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Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed

Abstract

Here, we provide comprehensive, highly efficient protocols for Agrobacterium tumefaciens-mediated transformation of a wide range of rice genotypes. Methods that use either immature embryos (japonica and indica rice) or calli (japonica cultivars and the indica cultivar, Kasalath) as a starting material for inoculation with Agrobacterium are described. Immature embryos are pretreated with heat and centrifugal force, which significantly enhances the efficiency of gene transfer, and then infected with Agrobacterium. Callus is induced from mature seeds and infected. Transformed cells proliferated from these tissues are selected on the basis of hygromycin resistance, and transgenic plants are eventually regenerated. A single immature japonica or Kasalath embryo will produce between 10 and 18 independent transgenic plants; for other non-Kasalath indica varieties, the number of transgenic plants expected will be between 5 and 13. For japonica and Kasalath, transformants should be obtained from between 50 and 90% of calli. From inoculation with Agrobacterium to transplanting to soil will take 55 d for japonica and Kasalath, and 74 d for indica other than Kasalath using the immature embryo method, and 50 d for japonica and Kasalath using the callus method.

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Figure 1: Timeline of rice-transformation protocol.
Figure 2: Schematic representation of plasmids.
Figure 3: Callus induction (japonica variety Yukihikari).
Figure 4: Size of immature embryos suitable for transformation.
Figure 5: Procedure for sectioning immature embryos after co-cultivation.
Figure 6: Sectioning of an immature embryo of japonica variety Yukihikari.

References

  1. Kloeti, A. & Potrykus, I. Rice improvement by genetic transformation. In Molecular Biology of Rice (ed. Shimamoto, K.) 283–301 (Springer-Verlag, Tokyo, Japan, 1999).

    Google Scholar 

  2. Shimamoto, K., Terada, R., Izawa, T. & Fujimoto, H. Fertile transgenic rice plants regenerated from transformed protoplasts. Nature 338, 274–276 (1989).

    Article  CAS  Google Scholar 

  3. Datta, S.K., Peterhans, A., Datta, K. & Potrykus, I. Genetically engineered fertile indica-rice recovered from protoplasts. Bio/Technol. 8, 736–740 (1990).

    CAS  Google Scholar 

  4. Christou, P., Ford, T.L. & Kofron, M. Production of transgenic rice (Oryza sativa L.) plants from agronomically important indica and japonica varieties via electric discharge particle acceleration of exogenous DNA into immature zygotic embryos. Bio/Technol. 9, 957–962 (1991).

    Article  Google Scholar 

  5. Hiei, Y., Ohta, S., Komari, T. & Kumashiro, T. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 6, 271–282 (1994).

    Article  CAS  PubMed  Google Scholar 

  6. Hiei, Y., Ishida, Y., Kasaoka, K. & Komari, T. Improved frequency of transformation in rice and maize by treatment of immature embryos with centrifugation and heat prior to infection with Agrobacterium tumefaciens. Plant Cell Tissue Organ Cult. 87, 233–243 (2006).

    Article  Google Scholar 

  7. Hiei, Y. & Komari, T. Improved protocols for transformation of indica rice mediated by Agrobacterium tumefaciens. Plant Cell Tissue Organ Cult. 85, 271–283 (2006).

    Article  CAS  Google Scholar 

  8. Toki, S. et al. Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice. Plant J. 47, 969–976 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. Hervé, P. & Kayano, T. Japonica rice varieties (Oryza sativa, Nipponbare, and others). Methods Mol. Biol. 343, 213–222 (2006).

    PubMed  Google Scholar 

  10. Park, M.C. et al. High-frequency Agrobacterium-mediated genetic transformation of Tongil rice varieties. J. Plant Biol. 46, 23–30 (2003).

    Article  CAS  Google Scholar 

  11. Rachmawati, D., Hosaka, T., Inoue, E. & Anzai, H. Agrobacterium-mediated transformation of Javanica rice cv. Rojolele. Biosci. Biotechnol. Biochem. 68, 1193–1200 (2004).

    Article  CAS  PubMed  Google Scholar 

  12. Hoque, M.E., Mansfield, J.W. & Bennett, M.H. Agrobacterium-mediated transformation of Indica rice genotypes: an assessment of factors affecting the transformation efficiency. Plant Cell Tissue Organ Cult. 82, 45–55 (2005).

    Article  CAS  Google Scholar 

  13. Kumar, K.K., Maruthasalam, S., Loganathan, M., Sudhakar, D. & Balasubramanian, P. An improved Agrobacterium-mediated transformation protocol for recalcitrant elite indica rice cultivars. Plant Mol. Biol. Rep. 23, 67–73 (2005).

    Article  CAS  Google Scholar 

  14. Lin, Y.J. & Zhang, Q. Optimising the tissue culture conditions for high efficiency transformation of indica rice. Plant Cell Rep. 23, 540–547 (2005).

    Article  CAS  PubMed  Google Scholar 

  15. Datta, K. & Datta, S.K. Indica rice (Oryza sativa, BR29 and IR64). Methods Mol. Biol. 343, 201–212 (2006).

    PubMed  Google Scholar 

  16. Martinez-Trujillo, M. & Cabrera-Ponce, J.L. & Herrera-Estrella, L. Improvement of rice transformation using bombardment of scutellum-derived calli. Plant Mol. Biol. Rep. 21, 429–437 (2003).

    Article  CAS  Google Scholar 

  17. Visarada, K.B.R.S. & Sarma, N.P. Transformation of indica rice through particle bombardment: factors influencing transient expression and selection. Biol. Plant. 48, 25–31 (2004).

    Article  CAS  Google Scholar 

  18. Lucca, P., Ye, X. & Potrykus, I. Effective selection and regeneration of transgenic rice plants with mannose as selective agent. Mol. Breed. 7, 43–49 (2001).

    Article  CAS  Google Scholar 

  19. Toriyama, K., Arimoto, Y., Uchimiya, H. & Hinata, K. Transgenic rice plants after direct gene transfer into protoplasts. Bio/Technol. 6, 1072–1074 (1988).

    CAS  Google Scholar 

  20. Huang, J.Q., WeI, Z.M., An, H.L. & Zhu, Y.X. Agrobacterium tumefaciens-mediated transformation of rice with the spider insecticidal gene conferring resistance to leaffolder and striped stem borer. Cell Res. 11, 149–155 (2001).

    Article  CAS  PubMed  Google Scholar 

  21. Enríquez-Obregón, G.A. et al. Agrobacterium-mediated Japonica rice transformation: a procedure assisted by an antinecrotic treatment. Plant Cell Tissue Organ Cult. 59, 159–168 (1999).

    Article  Google Scholar 

  22. Ogawa, T., Fukuoka, H., Yano, H. & Ohkawa, Y. Relationships between nitrite reductase activity and genotype-dependent callus growth in rice cell cultures. Plant Cell Rep. 18, 576–581 (1999).

    Article  CAS  Google Scholar 

  23. Ozawa, K. & Kawahigashi, H. Positional cloning of the nitrite reductase gene associated with good growth and regeneration ability of calli and establishment of a new selection system for Agrobacterium-mediated transformation in rice (Oryza sativa L.). Plant Sci. 170, 384–393 (2006).

    Article  CAS  Google Scholar 

  24. Glaszmann, J.C. Isozymes and classification of Asian rice varieties. Theor. Appl. Genet. 74, 21–30 (1987).

    Article  CAS  PubMed  Google Scholar 

  25. Ohta, S., Mita, S., Hattori, T. & Nakamura, K. Construction and expression in tobacco of a β-glucuronidase (GUS) reporter gene containing an intron within the coding sequence. Plant Cell Physiol. 31, 805–813 (1990).

    CAS  Google Scholar 

  26. Hood, E.E., Gelvin, S.B., Melchers, L.S. & Hoekema, A. New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res. 2, 208–218 (1993).

    Article  CAS  Google Scholar 

  27. Wang, M., Li, Z.Y., Upadhyaya, N.M., Brettel, R.I.S. & Waterhouse, P.M. Intron-mediated improvement of a selectable marker gene for plant transformation using Agrobacterium tumifaciens. J. Genet. Plant Breed. 51, 325–334 (1997).

    CAS  Google Scholar 

  28. Chilton, M.D. et al. Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumors. Proc. Natl. Acad. Sci. USA 71, 3672–3676 (1974).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Chu, C.-C. The N6 medium and its application to anther culture of cereal crops. In Proceedings of the Symposium on Plant Tissue Culture 43–50 (Science Press, Peking, 1978).

    Google Scholar 

  30. Murashige, T. & Skoog, F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant 15, 473–497 (1962).

    Article  CAS  Google Scholar 

  31. Potrykus, I., Harms, C.T. & Lorz, H. Callus formation from cell culture protoplasts of corn (Zea mays L.). Theor. Appl. Genet. 54, 209–214 (1979).

    Article  CAS  PubMed  Google Scholar 

  32. Toriyama, K. & Hinata, K. Cell suspension and protoplast culture in rice. Plant Sci. 41, 179–183 (1985).

    Article  CAS  Google Scholar 

  33. Gamborg, O.L., Miller, R.A. & Ojima, K. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50, 151–158 (1968).

    Article  CAS  PubMed  Google Scholar 

  34. Hood, E.E. et al. Restriction endonuclease map of pTiBo542, a potential Ti plasmid vector for genetic engineering of plants. Bio/Technol. 2, 702–709 (1984).

    CAS  Google Scholar 

  35. Komari, T., Halperin, W. & Nester, E.W. Physical and functional map of supervirulent Agrobacterium tumefaciens tumor-inducing plasmid pTiBo542. J. Bacteriol. 166, 88–94 (1986).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank Ms. Sachiyo Higashi and Ms. Yuki Nakazaki for skillful assistance.

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Correspondence to Yukoh Hiei.

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Hiei, Y., Komari, T. Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed. Nat Protoc 3, 824–834 (2008). https://doi.org/10.1038/nprot.2008.46

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