Plant Cell Reports

, Volume 25, Issue 4, pp 257–264 | Cite as

Production of doubled haploids in durum wheat (Triticum turgidum L.) through isolated microspore culture

  • L. Cistué
  • M. Soriano
  • A. M. Castillo
  • M. P. Vallés
  • J. M. Sanz
  • B. Echávarri
Cell Biology and Morphogenesis


The objective of this work was to produce doubled haploid plants from durum wheat through the induction of androgenesis. A microspore culture technique was developed and used to produce fertile doubled haploid plants of agronomic interest. Five cultivars, one selected line, plus a collection of 20 F1 crosses between different genotypes of high breeding value were used. Studies on several factors such as pre-treatments and media components were carried out in order to develop a protocol to regenerate green haploid plantlets. Anthers were pre-treated in 0.7 M mannitol. Microspores, from anther maceration, were plated on a C17 induction culture medium with ovary co-culture. The optimum regeneration medium J25–8 was used. From 35 microspore isolations, 407 green plantlets were obtained. With this technique mature embryos were obtained. Green plants were regenerated from all genotypes used and approximately 67% of them were spontaneously doubled haploids. Some haploids and a very few polyploids plants were obtained. From the 407 plants, 275 were completely fertile and gave enough seeds to be assayed in the field. This protocol could be used complementary to or instead of the intergeneric crossing with maize as an economically feasible method to obtain doubled haploids from most durum wheat genotypes.


