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High-efficiency transformation by biolistics of soybean, common bean and cotton transgenic plants

Nature Protocols volume 3, pages 410–418 (2008)Cite this article

Abstract

This protocol describes a method for high-frequency recovery of transgenic soybean, bean and cotton plants, by combining resistance to the herbicide imazapyr as a selectable marker, multiple shoot induction from embryonic axes of mature seeds and biolistics techniques. This protocol involves the following stages: plasmid design, preparation of soybean, common bean and cotton apical meristems for bombardment, microparticle-coated DNA bombardment of apical meristems and in vitro culture and selection of transgenic plants. The average frequencies (the total number of fertile transgenic plants divided by the total number of bombarded embryonic axes) of producing germline transgenic soybean and bean and cotton plants using this protocol are 9, 2.7 and 0.55%, respectively. This protocol is suitable for studies of gene function as well as the production of transgenic cultivars carrying different traits for breeding programs. This protocol can be completed in 7–10 months.

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Figure 1: Positioning of embryonic axes.
Figure 2
Figure 3: Diagram of the high-pressure helium microparticle acceleration system.
Figure 4: Scanning electron micrographs showing the morphology of the vegetative apical meristematic region (m) after the removal of the embryonic axes' primary leaves (p) of common bean, soybean and cotton.
Figure 5: Stages in the biolistic-mediated transformation of common bean, soybean and cotton embryonic axes.

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References

  1. McElroy, D. Valuing the product development cycle in agricultural biotechnology—what's in a name. Nat. Biotechnol. 22, 817–822 (2004).

    Article  CAS  Google Scholar 

  2. Hinchee, M.A.W. et al. Production of transgenic soybean plants using Agrobacterium-mediated DNA transfer. Biotechnology 6, 915–922 (1988).

    CAS  Google Scholar 

  3. McCabe, D.E., Swain, W.F., Martinell, B.J. & Christou, P. Stable transformation of soybean (Glycine max) by particle acceleration. Biotechnology 6, 923–926 (1988).

    Google Scholar 

  4. Christou, P., Swain, W.F., Yang, N.S. & McCabe, D.E. Inheritance and expression of foreign genes in transgenic soybean plants. Proc. Natl. Acad. Sci. USA 86, 7500–7504 (1989).

    Article  CAS  Google Scholar 

  5. Torisky, R.S. et al. Development of a binary vector system for plant transformation based on the supervirulent Agrobacterium tumefaciens strain Chry5. Plant Cell Rep. 17, 102–108 (1997).

    Article  CAS  Google Scholar 

  6. Meurer, C.A., Dinkins, R.D. & Collins, G.B. Factors affecting soybean cotyledonary node transformation. Plant Cell Rep. 18, 180–186 (1998).

    Article  CAS  Google Scholar 

  7. Maughan, P.J., Philip, R., Cho, M.J., Widholm, J.M. & Vodkin, L.O. Biolistic transformation, expression and inheritance of bovine b-casein in soybean (Glycine max). In Vitro Cell. Dev. Biol. 35, 334–349 (1999).

    Article  Google Scholar 

  8. Santarem, E.R. & Finer, J.J. Transformation of soybean (Glycine max (L.) Merrill) using proliferative embryogenic tissue maintained on semi-solid Medium. In Vitro Cell. Dev. Biol. Plant 35, 451–455 (1999).

    Article  Google Scholar 

  9. Zhang, Z.Y., Xiang, A.Q. & Staswick, Q. The use of glufosinate as a selective agent in Agrobacterium-mediated transformation of soybean. Plant Cell Tiss. Organ Cult. 56, 37–46 (1999).

    Article  CAS  Google Scholar 

  10. Olhoft, P.M., Flagel, L.E., Donovan, C.M. & Somers, D.A. Efficient soybean transformation using hygromycin B selection in the cotyledonary-node method. Planta 216, 723–735 (2003).

    CAS  PubMed  Google Scholar 

  11. Liu, H.K., Yang, C. & Wei, Z.M. Efficient Agrobacterium tumefaciens mediated transformation of soybeans using an embryonic tip regeneration system. Planta 219, 1042–1049 (2004).

    Article  CAS  Google Scholar 

  12. Paz, M.M., Martinez, J.C., Kalvig, A.B., Fonger, T.M. & Wang, K. Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation. Plant Cell Rep. 25, 206–213 (2005).

    Article  Google Scholar 

  13. Behrens, M.R. et al. Dicamba resistance: enlarging and preserving biotechnology-based weed management strategies. Science 316, 1185–1188 (2007).

