, 150:387 | Cite as

The Introduction of Transgenes to Control Blackheart in Pineapple (Ananas Comosus L.) cv. Smooth Cayenne by Microprojectile Bombardment

  • H. L. Ko
  • P. R. Campbell
  • M. P. Jobin-Décor
  • K. L. Eccleston
  • M. W. Graham
  • M. K. Smith


A transformation technique for the introduction of transgenes to control blackheart by particle bombardment has been developed for pineapple cv. Smooth Cayenne. Leaf callus cultures capable of high frequency organogenesis with a short regeneration time were used as explant material. Gus and gfp reporter genes were used to observe and determine transient and stable expression. The ppo gene, isolated from pineapple, was introduced to control blackheart. Co-transformation occurred with constructs containing the nptII gene conferring geneticin resistance. We have recovered 15 independent transgenic gus and gfp lines each from 8 separate experiments and 22 ppo lines from 11 experiments. Gus, gfp, ppo and nptII positive plants have been regenerated, which have been shown by Southern blot analysis to be stable transgenics containing multiple copies of the introduced genes. These results show that biolistic gene delivery in pineapple can be successfully achieved at an acceptable efficiency of 0.21–1.5% for genetic improvement of ’Smooth Cayenne’, the industry standard throughout the world.

Key words

Ananas microprojectile/particle bombardment pineapple polyphenol oxidase 



6-Benzyl amino purine


Green fluorescent protein




α-Naphthalene acetic acid


Neomycin phosphotransferase


Polyphenol oxidase


  1. Bachem, C.W.B., G.-J. Speckmann, P.C.G. van der Linder, F.T.M. Verheggen, M.D. Hunt, J.C. Steffens & M. Zabeau, 1994. Antisense expression of polyphenol oxidase genes inhibits enzymatic expression in potato tubers. Bio/Technology 12: 1101–1105.CrossRefGoogle Scholar
  2. Becker, D.K., B. Dugdale, M.K. Smith, R.M. Harding & J.L. Dale, 2000. Genetic transformation of Cavendish banana (Musa spp. AAA group) cv. Grand Nain via microprojectile bombardment. Plant Cell Rep 19: 229–234.CrossRefGoogle Scholar
  3. Birch, R.G. & R. Bower, 1994. Principles of gene transfer using particle bombardment. In: N.-S. Yang & P. Christou (Eds.) Particle bombardment technology for gene transfer. Oxford University press, New York/Oxford, pp. 3–37.Google Scholar
  4. Boevink, P., P.W.G. Chu & P. Keese, 1993. Sequence of subterranean stunt virus DNA: Affinities with the geminii virus. Virology 207: 354–361.CrossRefGoogle Scholar
  5. Bower, R., A.R. Elliott, B.A.M. Potier & R.G. Birch, 1996. High-efficiency, microprojectile-mediated cotransformation of sugarcane, using visible or selectable markers. Mol Breeding 2: 239–249.CrossRefGoogle Scholar
  6. Brettell, R.I.S. & F.R. Murray, 1995. DNA transfer and gene expression in transgenic cereals. Biotech Gen Engineering Rev 13: 315–334.Google Scholar
  7. Bucheli, C.S., I.B. Dry & S.P. Robinson, 1996. Isolation of a full-length cDNA encoding polyphenol oxidase from sugarcane, a C4 grass. Plant Mol Biol 31: 1233–1238.PubMedCrossRefGoogle Scholar
  8. Chalfie, M., Y. Tu, G. Euskirchen, W.W. Ward & D.C. Prasher, 1994. Green fluorescent protein as a marker for gene expression. Science 263: 802–805.PubMedCrossRefGoogle Scholar
  9. Christou, P., 1991. Particle bombardment-mediated transformation of organized tissue and its impact on agricultural biotechnology. IAPTC Newsletter 66: 2–14.Google Scholar
  10. Christou, P., T.L. Ford & M. Kofron, 1991. 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/Technology 9: 957–962.CrossRefGoogle Scholar
  11. Dugdale, B., P.R. Beetham, D.K. Becker, R.M. Harding & J.L. Dale, 1998. Promoter activity associated with the intergenic regions of banana bunchy top virus DNA-1 to -6 in transgenic tobacco and banana cells. J Gen Virology 79: 2301–2311.Google Scholar
  12. Elliott, A.R., J.A. Campbell, B. Dugdale, R.I.S. Brettell & C.P.L. Grof, 1999. Green-fluorescent protein facilitates rapid in vitro detection of genetically transformed plant cells. Plant Cell Rep 18: 707–714.CrossRefGoogle Scholar
  13. Finer, J.J., P. Vain, M.W. Jones & M.D. McMullen, 1992. Development of the particle inflow gun for DNA delivery to plant cells. Plant Cell Rep 11: 323–328.CrossRefGoogle Scholar
  14. Franks, T. & R.G. Birch, 1991. Gene transfer into intact sugarcane cells using microprojectile bombardment. Aust J Plant Physiol 18: 471–480.CrossRefGoogle Scholar
  15. Gleave, A.P., 1992. A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol Biol 20: 1203–1207.PubMedCrossRefGoogle Scholar
  16. Graham, G.C., P. Mayers & R.J. Henry, 1994. A simplified method for the preparation of fungal genomic DNA for PCR and RAPD analysis. BioTechniques 16(1): 48–50.PubMedGoogle Scholar
  17. Graham, M., L. Ko, V. Hardy, S. Robinson, B. Sawyer, T. O'Hare, M. Jobin, J. Dahler, S. Underhill & M. Smith, 2000. The development of blackheart resistant pineapples through genetic engineering. Acta Hort 529: 133–136.Google Scholar
  18. Haseloff, J. & B. Amos, 1995. GFP in plants. Trends in genetics 11: 328–329.PubMedCrossRefGoogle Scholar
  19. Hunt, M.D., N.T. Eannetta, Y. Haifeng, S.M. Newman & J.C. Steffens, 1993. cDNA cloning and expression of potato polyphenol oxidase. Plant Mol Biol 21: 59–68.PubMedCrossRefGoogle Scholar
  20. Jefferson, R.A., 1987. Assaying chimeric genes in plants: The GUS gene fusion system. Plant Mol Biol Rep 5: 387–405.CrossRefGoogle Scholar
  21. Jefferson, R.A., T.A. Kavanagh & M.W. Bevan, 1987. GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6(13): 3901–3907.PubMedGoogle Scholar
  22. Kartha, K.K., N.S. Nehra & R.N. Chibbar, 1994. Genetic engineering of wheat and barley. In: R.J. Henry & J.A. Ronalds (Eds.) Improvement of cereal quality by genetic engineering, Plenum Press, New York and London, pp. 21–30.Google Scholar
  23. Klein, T.M., R. Arentzen, P.A. Lewis & S. Fitzpatrick-McElligott, 1992. Transformation of microbes, plants and animals by particle bombardment. Bio/Technology 10: 286–291.CrossRefPubMedGoogle Scholar
  24. Ko, L. & M. Smith, 2005. In vitro conditions for pineapple biolistics transformation. Proceedings IAPTC&B Conference (Australian Branch), 21-24 September 2005, Perth (WA): 281.Google Scholar
  25. Livingstone, D.M. & R.G. Birch, 1999. Efficient transformation and regeneration of diverse cultivars of peanut (Arachis hypogea L.) by particle bombardment into embryogenic callus produced from mature seeds. Mol Breeding 5: 43–51.CrossRefGoogle Scholar
  26. Mathews, V.H. & T.S. Rangan, 1981. Growth and regeneration of plantlets in callus cultures of pineapple. Scientia Hort 14: 227–234.CrossRefGoogle Scholar
  27. Meyer, P., 1995. Variation of transgene expression in plants. Euphytica 85: 359–366.CrossRefGoogle Scholar
  28. Murashige, T. & F. Skoog, 1962. A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol Plant 15: 473–497.CrossRefGoogle Scholar
  29. Paull, R.E. & K.G. Rohrbach, 1985. Symptom development of chilling injury in pineapple fruit. J Amer Soc Hort Sci 110: 100–105.Google Scholar
  30. Rangan, T.S., 1982. Pineapple. In: D.A. Evan, W.R. Sharp, P.V. Ammirato & Y. Yamada (Eds.) Application of plant tissue culture: Methods for crop improvement, MacMillan, New York, pp. 373–382.Google Scholar
  31. Rohrbach, K.G., F. Leal & G. Coppens d'Eeckenbrugge, 2003. History, distribution and world production. In: D.P. Bartholomew, R.E. Paull & K.G. Rohrbach (Eds.) The Pineapple, botany, production and uses, CABI Publishing, pp. 1–12.Google Scholar
  32. Sambrook, J., E.F. Fritsch & T. Maniatis, 1989. Molecular cloning – a laboratory manual. Cold Spring Harbor Laboratory Press, New York.Google Scholar
  33. Sanewski, G.M. & J. Giles, 1997. Blackheart resistance in three clones of pineapple (Ananas comosus (L.) Merr.) in subtropical Queensland. Aust J Exp Agric 37: 459–461.CrossRefGoogle Scholar
  34. Sanford, J.C., F.D. Smith & J.A. Russell, 1993. Optimizing the biolistic process for different biological applications. Methods in Enzymology 217: 483–509.PubMedCrossRefGoogle Scholar
  35. Sheen, J., S. Hwang, Y. Niwa, H. Kobayashi & D.W. Galbraith, 1995. Green-fluorescent protein as a new vital marker in plant cells. Plant J 8(5): 777–784.PubMedCrossRefGoogle Scholar
  36. Smith, M.K., H.-L. Ko, S.D. Hamill & G.M. Sanewski, 2002. Pineapple transformation: Managing somaclonal variation. Acta Hort 575: 69–74.Google Scholar
  37. Smith, M.K., H-L Ko, S.D. Hamill, G.M. Sanewski & M.W. Graham, 2003. Biotechnology. In: D.P. Bartholomew, R.E. Paull & K.G. Rohrbach (Eds.) The Pineapple, botany, production and uses, CABI Publishing, pp. 57–68.Google Scholar
  38. Sripaoraya, S., R. Marchant, J.B. Power & M.R. Davey, 2001. Herbicide-tolerant transgenic pineapple (Ananas comosus) produced by microprojectile bombardment. Annals Bot 88: 597–603.CrossRefGoogle Scholar
  39. Stewart, R.J., B.J.B. Sawyer, C.S. Bucheli & S.P. Robinson, 2001. Polyphenol oxidase is induced by chilling and wounding in pineapple. Aust J Plant Physiol 28: 181–191.Google Scholar
  40. Thomson, D. & R. Henry, 1995. Single-step protocol for preparation of plant tissue for analysis by PCR. BioTechniques 19(3): 394–400.PubMedGoogle Scholar
  41. Vain, P., N. Keen, J. Murillo, C. Rathus, C. Nemes & J.J. Finer, 1993a. Development of the particle inflow gun. Plant Cell Tiss Org Cult 33: 237–246.CrossRefGoogle Scholar
  42. Vain, P., M.D. McMullen & J.J. Finer, 1993b. Osmotic treatment enhances particle bombardment-mediated transient and stable transformation of maize. Plant Cell Rep 12: 84–88.CrossRefGoogle Scholar
  43. Vasil, V., A.M. Castillo, M.E. Fromm & I.K. Vasil, 1992. Herbicide resistant fertile transgenic wheat plants obtained by microprojectile bombardment of regenerable embryogenic callus. Bio/Technology 10: 667–674.CrossRefGoogle Scholar
  44. Wakasa, K., 1989. In: Y.P.S. Bajaj (Ed.) Biotechnology in Agriculture and Forestry 5: Trees II, Springer-Verlag, London, pp. 13–29.Google Scholar

Copyright information

© SpringerScience + Business Media, Inc. 2006

Authors and Affiliations

  • H. L. Ko
    • 1
  • P. R. Campbell
    • 1
  • M. P. Jobin-Décor
    • 1
  • K. L. Eccleston
    • 1
  • M. W. Graham
    • 2
  • M. K. Smith
    • 1
  1. 1.Department of Primary Industries & FisheriesMaroochy Research StationQueenslandAustralia
  2. 2.Benitec Australia Ltd.St. LuciaAustralia

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