Skip to main content
SpringerLink
Log in

Transformation of Oats and Its Application to Improving Osmotic Stress Tolerance

  • Protocol
  • First Online:
Transgenic Wheat, Barley and Oats

Part of the book series: Methods in Molecular Biologyâ„¢ ((MIMB,volume 478))

Abstract

Oat (Avena sativa L.), a worldwide temperate cereal crop, is deficient in tolerance to osmotic stress due to drought and/or salinity. To genetically transform the available commercial oat cultivars, a genotype-independent and efficient regeneration system from shoot apical meristems was developed using four oat cultivars: Prairie, Porter, Ogle, and Pacer. All these oat cultivars generated a genotype-independent in vitro differentiated multiple shoots from shoot apical meristems at a high frequency. Using this system, three oat cultivars were genetically co-transformed with pBY520 (containing hva1 and bar) and pAct1-D (containing gus) using biolisticâ„¢ bombardment. Transgenic plants were selected and regenerated using herbicide resistance and GUS as a marker. Molecular and biochemical analyses of putative transgenic plants confirmed the co-integration of hva1 and bar genes with a frequency of 100%, and 61.6% of the transgenic plants carried all three genes (hva1, bar and gus). Further analyses of R0, R1, and R2 progenies confirmed stable integration, expression, and Mendalian inheritance for all transgenes. Histochemical analysis of GUS protein in transgenic plants showed a high level of GUS expression in vascular tissues and in the pollen grains of mature flowers. Immunochemical analysis of transgenic plants indicated a constitutive expression of hva1 at all developmental stages. However, the level of HVA1 was higher during the early seedling stages.

The characteristic of HVA1 expression for osmotic tolerance in transgenic oat progeny was analyzed in vitro as well as in vivo. Transgenic plants exhibited significantly (P <0.05) increased tolerance to stress conditions than non-transgenic control plants. The symptoms of wilting or death of leaves as observed in 80% of non-transgenic plants due to osmotic stress was delayed and detected only in less than 10% of transgenic plants. These observations confirmed the characteristic of HVA1 protein as providing or enhancing the osmotic tolerance in transgenic plants against salinity and possible water-deficiency stress conditions.

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 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
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. Hitchcock, A. S. (1971) Manual of the grasses of the United States. Originally published in 1950 as U.S.D.A. Miscellaneous publication No. 200. Dover, Second edition revised by Chase, A.New York.

    Google Scholar 

  2. Munz, P. A. and Keck, D. D. (1973) A California Flora (with Supplement by P. A. Munz). University of California Press, Berkeley, California.

    Google Scholar 

  3. Madson, B. A. (1951) Winter Covercrops. Circular 174, California Agricultural Extension Service, College of Agriculture, University of California, June 1951.

    Google Scholar 

  4. Johnny’s selected seeds (1983) Green Manures – A Mini Manual. Johnny’s selected seeds, Albion, Maine 04910.

    Google Scholar 

  5. Verhallen, A., Hayes, A. and Taylor, T. (2003) Cover crops – Oats, Ministry of Agriculture, Food and Rural Affairs, Ontario, Canada.

    Google Scholar 

  6. Gibson, L. and Benson, G. (2002) Origin, history, and uses of oat (Avena sativa) and wheat (Triticum aestivum).Course Agronomy 212, Iowa State University, Department of Agronomy, Iowa.

    Google Scholar 

  7. http://interactive.usask.ca/Ski/agriculture/crops/cereals/oats.html. Agriculture crops cereals oats. Saskatchewan Interactive, last updated 14 Dec 2002.

    Google Scholar 

  8. Forsberg, R. A. and Shands, H. L. (1989) ( Janick, J., Oat breeding in Plant Breeding Reviews. Vol. 6,Timber Press, Portland, OR, pp167–207.

    Google Scholar 

  9. Boyer, J. S. (1982) Plant productivity and environment. Science 218,444–448.

    Article  Google Scholar 

  10. Roy, M. Wu, R. (2002) Overexpression of S-adenosylmethionine decarboxylase gene in rice increases polyamine level and enhances sodium chloride-stress tolerance. Plant Science 163,987–992.

    Article  CAS  Google Scholar 

  11. Apse, M. P., Aharon, G. S., Snedden, W. A. and Blumwald, E. (1999) Salt tolerance conferred by over expression of a vacuolar Na+/H+ antiport in Arabidopsis Science 285,1256–1258.