    Article  CAS  Google Scholar 

  14. Dang, W. & Wei, Z. An optimized Agrobacterium-mediated transformation for soybean for expression of binary insect resistance genes. Plant Sci. 173, 381–389 (2007).

    Article  CAS  Google Scholar 

  15. Aragão, F.J.L. et al. Inheritance of foreign genes in transgenic bean (Phaseolus vulgaris L.) co-transformed via particle bombardment. Theor. Appl. Genet. 93, 142–150 (1996).

    Article  Google Scholar 

  16. Aragão, F.J.L. & Rech, E.L. Morphological factors influencing recovery of transgenic bean plants (Phaseolus vulgaris L.) of a Carioca cultivar. Int. J. Plant Sci. 158, 157–163 (1997).

    Article  Google Scholar 

  17. Aragão, F.J.L., Vianna, G.R., Albino, M.M.C. & Rech, E.L. Transgenic dry bean tolerant to the herbicide glufosinate ammonium. Crop. Sci. 42, 1298–1302 (2002).

    Article  Google Scholar 

  18. Vianna, G.R. et al. Fragment DNA as vector for genetic transformation of bean (Phaseolus vulgaris L.). Sci. Hortic. 99, 371–378 (2004).

    Article  CAS  Google Scholar 

  19. Liu, Z., Park, B.J., Kanno, A. & Kameya, T. The novel use of a combination of sonication and vacuum infiltration in Agrobacterium-mediated transformation of kidney bean (Phaseolus vulgaris L.) with lea gene. Mol. Breed. 16, 189–197 (2005).

    Article  CAS  Google Scholar 

  20. Umbeck, P., Johnson, G., Barton, K. & Swain, W. Genetically transformed cotton (Gossypium hirsutum L.) plants. Biotechnology 5, 263–266 (1987).

    Article  CAS  Google Scholar 

  21. Keller, G. et al. Transgenic cotton resistant to herbicide bialaphos. Transgenic Res. 6, 385–392 (1997).

    Article  CAS  Google Scholar 

  22. Bayley, C. et al. Engineering 2,4-D resistance into cotton. Theor. Appl. Genet. 83, 645–649 (1992).

    Article  CAS  Google Scholar 

  23. Thomas, J.C. et al. Protease inhibitors of Manduca sexta expressed in transgenic cotton. Plant Cell Rep. 14, 758–762 (1995).

    Article  CAS  Google Scholar 

  24. Agrawal, D.C. et al. In vitro induction of multiple shoots and plant regeneration in cotton (Gossypium hirsutum L.). Plant Cell Rep. 16, 647–652 (1997).

    Article  CAS  Google Scholar 

  25. Sunilkumar, G. & Rathore, K.S. Transgenic cotton: factors influencing Agrobacterium-mediated transformation and regeneration. Mol. Breed. 8, 37–52 (2001).

    Article  CAS  Google Scholar 

  26. Chaudhary, B. et al. Slow desiccation leads to high-frequency shoot recovery from transformed somatic embryos of cotton (Gossypium hirsutum L. cv. Coker 310 FR). Plant Cell Rep. 21, 955–960 (2003).

    Article  CAS  Google Scholar 

  27. Leelavathi, S. et al. A simple and rapid Agrobacterium-mediated transformation protocol for cotton (Gossypium hirsutum L.): embryogenic calli as a source to generate large numbers of transgenic plants. Plant Cell Rep. 22, 465–470 (2004).

    Article  CAS  Google Scholar 

  28. Sakhanokho, H.F. & Chee, P.W. The current status of gene transformation in cotton, SAAS Bull. Biochem. Biotech. 15, 34–46 (2002).

    Google Scholar 

  29. Rajasekaran, K., Hudspeth, R.L., Cary, J.W., Anderson, D.M. & Cleveland, T.E. High-frequency stable transformation of cotton (Gossypium hirsutum L.) by particle bombardment of embryogenic cell suspension cultures. Plant Cell Rep. 19, 539–545 (2000).

    Article  CAS  Google Scholar 

  30. McCabe, D.E. & Martinell, B.J. Transformation of elite cotton cultivars via particle bombardment of meristems. Biotechnology 11, 596–598 (1993).

    Google Scholar 

  31. Aragão, F.J.L., Sarokin, L., Vianna, G.R. & Rech, E.L. Selection of transgenic meristematic cells utilizing a herbicidal molecule results in the recovery of fertile transgenic soybean (Glycine max (L.) Merril) plants at a high frequency. Theor. Appl. Genet. 101, 1–6 (2000).

    Article  Google Scholar 

  32. Bonfim, K. et al. RNAi-mediated resistance to Bean golden mosaic virus in genetically engineered common bean (Phaseolus vulgaris). Mol. Plant Microbe Interact. 20, 717–726 (2007).

