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

  • Cell Biology and Morphogenesis
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Abstract

The objective of this study was to produce durum wheat doubled haploid (DH) plants through the induction of microspore embryogenesis. The microspore culture technique was improved to maximize production of green plants per spike using three commercial cultivars. Studies on factors such as induction media composition, induction media support and the stage and growth of donor plants were carried out in order to develop an efficient protocol to regenerate green and fertile DH plants. Microspores were plated on a C17 induction culture medium with ovary co-culture and a supplement of glutathione plus glutamine; 300 g/l Ficoll Type-400 was incorporated to the induction medium support. Donor plants were fertilized with a combination of macro and microelements. With the cultivars ‘Ciccio’ and ‘Claudio’ an average of 36.5 and 148.5 fertile plants were produced, respectively, from 1,000 anthers inoculated. This technique was then used to produce fertile DH plants of potential agronomic interest from a collection of ten F1 crosses involving cultivars of high breeding value. From these crosses 849 green plants were obtained and seed was harvested from 702 plants indicating that 83% of green plants were fertile and therefore were spontaneously DHs. No aneuploid plant was obtained. The 702 plants yielded enough seeds to be field tested. One of the DH lines obtained by microspore embryogenesis, named ‘Lanuza’, has been sent to the Spanish Plant Variety Office for Registration by the Batlle Seed Company. This protocol can be used instead of the labor-intensive inter-generic crossing with maize as an economically feasible method to obtain DHs for most crosses involving the durum wheat cultivars grown in Spain.

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Abbreviations

NAA:

Naphthalene acetic acid

BAP:

6-Benzylaminopurine

PEG:

Polyethylene glycol

ELS:

Embryo like structure

References

  • Arnison PG, Donaldson P, Jacson A, Ch Semple, Keller WA (1990) Genotype-specific response of cultured broccoli (Brassica oleracea var. Italica) anthers to cytokinins. Plant Cell Tissue Organ Cult 20:217–222

    CAS  Google Scholar 

  • Bakos F, Fábián A, Barnabás B (2007) Isolated microspore cultures of Hungarian durum wheat (Triticum turgidum L.) cultivar Martondur 1. Acta Agron Hung 55(2):157–164

    Article  Google Scholar 

  • Ballesteros J, García-Llamas C, Ramirez MC, Martin A (2003) Low relative humidity increases haploid production in durum wheat × maize crosses. Plant Breed 122(3):276–278

    Article  Google Scholar 

  • Belmonte MF, Macey J, Yeung EC, Stasolla C (2005) The effect of osmoticum on ascorbate and glutathione metabolism during white spruce (Picea glauca) somatic embryo development. Plant Physiol Biochem 43:337–346

    PubMed  CAS  Google Scholar 

  • Belmonte MF, Ambrose SJ, Ross ARS, Abrams SR, Stasolla C (2006) Improved development of microspore-derived embryo cultures of Brassica napus cv Topaz following changes in glutathione metabolism. Physiol Plant 127:690–700

    Article  CAS  Google Scholar 

  • Boutilier K, Offringa R, Sharma VK, Kieft H, Ouellet T, Zhang L, Hattori J, Ch-M Liu, van Lammeren AAM, Miki BLA, Custers JBM, n Campagne MM (2002) Ectopic expression of BABY BOOM triggers a conversion from vegetative to embryonic growth. Plant Cell 14:1737–1749

    Article  PubMed  CAS  Google Scholar 

  • Campbell AW, Griffin WB, Conner AJ, Rowarth JS, Burrit DJ (1998) The effects of temperature and light intensity on embryo numbers in wheat doubled haploid production through wheat × maize crosses. Ann Bot 82:29–33

    Article  Google Scholar 

  • Cattaneo M, Qiao YM, Pogna NE (1991) Embryoid induction and green plant regeneration from cultured anthers in a durum wheat line homozygous for the 1BL/1RS translocation. J Genet Breed 45:369–372

    Google Scholar 

  • Cistué L, Kasha KJ (2005) Gametic embryogenesis in Triticum: a study of some critical factors in haploid (microspore) embryogenesis. In: Mujib A, Samaj J (eds) Somatic embryogenesis plant cell monograph, vol 2. Springer, Berlin, pp 321–342

