Fertile allohexaploid Brassica hybrids obtained from crosses between B. oleracea and B. juncea via ovule rescue and colchicine treatment of cuttings

  • Margaret W. Mwathi
  • Mehak Gupta
  • Daniela Quezada-Martinez
  • Aneeta Pradhan
  • Jacqueline Batley
  • Annaliese S. MasonEmail author
Original Article


An allohexaploid Brassica crop (2n = AABBCC) does not exist naturally, but is of interest for its potential to combine useful traits found in the six cultivated Brassica species which share combinations of the A, B and C genomes with additional allelic heterosis. In this study, we aimed to produce 2n = AABBCC hybrids by crosses between B. juncea and a number of Brassica C genome species. We used ovule rescue to overcome hybridization barriers and different colchicine treatment methods to induce chromosome doubling of ABC hybrids to AABBCC allohexaploids, thus restoring fertility. Only the cross B. oleracea × B. juncea was successful, with six triploid hybrids produced from one genotype combination. Colchicine-containing regeneration media was unsuccessful in doubling chromosome number in these hybrids, but treatment of cuttings with 0.05 to 0.25% colchicine successfully produced ~ 200 S1 allohexaploid seeds. The S1 plants produced 7–84% viable pollen and set 0–390 seeds per plant, with 23–27 bivalents and 0–3 univalents during metaphase I of meiosis. Our results highlight the difficulties in working with the wild C genome species, but showed that our methods have utility for producing euploid, chromosome-doubled progeny in this cross combination. Further, Brassica oleracea × B. juncea allohexaploid hybrids may contain useful genetic factors for improved meiotic stability and fertility in allohexaploid germplasm pools.

Key message

Ovule rescue followed by 0.05–0.25% colchicine treatment of cuttings successfully produces fertile, partially stable allohexaploid Brassica from the cross B. juncea × B. oleracea.


Ovule culture Brassica Interspecific hybridization Chromosome doubling Colchicine Allohexaploid 



MWM and this project were supported by Australia-India Strategic Research Fund: Biotechnology grant AISRF06520. ASM was supported by Emmy Noether DFG grant MA6473/1-1, and DQM was supported by DFG Sino-German Centre grant MA6473/3-1. Travel and support of MG in Germany were funded by a DFG Initiation of Collaboration Grant (MA6473/10-1) and the INSA-DFG Bilateral Exchange Program-2019.

Author contributions

MWM carried out the experiments at UWA and drafted the paper. MG undertook the cytogenetic analysis, and DQM grew the plants at JLU, collected samples and measured their fertility traits. AP assisted with multiplication and colchicine treatment of the hybrid plants at UWA. ASM assisted with writing, presentation and interpretation of the results. MWM, JB and ASM designed the experiments and JB and ASM supervised MWM.


  1. Angadi SV, Cutforth HW, Miller PR, McConkey BG, Entz MH, Brandt SA, Volkmar KM (2000) Response of three Brassica species to high temperature stress during reproductive growth Can J. Plant Sci 80:693–701Google Scholar
  2. Arumugam N, Mukhopadhyay A, Gupta V, Pental D, Pradhan AK (1996) Synthesis of hexaploid (AABBCC) somatic hybrids: a bridging material for transfer of ‘tour’ cytoplasmic male sterility to different Brassica species. Theor Appl Genet 92:762–768CrossRefGoogle Scholar
  3. Banga SS, Bhaskar PB, Ahuja I (2003) Synthesis of intergeneric hybrids and establishment of genomic affinity between Diplotaxis catholica and crop Brassica species. Theor Appl Genet 106:1244–1247. CrossRefPubMedGoogle Scholar
  4. Bhat S, Sarla N (2004) Identification and overcoming barriers between Brassica rapa L. em. Metzg. and B. nigra (L.) Koch crosses for the resynthesis of B. juncea (L.) Czern. Genet Resour Crop Ev 51:455–469. CrossRefGoogle Scholar
  5. Branca F, Cartea E (2011) Brassica. In: Kole C (ed) Wild crop relatives: genomic and breeding resources. Springer, BerlinGoogle Scholar
  6. Busso C, Attia T, Röbbelen G (1987) Trigenomic combinations for the analysis of meiotic control in the cultivated Brassica species. Genome 29:331–333CrossRefGoogle Scholar
  7. Chen LP, Zhang MF, Li CS, Hirata Y (2005) Production of interspecific somatic hybrids between tuber mustard (Brassica juncea) and red cabbage (Brassica oleracea). Plant Cell Tiss Org 80:305–311. CrossRefGoogle Scholar
  8. Chen L-P, Ge Y-M, Zhu X-Y (2006) Artificial synthesis of interspecific chimeras between tuber mustard (Brassica juncea) and cabbage (Brassica oleracea) and cytological analysis. Plant Cell Rep 25:907–913CrossRefGoogle Scholar
  9. Chen S et al (2011) Trigenomic bridges for Brassica improvement. Crit Rev Plant Sci 30:524–547CrossRefGoogle Scholar
  10. Chèvre AM, Barret P, Eber F, Dupuy P, Brun H, Tanguy X, Renard M (1997) Selection of stable Brassica napus-B. juncea recombinant lines resistant to blackleg (Leptosphaeria maculans). 1. Identification of molecular markers, chromosomal and genomic origin of the introgression. Theor Appl Genet 95:1104–1111CrossRefGoogle Scholar
  11. Cousin A, Heel K, Cowling WA, Nelson MN (2009) An efficient high-throughput flow cytometric method for estimating DNA ploidy level in plants. Cytometry A 75:1015–1019. CrossRefPubMedGoogle Scholar
  12. Dhooghe E, Van Laere K, Eeckhaut T, Leus L, Van Huylenbroeck J (2011) Mitotic chromosome doubling of plant tissues in vitro. Plant Cell Tissue Org 104:359–373. CrossRefGoogle Scholar
  13. FitzJohn RG, Armstrong TT, Newstrom-Lloyd LE, Wilton AD, Cochrane M (2007) Hybridisation within Brassica and allied genera: evaluation of potential for transgene escape. Euphytica 158:209–230CrossRefGoogle Scholar
  14. Gaebelein R, Mason AS (2018) Allohexaploids in the genus Brassica. Crit Rev Plant Sci 37:422–437CrossRefGoogle Scholar
  15. Gaebelein R, Alnajar D, Koopmann B, Mason AS (2019a) Hybrids between Brassica napus and B nigra show frequent pairing between the B and A/C genomes and resistance to blackleg. Chromosome Res. CrossRefPubMedGoogle Scholar
  16. Gaebelein R, Schiessl SV, Samans B, Batley J, Mason AS (2019b) Inherited allelic variants and novel karyotype changes influence fertility and genome stability in Brassica allohexaploids. New Phytol 223:965–978. CrossRefPubMedGoogle Scholar
  17. Garg H, Banga S, Bansal P, Atri C, Banga SS (2007) Hybridizing Brassica rapa with wild crucifers Diplotaxis erucoides and Brassica maurorum. Euphytica 156:417–424. CrossRefGoogle Scholar
  18. Geng XX et al (2013) Doubled haploids of novel trigenomic Brassica derived from various interspecific crosses. Plant Cell Tiss Org 113:501–511. CrossRefGoogle Scholar
  19. Gupta M, Atri C, Agarwal N, Banga SS (2016) Development and molecular-genetic characterization of a stable Brassica allohexaploid. Theor Appl Genet 129:2085–2100. CrossRefPubMedGoogle Scholar
  20. Hilgert-Delgado A, Klima M, Viehmannova I, Urban MO, Fernandez-Cusimamani E, Vyvadilova M (2015) Efficient resynthesis of oilseed rape (Brassica napus L.) from crosses of winter types B. rapa x B. oleracea via simple ovule culture and early hybrid verification. Plant Cell Tiss Org 120:191–201. CrossRefGoogle Scholar
  21. Katche E, Quezada-Martinez D, Katche EI, Vasquez-Teuber P, Mason AS (2019) Interspecific hybridization for Brassica crop improvement Crop Breeding. Genet Genomics 1:e190007Google Scholar
  22. Kinian SF, Quiros CF (1992) Trait inheritance, fertility, and genomic relationships of some n = 9 Brassica species. Genet Resour Crop Ev. 39:165–175Google Scholar
  23. Lannér C, Bryngelsson T, Gustafsson M (1997) Relationships of wild Brassica species with chromosome number 2n = 18, based on RFLP studies. Genome 40:302–308. CrossRefPubMedGoogle Scholar