Doubled haploid Microspore culture Durum wheat Triticum turgidum 



Doubled haploid


2,4-dic-hlorophenoxyacetic acid






Naphthalene acetic acid


Phenylacetic acid


  1. Barnabás B, Szakács Ë, Karsai I, Bedö Z (2001) In vitro androgenesis of wheat: from fundamentals to practical application. Euphytica 119:211–216CrossRefGoogle Scholar
  2. Castillo AM, Cistué L (1993) Production of gynogenic haploids of Hordeum vulgare. Plant Cell Rep 12:139–143CrossRefGoogle Scholar
  3. Castillo AM, Vallés MP, Cistué L (2000) Comparison of anther and isolated microspore cultures in barley. Effects of culture density and regeneration medium. Euphytica 111:1–8CrossRefGoogle Scholar
  4. Cattaneo M, Qiao YM, Pogna NE (1991) Embryoid induction and green plant regeneration from cultured anthers in a durum wheat line homozygous for the 1 BL/1RS translocation. J Genet Breed 45:369–372Google Scholar
  5. Cho U-H (1991) Hormonal aspects of androgenic induction in barley (Hordeum vulgare L.). PhD Thesis, University of Guelph, CanadaGoogle Scholar
  6. Cistué L, Ramos A, Castillo AM (1999) Influence of anther pre-treatment and culture medium composition on the production of barley doubled haploids from model and low responding cultivars. Plant Cell Tissue Organ Cult 55:159–166CrossRefGoogle Scholar
  7. Cistué L, Ramos A, Castillo AM, Romagosa I (1994) Production of large number of doubled haploid plants from barley anthers pre-treated with high concentrations of mannitol. Plant Cell Rep 13:709–712CrossRefGoogle Scholar
  8. Cistué L, Ziauddin A, Simion E, Kasha KJ (1995) Effects of culture conditions on isolated microspore response of barley cultivar Igri. Plant Cell Tissue Organ Cult 42:163–169CrossRefGoogle Scholar
  9. Devaux P, Li H (2001) Enhancement of microspore culture efficiency of recalcitrant barley genotypes. Plant Cell Rep 20:475–481CrossRefGoogle Scholar
  10. Dogramaci-Altuntepe M, Peterson TS, Jauhar PP (2001) Anther culture-derived regenerants of durum wheat and their cytological characterization. J Heredity 192(1):56–64CrossRefGoogle Scholar
  11. Foroughi-Wher B, Zeller FJ (1990) In vitro microspore reaction of different German wheat cultivars. Theor Appl Genet 79:77–80CrossRefGoogle Scholar
  12. Ghaemi M, Sarrafi A, Alibert G (1993) Influence of genotype and culture conditions on the production of embryos from anthers of tetraploid wheat (Triticum turgidum). Euphytica 65:81–85CrossRefGoogle Scholar
  13. Ghaemi M, Sarrafi A, Alibert G (1994) The effects of silver nitrate, colchicine, cupric sulfate and genotype on the production of embryoids from anthers of tetraploid wheat (Triticum turgidum). Plant Cell Tissue Organ Cult 36:355–359CrossRefGoogle Scholar
  14. Ghaemi M, Sarrafi A, Alibert G (1995) Influence of genotype, media composition, cold pre-treatment and their interactions on androgenesis in durum wheat (Triticum turgidum). Cereal Res Commun 23(3):215–227Google Scholar
  15. Gustafson VD, Baezinger PS, Wright MS, Sreoup WW, Yen Y (1995) Isolated wheat microspore culture. Plant Cell Tissue Organ Cult 42:207–213CrossRefGoogle Scholar
  16. Hadwiger MA, Heberle-Bors E (1986) Pollen plant production in Triticum turgidum ssp. durum. In: Genetic manipulación in plant breeding. Walter de Gruyter and Co., Berlin, pp 303–305Google Scholar
  17. Hu TC, Kasha KJ(1997) Improvement of isolated microspore culture of wheat (Triticum aestivum L.) through ovary co-culture. Plant Cell Rep 16:520–525CrossRefGoogle Scholar
  18. Hu TC, Ziauddin A, Simion E, Kasha KJ (1995) Isolated microspore culture of wheat (Triticum aestivum L.) in a defined media: I. Effects of pretreatment, isolation methods, and hormones. In Vitro Cell Dev Biol 31:79–83CrossRefGoogle Scholar
  19. Immonen S, Robinson J (2000) Stress treatments and ficoll for improving green plant regeneration in triticale anther culture. Plant Sci 150:77–84CrossRefGoogle Scholar
  20. Jähne A, Lorz H (1995) Cereal microspore culture. Plant Sci 109:1–12CrossRefGoogle Scholar
  21. Jauhar PP (2003a) Haploid and doubled haploid production in durum wheat by wide hybridization. In: Maluszynski M et al (eds) Doubled haploid production in crop plants. Kluwer, pp 161–166Google Scholar
  22. Jauhar PP (2003b) Haploid and doubled haploid production in durum wheat by anther culture. In: Maluszynski M et al (eds) Doubled haploid production in crop plants. Kluwer, pp 167–172Google Scholar
  23. J'Aiti F, Benlhabib O, Sharma HC, El Jaafari S, El Hadrami I (1999) Genotypic variation in anther culture and effect of ovary coculture in durum wheat. Plant Cell Tissue Organ Cult 59:71–76CrossRefGoogle Scholar
