Agrobacterium-Mediated Transformation of Sorghum bicolor Using Immature Embryos

  • Songul Gurel
  • Ekrem Gurel
  • Tamara I. Miller
  • Peggy G. Lemaux
Part of the Methods in Molecular Biology book series (MIMB, volume 847)


Successful efforts describing in vitro culturing, regeneration, and transformation of grain sorghum were reported, using particle bombardment, as early as 1993, and with Agrobacterium tumefaciens in 2000. Reported transformation efficiencies via Agrobacterium routinely range from 1 to 2%. Recently, such efficiencies via Agrobacterium in several plant species were improved with the use of heat and centrifugation treatments of explants prior to infection. Here, we describe the successful use of heat pretreatment of immature embryos (IEs) prior to Agrobacterium inoculation to increase routine transformation frequencies of a single genotype, P898012, to greater than 7%. This reproducible frequency was calculated as numbers of independently transformed IEs, confirmed by PCR, western, and DNA hybridization analysis, that produced fertile transgenic plants, divided by total numbers of infected IEs.

Key words

Agrobacterium tumefaciens GFP Heat treatment Immature embryos Phosphomannose isomerase Sorghum Transformation 



The authors would like to thank Joshua Wong for technical advice and support, Han-Qi Tan and Katrina Linden for their participation in the experimental aspects of the work, and Barbara Alonso for graphic and editing support.