    Chapter  Google Scholar 

  • 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 13:709–712

    Article  Google Scholar 

  • 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 42:163–169

    Article  Google Scholar 

  • Cistué L, Vallés MP, Echávarri B, Sanz M, Castillo AM (2004) Production of barley doubled haploids by anther and microspore culture. In: Mujib A, Cho M-J, Predieri S, Banerjee S (eds) In vitro application in crop improvement. Science Publishers, Inc., Enfield, pp 1–17

    Google Scholar 

  • Cistué L, Soriano M, Castillo AM, Vallés MP, Sanz JM, Echávarri B (2006) Production of doubled haploids in durum wheat (Triticum turgidum L.) through isolated microspore culture. Plant Cell Rep 25:257–264

    Article  PubMed  CAS  Google Scholar 

  • Coventry J, Kott L, Beversdorf WD (1988) Manual for microspore culture technique for Brassica napus. Department of Crop Science Technical Bulletin, OAC Publication 0489, University of Guelph, Ontario

  • Dahleen LS (1999) Donor-plant environment effects on regeneration from barley embryo-derived callus. Crop Sci 39:682–685

    Article  Google Scholar 

  • Devaux P, Li H (2001) Enhancement of microspore culture efficiency of recalcitrant barley genotypes. Plant Cell Rep 20:475–481

    Article  CAS  Google Scholar 

  • Devaux P, Pickering RA (2005) Haploids in the improvement of Poaceae. In: Palmer CE, Keller WE, Kasha KJ (eds) Biotechnology in agriculture and forestry, vol 56. Haploids in crop improvement II. Springer, Berlin, pp 215–242

    Chapter  Google Scholar 

  • Dogramaci-Altuntepe M, Peterson TS, Jauhar PP (2001) Anther culture-derived regenerants of durum wheat and their cytological characterization. J Hered 192(1):56–64

    Article  Google Scholar 

  • Fletcher R, Coventry J, Kott L (1998) Manual for microspore culture technique for Brassica napus. Department of Plant Agriculture, Technical Bulletin, University of Guelph, Ontario

    Google Scholar 

  • Foroughi-Wehr B, Zeller FJ (1990) In vitro microspore reaction of different German wheat cultivars. Theor Appl Genet 79:77–80

    Article  Google Scholar 

  • Forster BP, Heberle-Bors E, Kasha KJ, Touraev A (2007) The resurgence of haploids in higher plants. Trends Plant Sci 12(8):368–375

    Article  PubMed  CAS  Google Scholar 

  • 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 65:81–85

    Article  Google Scholar 

  • 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 36:355–359

    Article  CAS  Google Scholar 

  • Ghaemi M, Sarrafi A, Alibert G (1995) Influence of genotype, media composition, cold pre-treatment and their interactions on microspore embryogenesis in durum wheat (Triticum turgidum). Cereal Res Commun 23(3):215–227

    Google Scholar 

  • Górecka K, Krzyzanowska D, Górecki R (2005) The influence of several factors on the efficiency of microspore embryogenesis in carrot. J Appl Genet 46(3):265–269

    PubMed  Google Scholar 

  • Guzy-Wróbelska J, Labocha-Pawlowska Kwasniewski, Szarejko I (2007) Different recombination frequencies in wheat doubled haploid populations obtained through maize pollination and anther culture. Euphytica 156:173–183

    Article  Google Scholar 

  • 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 303–305

    Google Scholar 

  • Hosp J, Maraschin SF, Touraev A, Boutilier K (2007) Functional genomics of microspore embryogenesis. Euphytica 158:275–285

    Article  Google Scholar 

  • Hu TC, Kasha KJ (1997) Improvement of isolated microspore culture of wheat (Triticum aestivum L.) through ovary co-culture. Plant Cell Rep 16:520–525

    Article  CAS  Google Scholar 

  • Ilic-Grubor K, Attree SM, Fowke LC (1998a) Induction of microspore-derived embryos of Brassica napus L. with polyethylene glycol (PEG) as osmoticum in a low sucrose medium. Plant Cell Rep 17:329–333

