Skip to main content
SpringerLink
Log in

Doubled Haploid Transgenic Wheat Lines by Microspore Transformation

  • Protocol
  • First Online:
Wheat Biotechnology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1679))

Abstract

Microspores are preferred explant choice for genetic transformation, as their use shortens the duration of obtaining homozygous transformants. All established gene-delivery methods of particle bombardment, electroporation, and cocultivation with Agrobacterium tumefaciens were optimized on androgenic microspores or derived tissues. In the biolistic gene delivery method 35–40 days old haploid microspore embryoids were used for genetic transformation, whereas freshly isolated androgenic microspores were used for genetic transformation in the electroporation and Agrobacterium cocultivation-based methods. The genetic transformation methods of biolistic gene-delivery and electroporation gave rise to the chimeric plants, whereas the method involving cocultivation with Agrobacterium yielded homozygous transformants. These methods were tested on a large number of cultivars belonging to different market classes of wheat, and found to be fairly independent of the explant genotype. Other benefits of using microspores or derived tissues for transformation are: (1) a few explant donors are required to obtain desired transformants and (2) the time required for obtaining homozygous transformants is about 8 months in case of spring wheat genotypes and about a year in case of winter wheat genotypes.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Ortiz R, Sayre KD, Govaerts B, Gupta R, Subbarao GV, Ban T, Hodson D, Dixon JM, Ortiz-Monasterio JI, Reynolds M (2008) Climate change: can wheat beat the heat? Agr Ecosyst Environ 126:46–58

    Article  Google Scholar 

  2. Brenchley R, Spannagl M, Pfeifer M, Barker GL, D’Amore R, Allen AM, McKenzie N, Kramer M, Kerhornou A, Bolser D, Kay S, Waite D, Trick M, Bancroft I, Gu Y, Huo N, Luo MC, Sehgal S, Gill B, Kianian S, Anderson O, Kersey P, Dvorak J, McCombie WR, Hall A, Mayer KF, Edwards KJ, Bevan MW, Hall N (2012) Analysis of the bread wheat genome using whole-genome shotgun sequencing. Nature 491:705–710

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Piironen V, Lampi A-M, Ekholm P, Salmenkallio-Marttila M, Liukkonen K-H (2009) Micronutrients and phytochemicals in wheat grain. In: Khan K, Shewry PR (eds) Wheat: chemistry and technology, 4th edn. AACC International Inc., St Paul, MN, pp 179–222

    Chapter  Google Scholar 

  4. Shewry PR (2013) Improving grain quality: wheat. In: Becraft PW (ed) Seed genomics. Wiley-Blackwell, Oxford, pp 159–178

    Chapter  Google Scholar 

  5. Moore J, Hao J (2012) Antioxidant and health promoting properties of wheat (Triticum spp.) In: Yu L, Tsao R, Shahidi F (eds) Cereals and pulses: nutraceutical properties and health benefits. Wiley-Blackwell, Oxford, pp 113–130

    Chapter  Google Scholar 

  6. Andersson AAM, Dimberg L, Åman P, Landberg R (2014) Recent findings on certain bioactive components in whole grain wheat and rye. J Cereal Sci 59:294–311

    Article  CAS  Google Scholar 

  7. Tatham AS, Shewry PR (2008) Allergens to wheat and related cereals. Clin Exp Allergy 38:1712–1726

    CAS  PubMed  Google Scholar 

  8. Sapone A, Bai JC, Ciacci C, Dolinsek J, Green PH, Hadjivassiliou M, Kaukinen K, Rostami K, Sanders DS, Schumann M, Ullrich R, Villalta D, Volta U, Catassi C, Fasano A (2012) Spectrum of gluten-related disorders: consensus on new nomenclature and classification. BMC Med 10:13

    Article  PubMed  PubMed Central  Google Scholar 

  9. Rustgi S (2013) Engineering wheat genotypes compatible for gluten sensitive, allergenic and intolerant individuals. Int J Plant Biol Res 1:1003