  1. 1.
    Cai T, Daly, B., Butler, L. (1987) Callus induction and plant regeneration from shoot portions of mature embryos of high tannin sorghum. Plant Cell Tiss. Org. Cult. 9, 245–252.CrossRefGoogle Scholar
  2. 2.
    Cai T, Butler, L. (1990) Plant regeneration from embryogenic callus initiated from ­immature inflorescences of several high-tannin sorghums. Plant Cell Tiss. Org. Cult. 20, 101–110.CrossRefGoogle Scholar
  3. 3.
    Casas AM, Kononowicz, A.K., Zehr, U.B., Tomes, D.T., Axtell, J.D., Butler, L.G., Bressan, R.A., Hasegawa, P.M. (1993) Transgenic sorghum plants via microprojectile bombardment. Proc. Natl. Acad. Sci. USA 90, 11212–11216.CrossRefGoogle Scholar
  4. 4.
    Casas AM, Kononowicz, A.K., Haan, T.G., Zhang, L., Tomes, D.T., Bressan, R.A., Hasegawa, P.M. (1997) Transgenic sorghum plants obtained after microprojectile bombardment of immature inflorescences. In Vitro Cell. Dev. Biol-Plant. 33, 92–100.CrossRefGoogle Scholar
  5. 5.
    Emani C, Sunilkumar, G., Rathore, K.S. (2002) Transgene silencing and reactivation in sorghum. Plant Sci. 162, 181–192.CrossRefGoogle Scholar
  6. 6.
    Gao Z, Jayaraj, J., Muthukrishnan, S., Claflin, L., Liang, G.H. (2005a) Efficient genetic transformation of sorghum using a visual screening marker. Genome 48, 321–333.PubMedCrossRefGoogle Scholar
  7. 7.
    Gao Z, Xie, X., Ling, Y., Muthukrishnan, S., Liang, G.H. (2005b) Agrobacterium tumefaciens-mediated sorghum transformation using a mannose selection system. Plant Biotechnol. J. 3, 591–599.PubMedCrossRefGoogle Scholar
  8. 8.
    Gurel S, Gurel, E., Kaur, R., Wong, J., Meng, L., Tan, H.-Q., Lemaux, P.G. (2009) Efficient, reproducible Agrobacterium-mediated transformation of sorghum using heat treatment of immature embryos. Plant Cell Rep. 28, 429–444.PubMedCrossRefGoogle Scholar
  9. 9.
    Hagio T, Blowers, A.D., Earle, E.D. (1991) Stable transformation of sorghum cell cultures after bombardment with DNA-coated microprojectiles. Plant Cell Rep. 10, 260–264.CrossRefGoogle Scholar
  10. 10.
    Howe A, Sato, S., Dweikat, I., Fromm, M., Clemente, T. (2006) Rapid and reproducible Agrobacterium-mediated transformation of sorghum. Plant Cell Rep. 25, 784–791.PubMedCrossRefGoogle Scholar
  11. 11.
    Kaeppler HF, Pederson, JF (1997) Evaluation of 41 elite and exotic inbred sorghum genotypes for high quality callus production. Plant Cell Tiss. Org. Cult. 48, 71–75.CrossRefGoogle Scholar
  12. 12.
    Masteller VJ, Holden, D.J. (1970) The growth of and organ formation from callus tissue of sorghum. Plant Physiol. 45, 362–364.PubMedCrossRefGoogle Scholar
  13. 13.
    Nguyen TV, Thu, T.T., Claeys, M., Angenon, G. (2007) Agrobacterium-mediated transformation of sorghum (Sorghum bicolor (L.) Moench) using an improved in vitro regeneration system. Plant Cell Tiss. Org. Cult. 91, 155–164.CrossRefGoogle Scholar
  14. 14.
    Zhao ZY, Cai, T., Tagliani, L., Miller, M., Wang, N., Pang, H., Rudert, M., Schroeder, S., Hondred, D., Seltzer, J., Pierce, D. (2000) Agrobacterium-mediated sorghum transformation. Plant Mol. Biol. 44, 789–798.PubMedCrossRefGoogle Scholar
  15. 15.
    Zhu H, Muthukrishnan, S., Krishnaveni, S., Wilde, G., Jeoung, J.M., Liang, G.H.. (1998) Biolistic transformation of sorghum using a rice chitinase gene. J. Genet. Breed. 52, 243–252.Google Scholar
  16. 16.
    Carvalho CHS, Zehr, U.B., Gunaratna, N., Anderson, J., Kononowicz, H.H., Hodges, T.K., Axtell, J.D. (2004) Agrobacterium-mediated transformation of sorghum: factors that affect transformation efficiency. Genet. Mol. Biol. 27, 259–269.CrossRefGoogle Scholar
  17. 17.
    Tadesse Y, Sagi, L., Swennen, R., Jacobs, M. (2003) Optimization of transformation conditions and production of transgenic sorghum (Sorghum bicolor) via microprojectile bombardment. Plant Cell Tiss. Org. Cult. 75, 1–18.CrossRefGoogle Scholar
  18. 18.
    O’Kennedy MM, Grootboom, A., Shewry, P.R. (2006) Harnessing sorghum and millet biotechnology for food and health. J. Cereal Sci. 44, 224–235.CrossRefGoogle Scholar
  19. 19.
    Zhao ZY. (2006) Sorghum (Sorghum bicolor L.), in Methods in Molecular Biology. Agrobacterium protocols 2/e, vol 1. (Wang K, Ed.), pp 233–244, Humana Press, Totowa.CrossRefGoogle Scholar
  20. 20.
    Khanna H, Becker, D., Kleidon, J., Dale, J. (2004) Centrifugation-assisted Agrobacterium tumefaciens-mediated transformation (CAAT) of embryogenic cell suspensions of banana (Musa spp. Cavendish AAA and Lady finger Aab). Mol. Breed. 14, 239–252.CrossRefGoogle Scholar
  21. 21.
    Hiei Y, Ishida, Y., Kasaoka, K., Komari, T. (2006) 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 Tiss. Org. Cult. 87, 233–243.CrossRefGoogle Scholar
  22. 22.
    Chiu W, Hiwa, Y., Zeng, W., Hirano, T., Kobayashi, H., Sheen, J., Chiu, W.L. (1996) Engineered GFP as a vital reporter in plants. Curr. Biol. 6, 325–330.PubMedCrossRefGoogle Scholar
  23. 23.
    Mayo JW, Anderson, R.L. (1968) Pathway of L-Mannose Degradation in Aerobacter aerogenes. J. Biologi Chem. 243, 6330–6333.PubMedGoogle Scholar
  24. 24.
    Hood EE, Helmer, G.D., Fraley, R.T., Chilton, M.D. (1986) The hypovirulence of Agrobac­terium tumefaciens A281 is encoded in the region of pTiBo542 outside the T-DNA. J. Bacteriology 168, 1291–1301.PubMedGoogle Scholar
  25. 25.
    Hoekema A, Hirsch, P.R., Hooykaas, P.J.J., Schilperoot, R.A. (1983) A binary plant vector strategy based on separation of vir- and T regions of the Agrobacterium tumefaciens Ti-plasmid. Nature 303, 179–180.CrossRefGoogle Scholar
  26. 26.
    Sato S, Clemente, T., Dweikat, I. (2004) Identification of an elite sorghum genotype with high in vitro performance capacity. In Vitro Cell. Dev. Biol-Plant 40, 57–60.Google Scholar
  27. 27.
    Paterson AH, Bowers, J.E., Bruggmann, R., Dubchak, I., Grimwood, J., Gundlach, H., Haberer, G., Hellsten, U., Mitros, T., Poliakov, A., Schmutz, J., Spannagl, M., Tang, H., Wang, X., Wicker, T., Bharti, A.K., Chapman, J., Feltus, F.A., Gowik, U., Grigoriev, I.V., Lyons, E., Maher, C.A., Martis, M., Narechania, A., Otillar, R.P., Penning, B.W., Salamov, A.A., Wang, Y., Zhang, L., Carpita, N.C., Freeling, M., Gingle, A.R., Hash, C.T., Keller, B., Klein, P., Kresovich, S., McCann, M.C., Ming, R., Peterson, D.G., Mehboob-ur-Rahman, Ware, D., Westhoff, P., Mayer, K.F.X., Messing, J., Rokhsar, D.S. (2009) The Sorghum bicolor genome and the diversification of grasses. Nature 457, 551–556.PubMedCrossRefGoogle Scholar
  28. 28.
    Pedersen JF, Toy, J.J. (1999) Registration of N246 and N247 Sorghum Germplasm R-Lines. Crop Sci. 39, 1263.Google Scholar
  29. 29.
    Fu D, Huang, B., Xiao, Y., Muthukrishnan, S., Liang, G.H. (2007) Overexpression of barley Hva1 gene in creeping bentgrass for improving drought tolerance. Plant Cell Rep. 26, 467–477.PubMedCrossRefGoogle Scholar
  30. 30.
    Hajdukiewicz P, Svab, Z., Maliga, P. (1994) The small, versatile pPZP family of Agrobac­terium binary vectors for plant transformation. Plant Mol. Biol. 25, 989–994.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Songul Gurel
    • 1
  • Ekrem Gurel
    • 1
  • Tamara I. Miller
    • 1
  • Peggy G. Lemaux
    • 1
  1. 1.Department of Plant and Microbial BiologyUniversity of CaliforniaBerkeleyUSA

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