    Article  CAS  Google Scholar 

  33. Aragão, F.J.L., Vianna, G.R., Carvalheira, S.B.R.C. & Rech,, E.L. Germ line genetic transformation in cotton (Gossypium hirsutum L.) by selection of transgenic meristematic cells with a herbicide molecule. Plant Sci. 168, 1227–1233 (2005).

    Article  Google Scholar 

  34. Veltcheva, M., Svetleva, D., Petkova, S. & Perl, A. In vitro regeneration and genetic transformation of common bean (Phaseolus vulgaris L.)-problems and progress. Sci. Hortic. 107, 2–10 (2005).

    Article  CAS  Google Scholar 

  35. Finer, J.J. & MacMullen, M.D. Transformation of cotton (Gossypium hirsutum L.) via particle bombardment. Plant Cell Rep. 8, 586–589 (1990).

    Article  CAS  Google Scholar 

  36. Nunes, A.C. et al. RNAi-mediated silencing of the myo-inositol-1-phosphate synthase gene (GmMIPS1) in transgenic soybean inhibited seed development and reduced phytate content. Planta 224, 125–132 (2006).

    Article  CAS  Google Scholar 

  37. Sanford, J. et al. An improved, helium-driven biolistic device. Technique 1, 3–16 (1991).

    Google Scholar 

  38. Shaner, D.L., Anderson, P.C. & Stidham, M.A. Imidazolinones: potent inhibitors of acetohydroxyacid synthase. Plant Physiol. 76, 534–546 (1984).

    Article  Google Scholar 

  39. Sathasivan, K., Haughn, G.W. & Murai, N. Nucleotide sequence of a mutant acetolactate synthase gene from an imidazolinone resistant Arabidopsis thaliana var. Columbia. Nucleic Acids Res. 18, 2888 (1990).

    Article  Google Scholar 

  40. Sambrook, J. & Russell, D.W. Commonly used techniques in molecular cloning. In Molecular Cloning: A Laboratory Manual 3rd edn. Vol. 3 (eds. Sambrook, J. & Russell, D.W.) Appendix 8-A8.20 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).

  41. Aragão, F.J.L., Grossi-de-Sá, M.F., Almeida, E.R., Gander, E.S. & Rech, E.L. Particle bombardment mediated expression of a Brazil nut methionine-rich albumin in bean (Phaseolus vulgaris L.). Plant Mol. Biol. 20, 357–359 (1992).

    Article  Google Scholar 

  42. Trick, H.N. & Finer, J.J. Sonication-assisted Agrobacterium mediated transformation of soybean (Glycine max (L.) Merril) embryogenic suspension culture tissue. Plant Cell Rep. 17, 482–488 (1998).

    Article  CAS  Google Scholar 

  43. Olhoft, P.M. & Somers, D.A. l-Cysteine increases Agrobacterium-mediated T-DNA delivery into soybean cotyledonary-node cells. Plant Cell Rep. 20, 706–711 (2001).

    Article  CAS  Google Scholar 

  44. Hoekema, A., Hirsch, P.R., Hooykaas, P.J.J. & Schilperoort, R.A. A binary plant vector strategy based on separation of the vir and T-region of the Agrobacterium tumefaciens Ti plasmid. Nature 303, 179–180 (1983).

    Article  CAS  Google Scholar 

  45. Birch, R.G. Plant transformation: problems and strategies for practical application. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 297–326 (1997).

    Article  CAS  Google Scholar 

  46. Altpeter, F. et al. Particle bombardment and the genetic enhancement of crops: myths and realities. Mol. Breed. 15, 305–327 (2005).

    Article  Google Scholar 

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Acknowledgements

The authors are grateful to Ana C.M.M. Gomes for scanning electron microscopy assistance and to Ivete M. Bernardes for diagram design. This study was supported by BASF and Empresa Brasileira de Pesquisa Agropecuária—EMBRAPA.

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Authors and Affiliations

  1. Embrapa Genetic Resources and Biotechnology, Laboratory of Gene Transfer, Biotechnology Unit, Parque Estação Biológica PqEB W5 Norte, Brasília, 70770-900, DF, Brazil

    Elibio L Rech, Giovanni R Vianna & Francisco J L Aragão

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  2. Giovanni R Vianna
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  3. Francisco J L Aragão
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Correspondence to Elibio L Rech or Francisco J L Aragão.

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Rech, E., Vianna, G. & Aragão, F. High-efficiency transformation by biolistics of soybean, common bean and cotton transgenic plants. Nat Protoc 3, 410–418 (2008). https://doi.org/10.1038/nprot.2008.9

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