    Google Scholar 

  10. Rosella CM, Barro F, Sousa C, Mena MC (2014) Cereals for developing gluten-free products and analytical tools for gluten detection. J Cereal Sci 49:354–364

    Article  Google Scholar 

  11. Bai JC, Fried M, Corazza GR, Schuppan D, Farthing M, Catassi C, Greco L, Cohen H, Ciacci C, Fasano A, González A, Krabshuis JH, LeMair A (2012) Celiac disease. World Gastroenterology Organisation Global Guidelines. http://www.nutritotal.com.br/diretrizes/files/266--2012_Celiac%20Disease_long_FINAL.pdf

  12. Koehler P, Wieser H (2013) Chemistry of cereal grains. In: Gobbetti M, Gänzle M (eds) Handbook on sourdough biotechnology, vol 11. Springer, New York, NY, pp 11–45

    Chapter  Google Scholar 

  13. Vasil V, Castillo AM, Fromm ME, Vasil IK (1992) Herbicide resistant fertile transgenic wheat plants obtained by microprojectile bombardment of regenerable embryogenic callus. BioTechnology 10:667–674

    CAS  Google Scholar 

  14. Vasil V, Srivastava V, Castillo AM, Fromm ME, Vasil IK (1993) Rapid production of transgenic wheat plants by direct bombardment of cultured immature embryos. BioTechnology 11:1553–1559

    Google Scholar 

  15. Cheng M, Fry JE, Pang S, Zhou H, Hironaka CM, Dunkan DR, Conner TW, Wan Y (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol 115:971–980

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Zhou H, Berg JD, Blank SE, Chay CA, Chen G, Eskelsen SR, Fry JE, Hoi S, Hu T, Isakson PJ, Lawton MB, Metz SG, Rempel CB, Ryerson DK, Sansone AP, Shook AL, Starke RJ, Tichota JM, Valenti SA (2003) Field efficacy assessment of transgenic roundup ready wheat. Crop Sci 43:1072–1075

    Article  CAS  Google Scholar 

  17. Pellegrineshi A, Noguera LM, Skovmand B, Brito RM, Velazquez L, Salgado MM, Hernandez R, Warburton M, Hoisington D (2002) Identification of highly transformable wheat genotypes for mass production of fertile transgenic plants. Genome 45:421–430

    Article  Google Scholar 

  18. Resch T, Touraev A (2011) Pollen transformation technology. In: Stewart NC, Touraev A, Citovsky V, Tzfira I (eds) Plant transformation technologies. Wiley-Blackwell, Chichester, pp 83–91

    Chapter  Google Scholar 

  19. Mentewab A, Letellier V, Marque C, Sarrafi A (1999) Use of anthocyanin biosynthesis stimulatory genes as markers for the genetic transformation of haploid embryos and isolated microspores in wheat. Cereal Res Commun 27:17–24

    CAS  Google Scholar 

  20. Folling L, Olesen A (2001) Transformation of wheat (Triticum aestivum L.) microspore-derived callus and microspores by particle bombardment. Plant Cell Rep 20:629–636

    Article  CAS  Google Scholar 

  21. Kumar M (2004) Genetic modification of wheat (Triticum aestivum L.) straw for potential use as a biofuel. M.Sc. thesis, University of Idaho, Moscow, ID

    Google Scholar 

  22. Chauhan H, Khurana P (2011) Use of doubled haploid technology for development of stable drought tolerant bread wheat (Triticum aestivum L.) transgenics. Plant Biotechnol J 9:408–417

    Article  CAS  PubMed  Google Scholar 

  23. Bilichak A, Luu J, Eudes F (2015) Intracellular delivery of fluorescent protein into viable wheat microspores using cationic peptides. Front Plant Sci 6:666. doi:10.3389/fpls.2015.00666

    PubMed  PubMed Central  Google Scholar 

  24. Liu W, Zheng M, Polle E, Konzak CF (2002) Highly efficient doubled-haploid production in wheat (Triticum aestivum L.) via induced microspore embryogenesis. Crop Sci 42:686–692