    Article  CAS  Google Scholar 

  • Ilic-Grubor K, Attree SM, Fowke LC (1998b) Comparative morphological study of zygotic and microspore-derived embryos of Brassica napus L. as revealed by scanning electron microscopy. Ann Bot 82:157–165

    Article  Google Scholar 

  • Immonen S, Robinson J (2000) Stress treatments and Ficoll for improving green plant regeneration in triticale anther culture. Plant Sci 150:77–84

    Article  CAS  Google Scholar 

  • Jacquard C, Asakaviciute R, Hamalian AM, Sangwan RS, Devaux P, Clément C (2006) Barley anther culture: effects of annual cycle and spike position on microspore embryogenesis and albinism. Plant Cell Rep 25:375–381

    Article  PubMed  CAS  Google Scholar 

  • J’Aiti F, Benlhabib O, Sharma HC, El Jaafari S, El Hadrami I (1999) Genotypic variation in anther culture and effect of ovary co-culture in durum wheat. Plant Cell Tissue Organ Cult 59:71–76

    Article  Google Scholar 

  • 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 149–152

  • 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 Academic Publishers, Dordrecht, pp 161–166

    Google Scholar 

  • 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 Academic Publishers, Dordrecht, pp 167–172

    Google Scholar 

  • Jensen CJ (1983) Producing haploid plants by chromosome elimination. In: Cell and tissue culture techniques for cereal crop improvement. Science Press, Bei**g, pp 55–79

    Google Scholar 

  • Kang T-J, Yang M-S, Deckard EL (2003) The effect of osmotic potential on anther culture in spring wheat (Triticum aestivum). Plant Cell Tissue Organ Cult 75:35–40

    Article  CAS  Google Scholar 

  • Kao KN (1981) Plant formation from barley anther cultures with Ficoll media. Z Pflanzenphysiol 103:437–443

    CAS  Google Scholar 

  • 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 Academic Publishers, Dordrecht, pp 1–4

    Google Scholar 

  • 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 52:1227–1238

    Article  PubMed  CAS  Google Scholar 

  • Knox RE, Clarke JM, DePauw RM (2000) Dicamba and growth condition effects on doubled haploid production in durum wheat crossed with maize. Plant Breed 119:289–298

    Article  Google Scholar 

  • Labbani Z, Richard N, De Buyser J, Pickard E (2005) Chlorophyllian durum wheat plants obtained by isolated microspores culture: importance of the pre-treatments. C R Biol 328(8):713–723

    Article  PubMed  Google Scholar 

  • Maluszynski M, Kasha KJ, Forster BP, Szarejko I (eds) (2003) Doubled haploid production in crop plants. A manual. Kluwer Academic Publisher, Dordrecht

    Google Scholar 

  • 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 51:59–62

    Google Scholar 

  • Mordhorst AP, Lörz H (1993) Embryogenesis and development of isolated barley (Hordeum vulgare L.) microspores are influenced by the amount and composition of nitrogen sources in culture media. J Plant Physiol 142:485–492

    CAS  Google Scholar 

  • Mordhorst AP, Toonen MAJ, de Vries SC (1997) Plant embryogenesis. Crit Rev Plant Sci 16(6):535–576

    Article  Google Scholar 

  • O’Donoughue LS, Bennett MD (1994a) Comparative responses of tetraploide wheats pollinated with Zea mays L. and Hordeum bulbosum L. Theor Appl Genet 87:673–680

    Google Scholar 

  • O’Donoughue LS, Bennett MD (1994b) Durum wheat haploid production using maize wide-crossing. Theor Appl Genet 89:559–566

    Google Scholar 

  • Olsen (1987) Induction of microspores embryogenesis in cultured anthers of Hordeum vulgare. The effects of ammonium nitrate, glutamine and asparagines as nitrogen sources. Calsberg Res Commun 52:393–404

  • Orlov PA, Mavrishcheva EB, Palilova N (1993) Estimation of the response to anther culturing in 60 genotypes of different wheat species. Plant Breed 111:339–342

