Transformation of Tomato (Lycopersicon esculentum Mill.) for Virus Disease Protection

  • H. Toyoda
Part of the Biotechnology in Agriculture and Forestry book series (AGRICULTURE, volume 23)


One of the major aims of plant biotechnology is to protect crop plants from diseases. Transforming plants with certain genes relating to disease resistance would be a direct and profitable strategy for producing transgenic disease-resistant plants. For completing this strategy, however, it would be important and may be essential to evaluate in advance the feasibility of a gene to be used for transformation. From this point of view, this chapter first describes an efficient system for transforming tomato callus cells to elucidate at a cellular level the effectiveness of transformation with respect to virus disease protection. A microinjection technique has been used for this purpose, since it has been well recognized that this technique is a reliable method for directly introducing foreign genes into the correct portions of target cells (Neuhaus and Spangenberg 1990; Potrykus 1990). Nevertheless, this system may not be necessarily effective for an efficient production of transgenic plants from transformed cells due to its complicated procedures and difficulty in plant regeneration. In order to promote and simplify production of transgenic plants, this chapter also describes an Agrobacterium-mediated gene transfer for an actual genetic transformation of tomato.


Hairy Root Coat Protein Tobacco Mosaic Virus Coat Protein Gene Agrobacterium Rhizogenes 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


