Organogenesis From Transformed Tomato Explants

  • Anne Frary
  • Joyce Van Eck
Part of the Methods in Molecular Biology™ book series (MIMB, volume 286)


Tomato was one of the first crops for which a genetic transformation system was reported involving regeneration by organogenesis from Agrobacterium-transformed explants. Since the initial reports, various factors have been studied that affect the efficiency of tomato transformation and the technique has been useful for the isolation and identification of many genes involved in plant disease resistance, morphology and development. In this method, cotyledon explants from in vitro-grown seedlings are precultured overnight on a tobacco suspension feeder layer. The explants are then inoculated with Agrobacterium and returned to the feeder layer for a 2-d period of cocultivation. After cocultivation, the explants are transferred to an MS-based selective regeneration medium containing zeatin. Regenerated shoots are then rooted on a separate selective medium. This protocol has been used with several tomato cultivars and routinely yields transformation efficiencies of 10–15%.

Key Words

Agrobacterium tumefaciens biotechnology gene transfer genetic engineering genetic transformation Lycopersicon esculentum morphogenesis regeneration transgenic plants 


  1. 1.
    Horsch, R. B., Fry, J. B., Hoffmann, N. L., et al. (1985) A simple and general method for transferring genes into plants. Science 227, 1229–1231.CrossRefGoogle Scholar
  2. 2.
    McCormick, S., Niedermeyer, J., Fry, J., Barnason, A., Horsch, R., and Fraley, R. (1986) Leaf disc transformation of cultivated tomato (Lycopersicon esculentum) using Agrobacterium tumefaciens. Plant Cell Rep. 5, 81–84.CrossRefGoogle Scholar
  3. 3.
    Toyoda, H., Matsuda, Y., Utsumi, R., and Ouchi, S. (1988) Intranuclear microinjection for transformation of tomato callus cultures. Plant Cell Rep. 7, 293–296.CrossRefGoogle Scholar
  4. 4.
    Xu, Y., Yu, H., and Hall, T.C. (1994) Rice triosephosphate isomerase gene 5′ sequence directs glucuronidase activity in transgenic tobacco but requires an intron for expression in rice. Plant Physiol. 106, 459–467.PubMedCrossRefGoogle Scholar
  5. 5.
    Van Eck, J. M., Blowers, A. D., and Earle, E. D. (1995) Stable transformation of tomato cell cultures after bombardment with plasmid and YAC DNA. Plant Cell Rep. 14, 299–304.CrossRefGoogle Scholar
  6. 6.
    Nakata, K., Tanaka, H., Yano, K., and Takagi, M. (1992) An effective transformation system for Lycopersicon peruvianum by electroporation. Jpn. J. Breed. 42, 487–495.Google Scholar
  7. 7.
    Koornneef, M., Hanhart, C., Jongsma, M., et al. (1986) Breeding of a tomato genotype readily accessible to genetic manipulation. Plant Sci. 45, 201–208.CrossRefGoogle Scholar
  8. 8.
    Tanksley, S. D., Ganal, M. W., Prince, J. P., et al. (1992) High density molecular linkage maps of the tomato and potato genomes. Genetics 132, 1141–1160.PubMedGoogle Scholar
  9. 9.
    Martin, G. B., Frary, A., Wu, T., et al. (1994) A member of the tomato Pto gene family confers sensitivity to fenthion resulting in rapid cell death. Plant Cell 6, 1543–1552.PubMedCrossRefGoogle Scholar
  10. 10.
    Frary, A., Nesbitt, T. C., Frary, A., et al. (2000) fw2.2: a quantitative trait locus key to the evolution of tomato fruit size. Science 289, 85–88.PubMedCrossRefGoogle Scholar
  11. 11.
    Jones, D. A., Thomas, C. M., Hammond-Kosack, K. E., Balint-Kurti, P. J., and Jones, J. D. (1994) Isolation of the tomato Cf-9 gene for resistance to Cladosporium fulvum by transposon tagging. Science 266, 789–793.PubMedCrossRefGoogle Scholar
  12. 12.
    Milligan, S. B., Bodeau, J., Yaghoobi, J., Kaloshian, I., Zabel, P., and Williamson, V. M. (1998) The root knot nematode resistance gene Mi from tomato is a member of the leucine zipper, nucleotide-binding leucine-rich repeat family of plant genes. Plant Cell 10, 1307–1319.PubMedCrossRefGoogle Scholar
  13. 13.
    Liu, J., Van Eck, J., Cong, B., and Tanksley, S. D. (2002) A new class of regulatory genes underlying the cause of pear-shaped tomato fruit. Proc. Natl. Acad Sci. USA 99, 13,302–13,306.PubMedCrossRefGoogle Scholar
  14. 14.
    Sheehy, R. E., Kramer, M., and Hiatt, W. R. (1988) Reduction of polygalacturonase activity in tomato fruit by antisense RNA. Proc. Nat. Acad. Sci. USA 85, 8805–8809.PubMedCrossRefGoogle Scholar
  15. 15.
    Frary, A. and Hamilton, C. M. (2001) Efficiency and stability of high molecular weight DNA transformation: an analysis in tomato. Transgenic Res. 10, 121–132.PubMedCrossRefGoogle Scholar
  16. 16.
    Chyi, Y. S. and Phillips, G. C. (1987) High efficiency Agrobacterium-mediated transformation of Lycopersicon based on conditions favorable for regeneration. Plant Cell Rep. 6, 105–108.Google Scholar
  17. 17.
    Fillatti, J. J., Kiser, J., Rose, R., and Comai, L. (1987) Efficient transfer of a glyphosate tolerance gene into tomato using a binary Agrobacterium tumefaciens vector. Biotechnology 5, 726–730.CrossRefGoogle Scholar
  18. 18.
    Hamza, S. and Chupeau, Y. (1993) Re-evaluation of conditions for plant regeneration and Agrobacterium-mediated transformation from tomato (Lycopersicon esculentum). J. Exp. Bot. 44, 1837–1845.CrossRefGoogle Scholar
  19. 19.
    Davis, M. E., Miller, A. R., and Lineberger, R. D. (1991) Temporal competence for transformation of Lycopersicon esculentum (L. Mill.) cotyledons by Agrobacterium tumefaciens: relation to wound-healing and soluble plant factors. J. Exp. Bot. 42, 359–364.CrossRefGoogle Scholar
  20. 20.
    van Roekel, J. S., Damm, B., Melchers, L. S., and Hoekema, A. (1993) Factors influencing transformation frequency of tomato (Lycopersicon esculentum). Plan Cell Rep. 12, 644–647.Google Scholar
  21. 21.
    Frary, A. and Earle, E. D. (1996) An examination of factors affecting the efficiency of Agrobacterium-mediated transformation of tomato. Plant Cell Rep. 16, 235–240.Google Scholar

Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Anne Frary
    • 1
  • Joyce Van Eck
    • 2
  1. 1.Department of Molecular Biology and GeneticsIzmir Institute of TechnologyIzmirTurkey
  2. 2.The Boyce Thompson Institute for Plant ResearchIthaca

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