    Article  CAS  Google Scholar 

  12. Schachtman, D. and Lui, W. (1999) Molecular pieces to the puzzle of the interaction between potassium and sodium uptake in plants. Trends in Plant Science 4,281–287.

    Article  Google Scholar 

  13. Qadir, M., Qureshi, R. H. and Ahmad, N. (1998) Horizontal flushing: a promising ameliorative technology for hard saline-sodic and sodic soils. Soil Tillage Research 45,119–131.

    Article  Google Scholar 

  14. Cherry, J. H., Locy, R. D. and Rychter, A. (1999) Proc. NATO Adv. Res. Workshop, Mragowa, Poland. 13–19 June. Kluwer, Amsterdam, The Netherlands.

    Google Scholar 

  15. Martin, R. J., Jamieson, P. D., Gillespie, R. N. and Maley, S. (2001) Effect of timing and intensity of drought on the yield of oats (Avena sativa L.). Proceeding of the 10th Australian Agronomy Conference, Hobart.

    Google Scholar 

  16. Murty, A. S., Misra, P. N. and Haider, M. M. (1984) Effect of different salt concentrations on seed germination and seedling development in few oat cultivars. Indian Journal of Agricultural Research 18,129–132.

    Google Scholar 

  17. Verma, O. P. S. and Yadava, R. B. R. (1986) Salt tolerance of some oats (Avena sativa L.) varieties at germination and seedling stage. Journal of Agronomy and Crop Science 156,123–127.

    Article  Google Scholar 

  18. Schonbeck, M. W. (1988) Cover Cropping and Green Manuring on Small Farms in New England and New York: An Informal Survey. Research Report 10, New Alchemy Institute, East Falmouth, MA 02536.

    Google Scholar 

  19. Koev, G., Mohan, B. R., Dinesh-Kumar, S. P., Torbert, K. A., Somers, D. A. and Miller, W. A. (1998) Extreme reduction of disease in oats transformed with the 5’ half of the barley yellow dwarf virus PAV genome. Phytopathology 88,1013–1019.

    Article  CAS  Google Scholar 

  20. Stoskopf, N. C. (1985 ( Stoskopf, N. C., ed.), ) Barley and Oat, in Cereal Grain Crops,Reston Publishing, Reston, pp. , Virginia444–458.

    Google Scholar 

  21. Cushman, J. C. and Bohnert, H. J. (2000) Genomic approach to plant stress tolerance. Current Opinions in Plant Biology 3,117–124.

    Article  CAS  Google Scholar 

  22. Abebe, T., Guenzi, A. C., Martin, B. and Cushman, J. C. (2003) Tolerance of mannitol-accumulating transgenic wheat to water stress and salinity. Plant Physiology 131,1748–1755.

    Article  CAS  Google Scholar 

  23. Epstein, E., Norlyn, J., Rush, D., Kingsbury, R., Kelley, D., Cunningham, G. and Wrona, A. (1980) Saline culture of crops: a genetic approach. Science 210,399–404.

    Article  CAS  Google Scholar 

  24. Ribaut, J. M. and Hoisington, D. A. (1998) Marker assisted selection: new tools and strategies. Trends in Plant Science 3,236–239.

    Article  Google Scholar 

  25. FAO. (1999). Biotechnology in food and agriculture. http://www.fao.org/unfao/bodies/COAG/COAG15/X0074E.htm.

    Google Scholar 

  26. Sharma, H. C., Crouch, J. H., Sharma, K. K., Seetharama, N. and Hash, C. T. (2002) Application of biotechnology for crop improvement: prospects and constraints. Plant Science 163,381–395.

    Article  CAS  Google Scholar 

  27. Mazur, B., Krebbers, E. and Tingey, S. (1999) Gene discovery and product development for grain quality traits. Science 285,372–375.

    Article  CAS  Google Scholar 

  28. Rines, H. W., Phillips, R. L. and Somers, D. A. (1992) Marshall, H. G., and Sorrels, M. E., eds.), Application of tissue culture to oat improvement, in Oat Science and Technology, (American Society of Agronomy and Crop Science Society, Madison WI, pp. 777–791.

    Google Scholar 

  29. Somers, D. A., Torbert, K. A., Pawlowski, W. P. and Rines, H. W. (1994) Henry, R. J. and Ronalds, J. A., eds.), Genetic engineering of oat, in Improvement of Cereal Quality by Genetic Engineering, (Plenum, New York, pp. 37–46.