  24. Lazáro A, Aguinagalde I (1998) Genetic diversity in Brassica oleracea L. (Cruciferae) and wild relatives (2n = 18) using isozymes. Ann Bot 82:821–828. CrossRefGoogle Scholar
  25. Li JX, Rao LL, Meng QF, Ghani MA, Chen LP (2015) Production of Brassica tri-genomic vegetable germplasm by hybridisation between tuber mustard (Brassica juncea) and red cabbage (B. oleracea). Euphytica 204:323–333. CrossRefGoogle Scholar
  26. Lian YJ, Lin GZ, Zhao XM, Lim HT (2011) Production and genetic characterization of somatic hybrids between leaf mustard (Brassica juncea) and broccoli (Brassica oleracea). Vitro Cell Dev 47:289–296. CrossRefGoogle Scholar
  27. Mallet J (2007) Hybrid speciation. Nat Rev 446:279–283Google Scholar
  28. Mason AS, Yan GJ, Cowling WA, Nelson MN (2012) A new method for producing allohexaploid Brassica through unreduced gametes. Euphytica 186:277–287. CrossRefGoogle Scholar
  29. Mason AS et al (2015) High-throughput genotyping for species identification and diversity assessment in germplasm collections. Mol Ecol Resour 15:1091–1101. CrossRefPubMedGoogle Scholar
  30. Mei J et al (2010) Genomic relationships between wild and cultivated Brassica oleracea L. with emphasis on the origination of cultivated crops. Genet Resour Crop Ev 57:687–692. CrossRefGoogle Scholar
  31. Morinaga T (1934) Interspecific hybridisation in Brassica VI: The cytology of F1 hybrids of B. juncea and B. nigra. Cytologia 6:62–67CrossRefGoogle Scholar
  32. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497. CrossRefGoogle Scholar
  33. Nagaharu U (1935) Genome-analysis in Brassica with special reference to the experimental formation of B. napus and peculiar mode of fertilization. Jpn J Bot 7:389–452Google Scholar
  34. Pradhan A, Plummer JA, Nelson MN, Cowling WA, Yan G (2010) Successful induction of trigenomic hexaploid Brassica from a triploid hybrid of B. napus L. and B. nigra (L.). Koch Euphytica 176:87–98CrossRefGoogle Scholar
  35. Saal B, Brun H, Glais I, Struss D (2004) Identification of a Brassica juncea-derived recessive gene conferring resistance to Leptosphaeria maculans in oilseed rape. Plant Breed 123:505–511CrossRefGoogle Scholar
  36. Snogerup S, Gustafsson M, von Bothmer R (1990) Brassica sect. Brassica (Brassicaceae) I. Taxonomy and variation. Willdenowia 19:271–365Google Scholar
  37. Snow AA, Moran-Palma P, Rieseberg LH, Wszelaki A, Seiler GJ (1998) Fecundity, phenology, and seed dormancy of F1 wild-crop hybrids in sunflower (Helianthus annuus, Asteraceae). Am J Bot 85:794–801. CrossRefPubMedGoogle Scholar
  38. Stewart A (2004) A review of crossing relationship between cultivated Brassica species. Crucif Newslett 25:25–26Google Scholar
  39. Tian E, Jiang Y, Chen L, Zou J, Liu F, Meng J (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. CrossRefPubMedGoogle Scholar
  40. Tonguc M, Griffiths PD (2004) Development of black rot resistant interspecific hybrids between Brassica oleracea L. cultivars and Brassica accession A 19182, using embryo rescue. Euphytica 136:313–318. CrossRefGoogle Scholar
  41. von Bothmer R, Gustafsson M, Snogerup S (1995) Brassica sect. Brassica (Brassicaceae). 2. Interspecific and intraspecific crosses with cultivars of Brassica oleracea. Genet Resour Crop Ev 42:165–178. CrossRefGoogle Scholar
  42. Weerakoon S (2011) Producing inter-specific hybrids between Brassica juncea (L.) Czern & Coss and B. oleracea (L.) to synthesize trigenomic (abc) Brassica. J Sci 6:13–34Google Scholar
  43. Wen J et al (2012) Characterization of interploid hybrids from crosses between Brassica juncea and B. oleracea and the production of yellow-seeded B. napus. Theor Appl Genet 125:19–32. CrossRefPubMedGoogle Scholar
  44. Yao XC, Ge XH, Li ZY (2012) Different fertility and meiotic regularity in allohexaploids derived from trigenomic hybrids between three cultivated Brassica allotetraploids and B. maurorum. Plant Cell Rep 31:781–788. CrossRefPubMedGoogle Scholar
  45. Zhang GQ, Zhou WJ, Gu HH, Song WJ, Momoh EJJ (2003) Plant regeneration from the hybridization of Brassica juncea and B. napus through embryo culture. J Agron Crop Sci 189:347–350. CrossRefGoogle Scholar
  46. Zhou JN, Tan C, Cui C, Ge XH, Li ZY (2016) Distinct subgenome stabilities in synthesized Brassica allohexaploids. Theor Appl Genet 129:1257–1271. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.School of Agriculture and Food SciencesUniversity of QueenslandBrisbaneAustralia
  2. 2.School of Biological SciencesThe University of Western AustraliaCrawleyAustralia
  3. 3.DBT Centre of Excellence on Brassicas, Department of Plant Breeding and GeneticsPunjab Agricultural UniversityLudhianaIndia
  4. 4.Department of Plant Breeding, Research Centre for BiosystemsLand Use and Nutrition, Justus Liebig UniversityGiessenGermany

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