  24. J'Aiti F, El Jaafari S, El Hadrami I, Nachit M, Baum M, De Buyser J, Picard E (2000) Problématique de la culture de microspores chez le blé dur Triticum turgidum L. var. Durum (2000). In: Durum wheat improvement in the Mediterranean region: New challenges. Instituto Agronómico Mediterráneo Zaragoza, 12–14 April 2000, pp 149–152Google Scholar
  25. Jensen CJ (1983) Producing haploid plants by chromosome elimination. Cell and tissue culture techniques for cereal crop improvement. Science Press, Beijing, China,, pp 55–79Google Scholar
  26. Kang T-J, Yang M-S, El Deckard (2003) The effect of osmotic potential on anther culture in spring wheat (Triticum aestivum). Plant Cell Tissue Organ Cult 75:35–40CrossRefGoogle Scholar
  27. Kasha KJ, Maluszynski M (2003) Production of doubled haploids in crop plants. An introduction. In: Maluszynski M et al (eds) Doubled haploid production in crop plants. Kluwer, pp 1–4Google Scholar
  28. Kasha KJ, Hu TC, Oro R, Simion E, Shim YS (2001) Nuclear fusion leads to chromosome doubling during mannitol pre-treatment of barley (Hordeum vulgare L.) microspores. J Exp Bot 52:1227–1238PubMedCrossRefGoogle Scholar
  29. Kasha KJ, Simion E, Miner M, Letarte J, Hu TC (2003) Haploid wheat isolated microspore culture protocol. In: Maluszynski M et al (eds) Doubled haploid production in crop plants. Kluwer, pp 77–81Google Scholar
  30. Mentewab A, Sarrafi A (1997) Influence of genotype and cold pre-treatment on the production of embryoids and their regeneration in tetraploid and hexaploid wheats. J Genet Breed 51:59–62Google Scholar
  31. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plantarum 15:473–497CrossRefGoogle Scholar
  32. Orlov PA, Mavrishcheva EB, Palilova N (1993) Estimation of the response to anther culturing in 60 genotypes of different wheat species. Plant Breed 111:339–342CrossRefGoogle Scholar
  33. Otani M, Shimada T (1994) Pollen embryo formation and plant regeneration from cultured anthers of tetraploid wheat. J Genet Breed 48:103–106Google Scholar
  34. Ouyang JW, Hu H, Chuang CC, Tseng CC (1973) Induction of pollen plants from anther of Triticum aestivum L. cultured in vitro. Sci Sin 16:79–95Google Scholar
  35. Roberts-Oehlschlager SL, Dunwell JM (1990) Barley anther culture: Pre-treatment on mannitol stimulates production of microspore-derived embryos. Plant Cell Tissue Organ Cult 20:235–240Google Scholar
  36. Saidi N, Cherkaoui S, Chlyah A, Chlyah H (1997) Embryo formation and regeneration in Triticum turgidum ssp. durum anther culture. Plant Cell Tissue Organ Cult 51:27–33CrossRefGoogle Scholar
  37. Shim YS, Kasha KJ (2003) The influence of pre-treatment on cell stage progression and the time of DNA synthesis in barley (Hordeum vulgare L.) uni-nucleate microspores. Plant Cell Rep 21:1065–1071PubMedCrossRefGoogle Scholar
  38. Silva-Romano MC, Canhoto JM (1999) Resposta de genótipos de Triticum aestivum e de Triticum durum a cultura in vitro de anteras. Melhoramento 36:5–14Google Scholar
  39. Thiebaut J, Kasha KJ, Tsai A (1979) Influence of plant development stage, temperature and plant hormones on chromosomes doubling of barley haploids using colchicine. Can J Bot 57:480–483CrossRefGoogle Scholar
  40. Tuvesson S, Ljungberg A, Johansson N, Karlsson KE, Suijs LW, Josset JP (2000) Large-scale production of wheat and triticale doubled haploids through the use of a single-anther culture method. Plant Breed 119:455–459CrossRefGoogle Scholar
  41. Wang P, Chen YR (1986) A study on the application of C17 medium for anther culture. Acta Bot Sin 28:38–45Google Scholar
  42. Zheng MY (2003) Microspore culture in wheat (Triticum aestivum) – doubled haploid production via induced embryogenesis. Plant Cell Tissue Organ Cult 73:213–230CrossRefGoogle Scholar
  43. Zheng MY, Liu W, Weng Y, Polle E, Konzak CF (2001) Culture of freshly isolated wheat (Triticum aestivum L.) microspores treated with inducer chemicals. Plant Cell Rep 20:685–690CrossRefGoogle Scholar
  44. Zheng MY, Weng Y, Liu W, Konzak CF (2002) The effect of ovary-conditioned medium on microspore embryogenesis in common wheat (Triticum aestivum L.). Plant Cell Rep 20:802–807CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • L. Cistué
    • 1
  • M. Soriano
    • 1
  • A. M. Castillo
    • 1
  • M. P. Vallés
    • 1
  • J. M. Sanz
    • 1
  • B. Echávarri
    • 1
  1. 1.Departamento de Genética y Producción VegetalEstación Experimental de Aula Dei, Consejo Superior de Investigaciones CientíficasZaragozaSpain

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