    Google Scholar 

  30. Cummings, D. P., Green, C. E. and Stuthman, D. D. (1976) Callus induction and plant regeneration in oats. Crop Science 16,465–470.

    Article  Google Scholar 

  31. Rines, H. W. and McCoy, T. J. (1981) Tissue culture initiation and plant regeneration in hexaploid species of oats. Crop Science 21,837–842.

    Article  Google Scholar 

  32. Bregitzer, P., Bushnell, W. R., Somers, D. A. and Rines, H. W. (1989) Development and characterization of friable, embryogenic oat callus. Crop Science 29,798–803.

    Article  Google Scholar 

  33. Rines, H. W. and Luke, H. H. (1985) Selection and regeneration of toxin insensitive plants from tissue cultures of oat (Avena sativa) susceptible to Helminthosporium victoriae. Theoretical and Applied Genetics 71,16–21.

    Article  Google Scholar 

  34. Somers, D. A., Rines, H. W., Gu, W., Kaeppler, H. F. and Bush-Nell, W. R. (1992) Fertile transgenic oat plants. Bio/Technology 10,1589–1594.

    Article  CAS  Google Scholar 

  35. Zhang, S., Zhong, H. and Sticklen, M. B. (1996) Production of multiple shoots from apical meristems of oat (Avena sativa L.). Journal of Plant Physiology 148,667–671.

    CAS  Google Scholar 

  36. Torbert, K. A., Rines, H. W. and Somers, D. A. (1998) Transformation of oat using mature embryo-derived tissue cultures. Crop Science 38,226–231.

    Article  Google Scholar 

  37. Cho, M. J., Jiang, W. and Lemaux, P. G. (1999) High frequency transformation of oat via microprojectile bombardment of seed-derived highly regenerative cultures. Plant Science 148,9–17.

    Article  CAS  Google Scholar 

  38. Gless, C., Lorz, H. and Jahne-Gartner, A. (1998) Establishment of a highly efficient regeneration system from leaf base segments of oat (Avena sativa L.). Plant Cell Reports 17,441–445.

    Article  CAS  Google Scholar 

  39. Kaeppler, H. F., Menon, G. K., Skadsen, R. W., Nuutila, A. M. and Carlson, A. R. (2000) Transgenic oat plants via visual selection of cells expressing green fluorescent protein. Plant Cell Reports 19,661–666.

    Article  CAS  Google Scholar 

  40. Somers, D. A. (1999) Vasil, I. and PhillipesR., eds.), Genetic engineering of oat, in Molecular Improvement of Cereal Crops, (Kluwer, Dordrecht, The Netherlands.

    Google Scholar 

  41. Choi, H. W., Lemaux, P. G. and Cho, M. J. (2001) High frequency of cytogenetic aberration in transgenic oat (Avena sativa L.) plants. Plant Science 160,761–762.

    Article  CAS  Google Scholar 

  42. Zhang, S., Cho, M. J., Koprek, T., Yun, R., Bregitzer, P. and Lemaux, P. G. (1999) Genetic transformation of commercial cultivars of oat (Avena sativa L.) and barley (Hordeum vulgare L.) using in vitro shoot meristematic cultures derived from germinated seedlings. Plant Cell Reports 18,959–966.

    Article  CAS  Google Scholar 

  43. Zhong, H., Srinivasan, C. and Sticklen, M. B. (1992) In vitro morphogenesis of corn (Zea mays L.). II. Differentiation of ear and tassel clusters from cultured shoot apices and immature inflorescences. Planta 187,483–489.

    CAS  Google Scholar 

  44. Zhong, H., Wang, W. and Sticklen, M. B. (1998) In vitro morphogenesis of Sorghum bicolor (L.) Moench: efficient plant regeneration from shoot apices. Journal of Plant Physiology 153,719–726.

    CAS  Google Scholar 

  45. Devi, P., Zhong, H. and Sticklen, M. B. (2000) In vitro morphogenesis of pearl millet (Pennisetum glaucum (L.) R. Br.): efficient production of multiple shoots and inflorescences from shoot apices. Plant Cell Reports 19,546–550.

    CAS  Google Scholar 

  46. Ahmad, A., Zhong, H., Wang, W. and Sticklen, M. B. (2001) Shoot apical meristem: In vitro plant regeneration and morphogenesis in wheat (Triticum aestivum L.). In Vitro Cellular and Developmen. Biology-Plant 38,163–167.