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  1. An G, Watson BD, Chiang CC (1986) Transformation of tobacco, tomato, potato, and Arabidopsis thaliana using a binary Ti vector system. Plant Physiol 81:301–305PubMedCrossRefGoogle Scholar
  2. Beachy RN, Loesch-Fries S, Turner NE (1990) Coat protein-mediated resistance against virus infection. Annu Rev Phytopathol 28:451–474CrossRefGoogle Scholar
  3. Bird CR, Ray JA, Fletcher JD, Boniwell JM, Bird AS, Teulieres C, Blain I, Bramley PM, Schuch W (1991) Using antisense RNA to study gene function: inhibition of carotenoid biosynthesis in transgenic tomatoes. Bio/Technol 9:635–639CrossRefGoogle Scholar
  4. Childs GV, Naor Z, Hazum E, Tibolt R, Westlund KN, Hancock MB (1983) Cytochemical characterization of pituitary target cells for biotinylated gonadotropin releasing hormone. Peptides 4:549–555PubMedCrossRefGoogle Scholar
  5. Childs GV, Lloyd JM, Unabia G, Gharib SD, Wierman ME, Chin WW (1987) Detection of luteinizing hormone p messenger ribonucleic acid (RNA) in individual gonadotropes after castration: use of a new in situ hybridization method with a photobiotinylated complementary RNA probe. Mol Endocrinol 1:926–932PubMedCrossRefGoogle Scholar
  6. Ditta G, Stanfield S, Corbin D, Helinski DR (1980) Broad host range DNA cloning system for gramnegative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci USA 77:7347–7351PubMedCrossRefGoogle Scholar
  7. Fan Y-S, Davis LM, Shows TB (1990) Mapping small DNA sequences by fluorescence in situ hybribization directly on banded metaphase chromosomes. Proc Natl Acad Sci USA 87:6223–6227PubMedCrossRefGoogle Scholar
  8. Forster AC, Mclnnes JL, Skingle DC, Symons RH (1985) Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin. Nucleic Acid Res 13:745–761PubMedCrossRefGoogle Scholar
  9. Gee CE, Robert JL (1983) In situ hybridization histochemistry: a technique for the study of gene expression in single cells. DNA 2:157–163PubMedCrossRefGoogle Scholar
  10. Hemenway C, Fang R-X, Kaniewski WK, Chua N-H, Turner NE (1988) Analysis of the mechanism of protection in transgenic plants expressing the potato virus X coat protein or its antisense RNA. EMBO J 7:1273–1280PubMedGoogle Scholar
  11. Hoefler H, Childers H, Montminy MR, Lechan RM, Goodman RH, Wolfe HJ (1986) In situ hybridization methods for the detection of somatostatin mRNA in tissue sections using antisense RNA probes. Histochem J 18:597–604Google Scholar
  12. Holbrook LA, Miki BL (1985) Brassica Crown gall tumourigenesis and in vitro of transformed tissue. Plant Cell Rep 4:329–332Google Scholar
  13. Jefferson RA (1987) Assaying chimeric genes in plants: The GUS gene fusion system. Plant Mol Biol Rep 5:387–405Google Scholar
  14. Jefferson RA, Burgess SM, Hirsh D (1986) β-Glucuronidase from Escherichia coli as a gene-fusion marker. Proc Natl Acad Sci USA 83:8447–8451Google Scholar
  15. Jongsma M, Koornneef M, Zabel P, Hille J (1987) Tomato protoplast DNA transformation: physical linkage and recombination of exogenous DNA sequences. Plant Mol Biol 8:383–394Google Scholar
  16. Joshi S, Vincentini AM (1990) Controlled cell wall regeneration for efficient microinjections of Nicotiana tabacum var. Carlson protoplasts. Plant Cell Rep 9:117–120Google Scholar
  17. Klee HJ, Yanofsky MF, Nester EW (1985) Vectors for transformation of higher plants. Bio/Technol 3:637–642Google Scholar
  18. Kolchinsky A, Kanazin V, Yakovleva E, Gazumyan A, Kole C, Ananiev E (1990) 5S-RNA genes of barley are located on the second chromosome. Theor Appl Genet 80:333–336Google Scholar
  19. Koornneef M, Hanhart C, Jongsma M, Toma I, Weider R, Zabel P, Hille J (1986) Breeding of a tomato genotype readily accessible to genetic manipulation. Plant Sci 45:201–208Google Scholar
  20. Koornneef M, Hanhart C, Jongsma M, Toma I, Weider R, Zabel P, Hille J (1986) Breeding of a tomato genotype readily accessible to genetic manipulation. Plant Sci 45:201–208Google Scholar
  21. Matsuda Y, Toyoda H, Ouchi S (1989) Resistance of tomato callus cells against tobacco mosaic virus. Plant Tissue Cult Lett 6:33–34Google Scholar
  22. Matsuda Y, Toyoda H, Morita M, Ikeda S, Ouchi S (1993) A novel method for in situ hybridization in fungal cells based on pricking introduction of photobiotin-labeled probes. J. Phytopathol (in press)Google Scholar
  23. McCormick S, Niedermeyer J, Fry J, Barnason A, Horsch R, Fraley R (1986) Leaf disc transformation of cultivated tomato (Lycopersicon esculentum) using Agrobacterium tumefaciens. Plant Cell Rep 5:81–84Google Scholar