    Article  Google Scholar 

  25. Liu W, Zheng M, Konzak CF (2002) Improving green plant production via isolated microspore culture in bread wheat (Triticum aestivum L.) Plant Cell Rep 20:821–824

    Article  CAS  Google Scholar 

  26. Zheng MY, Weng Y, Liu W, Konzak CF (2001) The effect of ovary-conditioned medium on microspore embryogenesis in common wheat (Triticum aestivum L.) Plant Cell Rep 20:802–807

    Google Scholar 

  27. Lazo GR, Stein PA, Ludwig RA (1991) A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. BioTechnology 9:963–967

    Article  CAS  PubMed  Google Scholar 

  28. Horvath H, Huang J, Wong OT, von Wettstein D (2002) Experiences with genetic transformation of barley and characteristics of transgenic plants. In: Slafer GA, Molina-Cano JL, Savin R, Araus JL, Romagosa J (eds) Barley science. Harworth Press, New York, NY, pp 143–176

    Google Scholar 

  29. Brew-Appiah RAT, Ankrah N, Liu W, Konzak CF, von Wettstein D, Rustgi S (2013) Generation of doubled haploid transgenic wheat lines by microspore transformation. PLoS One 8:e80155

    Article  PubMed  PubMed Central  Google Scholar 

  30. Liu D (2009) Design of gene constructs for transgenic maize. In: Paul Scott M (ed) Methods in molecular biology: transgenic maize, vol 526. Humana Press, Totowa, NJ, pp 3–20

    Chapter  Google Scholar 

  31. Kumlehn J (2009) Embryogenic pollen culture: a promising target for genetic transformation. In: Touraev A, Forster BP, Jain SM (eds.) Advances in haploid production in higher plants. Springer, New York, NY, pp 295–305

    Chapter  Google Scholar 

  32. Jacquard C, Mazeyrat-Gourbeyer F, Devaux P, Boutilier K, Baillieul F et al (2009) Microspore embryogenesis in barley: anther pre-treatment stimulates plant defense gene expression. Planta 229:393–402

    Article  CAS  PubMed  Google Scholar 

  33. Maraschin SF, de Priester W, Spaink HP, Wang M (2005) Androgenic switch: an example of plant embryogenesis from the male gametophyte perspective. J Exp Bot 56:1711–1726

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This project is supported by the funding from the State of Washington grant WNP00251 and the Life Sciences Discovery Fund grant 3143956-01.

Author information

Authors and Affiliations

  1. Department of Plant and Environmental Sciences, Clemson University Pee Dee Research and Education Center, 2200 Pocket Road, Florence, SC, 29506, USA

    Sachin Rustgi

  2. Department of Crop and Soil Sciences, Washington State University, Pullman, WA, 99164, USA

    Sachin Rustgi, Nii O. Ankrah, Rhoda A. T. Brew-Appiah, Yue Sun & Diter von Wettstein

  3. School of Biological Sciences, Washington State University, Pullman, WA, 99164, USA

    Nii O. Ankrah

  4. Syngenta, Research Triangle Park, Cary, NC, 27513, USA

    Weiguo Liu

Authors
  1. Sachin Rustgi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nii O. Ankrah
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rhoda A. T. Brew-Appiah
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yue Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Weiguo Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Diter von Wettstein
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sachin Rustgi .

Editor information

Editors and Affiliations

  1. Plant Molecular Biology and Biotechnology Laboratory, School of Agriculture and Food, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria, Australia

    Prem L. Bhalla

  2. Plant Molecular Biology and Biotechnology Laboratory, School of Agriculture and Food, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria, Australia

    Mohan B. Singh

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Rustgi, S., Ankrah, N.O., Brew-Appiah, R.A.T., Sun, Y., Liu, W., von Wettstein, D. (2017). Doubled Haploid Transgenic Wheat Lines by Microspore Transformation. In: Bhalla, P., Singh, M. (eds) Wheat Biotechnology. Methods in Molecular Biology, vol 1679. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7337-8_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7337-8_13

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7335-4

  • Online ISBN: 978-1-4939-7337-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation