Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Maternal doubled haploid production in interploidy hybridization between Brassica napus and Brassica allooctaploids


Main conclusion

We found a new in vivo route to produce maternal doubled haploid of Brassica napus . The pollen donor, an allooctaploid rapeseed, acts as a DH inducer.

Inbred line has a powerful advantage in cultivar breeding and genetic analysis. Compared to the traditional breeding methods, doubled haploid production can save years off the breeding process. Though genotype-dependent tissue culture methods are widely used in the Brassica crops, seed-based in vivo doubled haploid developing systems are rare in nature and in the laboratory. As interspecific cross and interploid hybridization play an important role in genome evolution and plant speciation, we created a new Brassica artificial hybrid, a Brassica allooctaploid (AAAACCCC, 2n = 8× = 76), by interspecific crossing and genome doubling. A homozygous line was observed at the third self-generation of a synthesized Brassica allohexaploid (AAAACC, 2n = 6× = 58). Crosses between B. napus as female and Brassica allooctaploid as pollen donor were conducted, which yielded maternal doubled haploid B. napus that were identified based on phenotype, ploidy, and molecular analysis. The Brassica octaploid acted as a maternal doubled haploid inducer and had a relatively high induction rate. Our research provides a new insight for generation of homozygous lines in vivo using a single-step approach, as well as promotes the understanding in breeding programs and genetic studies involving the Brassicas.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7



Doubled haploid


Haploid inducer


Pollen mother cells


Zhongshuang 11


  1. Adams KL, Wendel JF (2005) Polyploidy and genome evolution in plants. Curr Opin Plant Biol 8:135–141

  2. Boulos C, France D, Liu S et al (2014) Early allopolyploid evolution in the post-Neolithic Brassica napus oilseed genome. Science 345:950–953

  3. Chen S, Nelson MN, Chèvre A-M et al (2011) Trigenomic bridges for Brassica improvement. Crit Rev Plant Sci 30:524–547

  4. Coe EH (1959) A line of maize with high haploid frequency. Am Nat 93:381–382

  5. Comai L (2000) Genetic and epigenetic interactions in allopolyploid plants. Plant Mol Biol 43:387–399

  6. Comai L (2005) The advantages and disadvantages of being polyploid. Nat Rev Genet 6:836–846

  7. Diao W (2009) Creation and genetic analysis of cucumber (Cucumis sativus L.) materials with different ploidies. Dissertation, Nanjing Agricultural University

  8. Dorota G, Twan R, Alok V et al (2005) Uniparental chromosome elimination at mitosis and interphase in wheat and pearl millet crosses involves micronucleus formation, progressive heterochromatinization, and DNA fragmentation. Plant Cell 17:2431–2438

  9. Frédérique E, Letanneur J, Anne-Marie C (1997) Chromosome number of oilseed rape (Brassica napus)–wild radish (Raphanus raphanistrum) spontaneous hybrids and of their progeny estimated by flow cytometry. Cruciferae Newslett 19:17–18

  10. Ge K, Zhang A (1979a) Synthesis of a new species and cytogenetic studies in oleiferous Brassica. V. The cytogenetic studies in auto-allooctoploidy of Brassica napus L. var. Oleifera. Acta Agron Sin 5:13–20

  11. Ge K, Zhang A (1979b) Synthesis of new species and cytogenetic studies in Brassica. V. Artificial synthesis of an autoallooctoploid in Brassica napus var Oleifera. Acta Genet Sin 6:62

  12. Geng X, Chen S, Astarini I et al (2013) Doubled haploids of novel trigenomic Brassica derived from various interspecific crosses. Plant Cell Tissue Organ Culture 113:501–511. doi:10.1007/s11240-013-0292-4

  13. Henry Y (1999) Origin of microspore-derived dihaploid and polyhaploid in vitro plants. Plant Tissue Culture Biotechnol 4:127–135

  14. Hoekstra S, van Zijderveld MH, Heidekamp F et al (1993) Microspore culture of Hordeum vulgare L.: the influence of density and osmolality. Plant Cell Rep 12:661–665

  15. Jakše M, Hirschegger P, Bohanec B et al (2010) Evaluation of gynogenic responsiveness and pollen viability of selfed doubled haploid onion lines and chromosome doubling via somatic regeneration. J Am Soc Hortic Sci 135:67–73

  16. Jiao Y, Wickett NJ, Saravanaraj A et al (2011) Ancestral polyploidy in seed plants and angiosperms. Nature 473:97–100

  17. Jugulam M, Ziauddin A, So KKY et al (2015) Transfer of dicamba tolerance from Sinapis arvensis to Brassica napus via embryo rescue and recurrent backcross breeding. PLoS ONE 10:e0141418

  18. Kasha KJ, Kao KN (1970) High frequency haploid production in barley (Hordeum vulgare L.). Nature 225:874–876

  19. Kelliher T, Starr D, Richbourg L et al (2017) MATRILINEAL, a sperm-specific phospholipase, triggers maize haploid induction. Nature 542:105

  20. Kidwell KK, Osborn TC (1992) Simple plant DNA isolation procedures. In: Beckman J, Osborn TC (eds) Plant genomes: methods for genetic and physical mapping. Kluwer Academic Publishers/Springer, Dordrecht, pp 1–13. doi:10.1007/978-94-011-2442-3

  21. Kuligowska K, Lütken H, Christensen B et al (2015) Evaluation of reproductive barriers contributes to the development of novel interspecific hybrids in the Kalanchoë genus. BMC Plant Biol 15:1–15

  22. Kuppu S, Tan EH, Nguyen H et al (2015) Point mutations in centromeric histone induce post-zygotic incompatibility and uniparental inheritance. PLoS Genet 11:e1005494

  23. Larter EN, Gustafson JP (1980) Triticale. Hybridization of crop plants. American Society of Agronomy/Crop Science Society of America, Madison

  24. Leflon M, Eber F, Letanneur JC et al (2006) Pairing and recombination at meiosis of Brassica rapa (AA) × Brassica napus (AACC) hybrids. Theor Appl Genet 113:1467–1480

  25. Li Y, Zhang HY, Wang XZ et al (2004) Identification and analysis of F2 stable population derived from the cross of triploid × diploid in rice. Acta Genet Sin 31:604–608

  26. Li H, Guo X, Wang C, Ji W (2015) Spontaneous and divergent hexaploid triticales derived from common wheat × rye by complete elimination of D-genome chromosomes. PLoS ONE 10:e0120421

  27. Lichter R (1982) Induction of haploid plants from isolated pollen of Brassica napus. Z Pflanzenphysiol 105:427–434

  28. Liu C, Douches DS (1993) Production of haploids of potato (Solanum tuberosum subsp. tuberosum) and their identification with electrophoretic analysis. Euphytica 70:113–126

  29. Liu B, Brubaker CL, Mergeai G et al (2001) Polyploid formation in cotton is not accompanied by rapid genomic changes. Genome 44:321–330

  30. Liu Z, Adamczyk K, Manzanaresdauleux M et al (2006) Mapping PrBn and other quantitative trait loci responsible for the control of homeologous chromosome pairing in oilseed rape (Brassica napus L.) haploids. Genetics 174:1583–1596

  31. Liu S, Liu Y, Yang X et al (2014) The Brassica oleracea genome reveals the asymmetrical evolution of polyploid genomes. Nat Commun 5:3930

  32. Mason AS, Nelson MN, Takahira J et al (2014) The fate of chromosomes and alleles in an allohexaploid Brassica population. Genetics 197:273–283

  33. Mcnaughton IH (1973) Brassica napocampestris L. (2n = 58). 1. Synthesis, cytology, fertility and general considerations. Euphytica 22:301–309

  34. Mo J, Li W, Yu Q et al (1995) Inheritance of the waxless character of Brassica napus Nilla glossy. Can J Plant Sci 75:893–894

  35. Nelson MN, Mason AS, Castello MC et al (2009) Microspore culture preferentially selects unreduced (2n) gametes from an interspecific hybrid of Brassica napus L. × Brassica carinata Braun. Theor Appl Genet 119:497–505

  36. Otto SP, Whitton J (2000) Polyploid incidence and evolution. Annu Rev Genet 34:401–437

  37. Pradhan A, Plummer JA, Nelson MN et al (2010) Trigenomic hybrids from interspecific crosses between Brassica napus and B. nigra. Crop Pasture Sci 61:464–474

  38. Ramsey J, Schemske DW (1998) Pathways, mechanisms, and rates of polyploid formation in flowering plants. Annu Rev Ecol Syst 29:467–501

  39. Ravi M, Chan SW (2010) Haploid plants produced by centromere-mediated genome elimination. Nature 464:615–618

  40. Ravi M, Marimuthu MPA, Tan EH et al (2014) A haploid genetics toolbox for Arabidopsis thaliana. Nat Commun 5:5334

  41. Rotarenco V, Dicu G, State D et al (2010) New inducers of maternal haploids in maize. Maize Genet Cooperation Newslett 84:1–7

  42. Sanei M, Pickering R, Kumke K et al (2011) Loss of centromeric histone H3 (CENH3) from centromeres precedes uniparental chromosome elimination in interspecific barley hybrids. Proc Natl Acad Sci USA 108:498–505

  43. Seymour DK, Filiault DL, Henry IM et al (2012) Rapid creation of Arabidopsis doubled haploid lines for quantitative trait locus mapping. Proc Natl Acad Sci USA 109:4227–4232

  44. Takeda T, Takahata Y (1996) Production of alloplasmic Chinese cabbage using synthesized trigenomic hexaploid (AABBCC) in Brassica. Acta Hortic 407:151–154

  45. Tatlioglu T (1989) Inheritance of waxlessness in kohlrabi (Brassica oleracea L. var. Gongyloides) and its utilization in hybrid seed production. Plant Breed 102:215–221

  46. Tian E, Jiang Y, Chen L et al (2010) Synthesis of a Brassica trigenomic allohexaploid (B. carinata × B. rapa) de novo and its stability in subsequent generations. Theor Appl Genet 121:1431–1440

  47. Tsai I, Chen P (1964) Synthesis of new species and cytogenetic studies in oleiferous Brassica—II. Synthesis of two auto-allohexaploids from Brassica napus L. and its two primary species. J Fudan Univ 9:413–428

  48. Uzunova MI, Ecke W (1999) Abundance, polymorphism and genetic mapping of microsatellites in oilseed rape (Brassica napus L.). Plant Breed 118:323–326

  49. Vieira P, Clercq AD, Stals H et al (2014) The cyclin-dependent kinase inhibitor KRP6 induces mitosis and impairs cytokinesis in giant cells induced by plant-parasitic nematodes in Arabidopsis. Plant Cell 26:2633

  50. Wang S, Zhang G, Zhang Y et al (2015) Comparative studies of mitochondrial proteomics reveal an intimate protein network of male sterility in wheat (Triticum aestivum L.). J Exp Bot 66:6191

  51. Warwick SI, Simard MJ, Legere A et al (2003) Hybridization between transgenic Brassica napus L. and its wild relatives: Brassica rapa L., Raphanus raphanistrum L., Sinapis arvensis L., and Erucastrum gallicum (Willd.) OE Schulz. Theor Appl Genet 107:528–539

  52. Wendel JF (2000) Genome evolution in polyploids. Plant Mol Biol 42:225

  53. Zhang L, Liu D, Yan Z et al (2004) Rapid changes of microsatellite flanking sequence in the allopolyploidization of new synthesized hexaploid wheat. Sci China Ser C 47:553–561

  54. Zhao X, Xu X, Xie H et al (2013) Fertilization and uniparental chromosome elimination during crosses with maize haploid inducers. Plant Physiol 163:721–731

Download references


This work was financially supported by the “13th Five-year” National Key Research Projects (China; Grant no. JFYS2016 ZY03002156), the “13th Five-year” National Key Research Projects “Utilization of heterosis technology in oilseed rape and creation of superior hybridization” (China; Grant no. 2016YFD0101300), Sichuan Province Breeding Project “Breakthrough innovation of oil crops materials and methods” (China; Grant no. 2016NYZ0031), and the project of Chengdu Academy of Agricultural and Forestry Sciences “Integrated application demonstration and scientific research innovation” (China; Grant no. 2017Y2500W06).

Author information

Correspondence to Jin Yang or Tingdong Fu.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 19 kb)

Supplementary material 2 (DOCX 74167 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Fu, S., Yin, L., Xu, M. et al. Maternal doubled haploid production in interploidy hybridization between Brassica napus and Brassica allooctaploids. Planta 247, 113–125 (2018).

Download citation


  • Artificial Brassica hybrid
  • Doubled haploid
  • Doubled haploid inducer
  • Genome doubling
  • Interploidy cross
  • In vivo breeding system
  • Polyploid