    Article  Google Scholar 

  • Otani M, Shimada T (1994) Pollen embryo formation and plant regeneration from cultured anthers of tetraploid wheat. J Genet Breed 48:103–106

    Google Scholar 

  • Ouyang JW, He HG, Feng GH, Jia SE (1987) The response of anther culture to culture temperature varies with growth conditions of anther-donor plants. Plant Sci 49:145–148

    Article  Google Scholar 

  • Patel M, Darvey NL, Marshall DR, Berry JO (2004) Optimization of culture conditions for improved plant regeneration efficiency from wheat microspore culture. Euphytica 140:197–204

    Article  CAS  Google Scholar 

  • Puolimatka M, Laine S, Pauk J (1996) Effect of ovary co-cultivation and culture medium on embryogenesis of directly isolated microspores of wheat. Cereal Res Commun 24:393–400

    Google Scholar 

  • SAS Institute (2000) The SAS system for windows, version 8.2. SAS Institute, Cary

    Google Scholar 

  • Silva-Romano MC, Canhoto JM (1999) Resposta de genótipos de Triticum aestivum e de Triticum durum a cultura in vitro de anteras. Melhoramento 36:5–14

    Google Scholar 

  • Stasolla C, Yeung EC (2003) Recent advances in conifer somatic embryogenesis: improving somatic embryo quality. Plant Cell Tissue Organ Cult 74:15–35

    Article  CAS  Google Scholar 

  • Stasolla C, Belmonte MF, van Zyl L, Craig DL, Liu W, Yeung EC, Sederoff RR (2004) The effect of reduced glutathione on morphology and gene expression of white spruce (Picea glauca) somatic embryos. J Exp Bot 55(397):695–709

    Article  PubMed  CAS  Google Scholar 

  • Supena EDJ, Winarto B, Riksen T, Dubas E, van Lammeren A, Offringa R, Boutilier K, Custers J (2008) Regeneration of zygotic-like microspore-derived embryos suggests an important role for the suspensor in early embryo patterning. J Exp Bot 59(4):803–814

    Article  PubMed  CAS  Google Scholar 

  • 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 57:480–483

    Article  CAS  Google Scholar 

  • Wang P, Chen YR (1986) A study on the application of C17 medium for anther culture. Acta Bot Sin 28:38–45

    CAS  Google Scholar 

  • Yeung EC (2002) The rapeseed microspore-derived embryo as a model system to study developmental processes in plants. J Plant Biol 45(3):119–133

    Article  Google Scholar 

  • 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 20:802–807

    Article  CAS  Google Scholar 

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Acknowledgments

The authors are grateful to Prof. Patrick Hayes for critical reading of the manuscript. The research was supported by Projects AGL2005-07195-C02 and AGL2008-05541-C02 from the Spanish Ministry of Science and Technology. We also want to thank Batlle Seeds Company S.A. and Agromonegros Seeds Company S.A. for their support of this research.

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Authors and Affiliations

  1. Departamento de Genética y Producción Vegetal, Estación Experimental de Aula Dei, Consejo Superior de Investigaciones Científicas, Avda. Montañana 1005, 50059, Zaragoza, Spain

    Luis Cistué & B. Echávarri

  2. Centre UdL-IRTA, Universitat de Lleida, Avda. Alcalde Rovira Roure 191, 25198, Lleida, Spain

    I. Romagosa

  3. Semillas Batlle S.A., Carretera Nacional II s/n, Bell-Lloc d’Urgel, 25220, Lleida, Spain

    F. Batlle

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  1. Luis Cistué
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  2. I. Romagosa
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  3. F. Batlle
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  4. B. Echávarri
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Correspondence to Luis Cistué.

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Communicated by P. Ozias-Akins.

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Cistué, L., Romagosa, I., Batlle, F. et al. Improvements in the production of doubled haploids in durum wheat (Triticum turgidum L.) through isolated microspore culture. Plant Cell Rep 28, 727–735 (2009). https://doi.org/10.1007/s00299-009-0690-6

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