis and that full spectrum high-intensity light supplementation boosted parthenogenesis, yielding hundreds of haploid seeds per head. Induction of doubled haploid plants can greatly accelerate plant breeding efficiency; however, despite successful engineering of haploid induction in many crops, few reported systems are commercially scalable1. Here we report efficient methods of chemical emasculation and genome doubling to produce fertile plants and enable a scalable sunflower doubled haploid system.
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Acknowledgements
We thank M. Marion, B. Lise, P. Joel and Y. Ma for whole-genome 33-SNP-maker analysis; J. Comadran Trabal and R. Sensolini for genetic distance check; J. Fabish for statistical support; W. Huang from the China Agriculture University and B. Ma for technical support; Y. Gao and F. Zhu for pipeline supports; V. Walbot for manuscript editing and consultation; A. Brosnan and M. Zong for ploidy analysis support; W. Shi and Y. Song from Zeiss for microcomputed tomography analysis; S. Tian and S. Ni for confocal microscopy studies; A. Bharti, A. Farmer and J. Curley for SY58 genome sequencing; E. Cheek for lighting infrastructure support; the Duke University Phytatron team, M. Betts, G. Piotrowski, A. Eddings and T. Smith; X. Zhang, B. Zhang, X. Chen, I. Jepson, C. Baxter, E. Dunder, R. Egger, J.-C. Rousseaux, C. Zuo, F. Kong, J. Zhang and O. Sauvageot for project guidance and support; B. Bao, Y. He, L. Li and M. Lopez-Sendon for sunflower lines; and C. Leming and M. Bublitz for intellectual property guidance. Syngenta Crop Protection provided internal funding.
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Contributions
J.L., D.L. and T.K. conceptualized the study. E.B. developed the emasculation method. V.M.T., D.S., P.W., X.C. and J.C. conducted the phenotyping trials in field locations. H.J., H.D. and E.B. undertook greenhouse plant care and phenotyping. B.C., P.W. and Z.L. conceptualized and performed the light treatments. Y.D., Changbao Li, Chao Li, R.C. and P.D. performed the tissue culture and genome doubling. C.P., A.T. and P.D. performed the marker analysis and fingerprinting. X.T. and J.L. performed the microscopy. B.S. and W.C. performed the seed lipid profile check. F.B. and V.M.T. provided guidance and methodological considerations. J.L. and T.K. wrote the original draft of the manuscript, and reviewed and edited the manuscript.
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J.L., D.L., T.K., E.B., Changbao Li, Y.D., F.B., V.M.T., C.P. and P.D. have filed the patent application PCT/CN2024/090146, which covers the haploid parthenogenesis phenomenon, information on germplasm testing, doubling methods, in-medium seed culture processes to enhance germination, and environmental response of haploid seed setting. Assignees of PCT/CN2024/090146 are Syngenta Group Co., Ltd and Syngenta Crop Protection AG. The status of the application is not yet published. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Biology and seed phenotypes of corn and sunflower haploids.
a, Typical corn colorless haploid and diploid embryos (center) dissected from haploid-induced ears (left) expressing purple anthocyanin pigment from transmission of the R1-nj marker in sperm. Haploid embryos are selected, genome-doubled, and cultured into DH plantlets (right). b, Disc floret from SY54 and SY54_CMS, after removing corolla. Anthers from SY54_CMS are shorter and not able to produce pollen, due to the CMS. c, Developing parthenogenic embryo in a SY54_CMS ovary ~20 days post-anthesis. d, An ovary from the same head lacking a parthenogenic embryo. Evidence of autonomous endosperm is not found in either image. e, SY54 (diploid) self-pollinated embryo with cellularized endosperm ~15 days post-anthesis. Results in c to e are representative of four to six replicate images of each. f, Normal diploid embryos from SY58 at 22 days post-anthesis. g, Normal diploid embryo from SY58 excised from the seed coat at 16 d post-anthesis. Two cotyledons are marked (^). h and i, Parthenogenic haploid embryos from SY58_CMS at 22 days post-anthesis. j and k, Transverse micro-computed tomography (microCT) sections of mature parthenogenic haploid (j) and normally fertilized diploid (k) seed from a SY54_CMS head. Subsets, green horizontal line identified the position of the section in the seed. l, Germplasm test in Toulouse field in 2021. SY54-CMS heads bagged as they start to flower. m, Close up image of the flowering stage with the outermost rings at pollination stage, surrounding the inner rings of immature flowers. n, Bagging and crossing plants in the field. PI, Propidium iodide stain; UV auto, autofluorescence visualized using UV light excitation (405 nm).
Extended Data Fig. 2 Parent emasculation and treatment to induce parthenogenic haploids.
a, R1 stage for GA3 treatment. b, R1 stage bud just following GA3 application. c, GA3-sterilized SY58 head showing absence of anther exertion and pollen shed. d, Parthenogenesis stimulation test (with maize pollen and other AIs treatment) with SY54_CMS sunflower head adding ZM01 maize pollen (e) painted onto the SY54 stigmas (f). g, Hormone was injected into the spongy layer of sunflower head (green arrow). h, Germination of SY58_CMS derived haploids in soil. Two hundred seeds yielded 80 seedings, of which roughly half were normal looking. i, and half were abnormal (j, k).
Extended Data Fig. 3 Parthenogenic haploid germination and doubling.
a-c, Impact of semi-solid formulation on tissue penetrance, effective treatment duration, and plant vigor. a, Liquid control formulation with DMSO and fluorescein showing some leaf penetration at 4 h but no penetration into the meristem (red circle). b, Semi-solid formulation with DMSO and fluorescein, showing deep tissue penetration at 5 h, and full saturation of the meristem region at 48 h (red circle). c, Liquid formulation with colchicine. d, Semi-solid formulation with colchicine, showing reduction of leaf growth compared to (c). This indicates that the colchicine is having a greater impact (limiting) growth in the semi-solid treatment. e and f, Growth of colchicine-induced doubled haploid plants (e) and leaves (f) derived from germinated parthenogenic haploid seeds. g, Spontaneous doubled haploids among parthenogenic (PRG) haploid progenies. Out of thirty-nine viable plants derived from the planting of 200 haploid seed, four appeared larger and scored as diploids in the ploidy analysis (flow cytometry) data. h, Sunflower parthenogenic doubled haploid (DH) process. Left, plants are emasculated manually or with a GA3 treatment. Center, seeds are separated using a blower and germinated in soil (bottom) or in tissue culture (top); a doubling treatment is provided to produce DH plants (right).
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Lv, J., Liang, D., Bumann, E. et al. Haploid facultative parthenogenesis in sunflower sexual reproduction. Nature 641, 732–739 (2025). https://doi.org/10.1038/s41586-025-08798-2
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DOI: https://doi.org/10.1038/s41586-025-08798-2