  24. Morgan AJ, Cox PN, Turner DA, Peel E, Daver MR, Gartland KMA, Mulligan BJ (1986) Transformation of tomato using an Ri plasmid vector. Plant Sci 49:37–49Google Scholar
  25. Mugnier J (1988) Establishment of new axenic hairy root lines by inoculation with Agrobacterium rhizogenes. Plant Cell Rep 7:9–12Google Scholar
  26. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio-assay with tobacco tissue cultures. Physiol Plant 15:473–497Google Scholar
  27. Neuhaus G, Spangenberg G (1990) Plant transformation by microinjection techniques. Physiol Plant 79:213–217Google Scholar
  28. Potrykus I (1990) Gene transfer to cereals: an assessment. Bio/Technol 8:535–542Google Scholar
  29. Shahin EA, Sukhapinda K, Simpson RB, Spivey R (1986) Transformation of cultivated tomato by a binary vector in Agrobacterium rhizogenes: transgenic plants with normal phenotypes harbor binary vector T-DNA, but no Ri-plasmid T-DNA. Theor Appl Genet 72:770–777Google Scholar
  30. Sheehy RE, Kramer M, Hiatt WR (1988) Reduction of polygalacturonase activity in tomato fruit by antisense RNA. Proc Natl Acad Sci USA 85:8805–8809Google Scholar
  31. Shiomi T, Shirakawa T, Takeuchi S, Oizumi T, Uematsu S (1987) Hairy root of melon caused by Agrobacterium rhizogenes Biovar 1. Ann Phytopathol Soc Jpn 53:454–459Google Scholar
  32. Smith CJS, Watson CF, Ray J, Bird CR, Morris PC, Schunch W, Grierson D (1988) Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomato. Nature 334:724–726Google Scholar
  33. Smith CJS, Watson CF, Morris PC, Bird CR, Seymour GB, Gray JE, Arnold C, Tucker GA, Schunch W, Harding S, Grierson D (1990) Inheritance and effect on ripening of antisense polygalacturonase genes in transgenic tomatoes. Plant Mol Biol 14:369–379Google Scholar
  34. Takamatsu N, Ohno T, Meshi T, Okada Y (1983) Molecular cloning and nucleotide sequence of the 30K and the coat protein cistron of TMV (tomato strain) genome. Nucleic Acid Res 11:3768–3778Google Scholar
  35. Toyoda H, Tanaka N, Hirai T (1984) Effects of the culture filtrate of Fusarium oxysporum f. sp. lycopersici on tomato callus growth and the selection of resistant callus cells to the filtrate. Ann Phytopathol Soc Jpn 50:53–62Google Scholar
  36. Toyoda H, Matsuda Y, Hirai T (1985) Resistance mechanism of cultured plant cells to tobacco mosaic virus (III). Efficient microinjection of tobacco mosaic virus into tomato callus cells. Ann Phytopathol Soc Jpn 51:32–38Google Scholar
  37. Toyoda H, Matsuda Y, Hirai T (1986) Multiplication and translocation of tobacco mosaic virus microinjected into cell-aggregates of tomato callus. Plant Tissue Cult Lett 3:22–27Google Scholar
  38. Toyoda H, Matsuda Y, Utsumi R, Ouchi S (1988) Intranuclear microinjection for transformation of tomato callus cells. Plant Cell Rep 7:293–296Google Scholar
  39. Toyoda H, Oki T, Matsuda Y, Katsuragi K, Nishiguchi T, Ouchi S (1989) Transformation of constituent cells of tomato callus aggregates by intranuclear microinjection. Plant Tissue Cult Lett 6:95–97CrossRefGoogle Scholar
  40. Toyoda H, Hosoi Y, Yamamoto A, Nishiguchi T, Maeda K, Takebayashi T, Shiomi T, Ouchi S (1991) Transformation of melon (Cucumis melo L.) with Agrobacterium rhizogenes. Plant Tissue Cult Lett 8:21–27CrossRefGoogle Scholar
  41. Trulson AJ, Simpson RB, Shahin EA (1986) Transformation of cucumber (Cucumis sativus L.) plants with Agrobacterium rhizogenes. Theor Appl Genet 73:11–15CrossRefGoogle Scholar
  42. Tsukada M, Kusano T, Kitagawa Y (1989) Introduction of foreign genes into tomato protoplasts by electroporation. Plant Cell Physiol 30:599–603Google Scholar
  43. Turner NE, O’Connell KM, Nelson RS, Sanders PR, Beachy RN, Fraley RT, Shah DM (1987) Expression of alfalfa mosaic virus coat protein gene confers cross-protection in transgenic tobacco and tomato plants. EMBO J 6:1181–1188Google Scholar
  44. Widholm JM (1972) The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Technol 47:189–194PubMedGoogle Scholar
  45. Yamamoto F, Furusawa M, Furusawa I, Obinata M (1982) The ‘pricking’ method. A new efficient technique for mechanically introducing foreign DNA into the nuclei of culture cells. Exp Cell Res 142:79–84PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1993

Authors and Affiliations

  • H. Toyoda
    • 1
  1. 1.Faculty of AgricultureKinki UniversityNaraJapan

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