    Article  Google Scholar 

  47. Maqbool, S. B., Zhong, H., El-Maghraby, Y., Ahmad, A., Chai, B., Wang, W., Sabzikar, R. and Sticklen, M. B. (2002) Competence of oat (Avena sativa L.) shoot apical meristems on integrative transformation, inherited expression, and osmotic tolerance of transgenic lines containing the hva1. Theoretical and Applied Genetics 105,201–208.

    Article  CAS  Google Scholar 

  48. Xu, D., Duan, X., Wang, B., Hong, B., Ho, T. and Wu, R. (1996) Expression of a late embryogenesis abundant protein gene, HVA1, from barley confers tolerance to water deficit and salt stress in transgenic rice. Plant Physiology 110,249–257.

    CAS  Google Scholar 

  49. Patnaik, D. and Khurana, P. (2003) Genetic transformation of Indian bread (T. aestivum L.) and pasta (T. durum L.) wheat by particle bombardment of mature embryo-derived calli. BMC Plant Biology 3,1–11.

    Google Scholar 

  50. Maqbool, S. B., Zhong, H. and Sticklen, M. B. (2004) Curtis, I. S., ed.), Genetic engineering of oat (Avena sativa L.) via the biolistic bombardment of shoot apical meristems, in Transgenic Crops of the World – Essential Protocols, Chap. 5, (Kluwer, Dordrecht, The Netherlands, pp. 63–78.

    Google Scholar 

  51. Oraby, H. F., Ransom, C. B., Kravchenko, A. N. and Sticklen, M. B. (2005) Barley HVA1 gene confers salt tolerance in R3 transgenic oat. Crop Science 45,2218–2227.

    Article  CAS  Google Scholar 

  52. Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiology Plant 15,473–497.

    Article  CAS  Google Scholar 

  53. Strickberger, M. W. (1985) Genetics, 3rd ed. Macmillan, New York, pp. 126–146.

    Google Scholar 

  54. Southern, E. M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98,503–517.

    Article  CAS  Google Scholar 

  55. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Lab, New York.

    Google Scholar 

  56. Bradford, M. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Annals of Biochemistry 72,248–254.

    Article  CAS  Google Scholar 

  57. Jafferson, R. A., Kavanagh, T. A. and Bevan, M. W. (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO Journal 6,3901–3907.

    Google Scholar 

  58. Lamb, C. R. C., Milach, S. C. K., Pasquali, G. and Barro, R. S. (2002) Somatic embryogenesis and plant regeneration derived from mature embryos of oat. Pesquisa Agropecuaria Brasileira 37,123–130.

    Google Scholar 

Download references

Acknowledgements

The MSU Plant Breeding and Genetics Program, the Northwest Plant Biotechnology Consortium, and Al-Azhar University, Assiut, Egypt, supported this research.

Author information

Authors and Affiliations

  1. Department of Biology, Syracuse University, 130 College Place, Syracuse, NY, 13244, USA

    Shahina B. Maqbool*

  2. SABRI, Research Triangle Park, NC, USA

    Heng Zhong

  3. Department of Horticulture, Michigan State University, East Lansing, MI, USA

    Hesham F. Oraby

  4. Department of Crop and Soil Sciences, Michigan State University, East Lansing, MI, USA

    Mariam B. Sticklen

Authors
  1. Shahina B. Maqbool*
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Heng Zhong
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hesham F. Oraby
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mariam B. Sticklen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mariam B. Sticklen .

Editor information

Editors and Affiliations

    Rights and permissions

    Reprints and permissions

    Copyright information

    © 2009 Humana Press, a part of Springer Science+Business Media, LLC

    About this protocol

    Cite this protocol

    Maqbool*, S., Zhong, H., Oraby, H., Sticklen, M. (2009). Transformation of Oats and Its Application to Improving Osmotic Stress Tolerance. In: Jones, H., Shewry, P. (eds) Transgenic Wheat, Barley and Oats. Methods in Molecular Biologyâ„¢, vol 478. Humana Press. https://doi.org/10.1007/978-1-59745-379-0_10

    Download citation

    • DOI: https://doi.org/10.1007/978-1-59745-379-0_10

    • Published:

    • Publisher Name: Humana Press

    • Print ISBN: 978-1-58829-961-1

    • Online ISBN: 978-1-59745-379-0

    • eBook Packages: Springer Protocols

    Publish with us

    Policies and ethics

    Search

    Navigation