Molecular Breeding

, Volume 7, Issue 1, pp 43–49 | Cite as

Effective selection and regeneration of transgenic rice plants with mannose as selective agent

  • Paola Lucca
  • Xudong Ye
  • Ingo Potrykus


A new method for the selection of transgenic rice plants without the use of antibiotics or herbicides has been developed. The phosphomannose isomerase (PMI) gene from Escherichia coli has been cloned and consitutively expressed in japonica rice variety TP 309. The PMI gene was transferred to immature rice embryos by Agrobacterium-mediated transformation, which allowed the selection of transgenic plants with mannose as selective agent. The integration and expression of the transgene was confirmed by Southern and northern blot analysis and the activity of PMI indirectly proved with the chlorophenol red assay. The results of genetic analysis showed that the transgenes were segregated in a Mendelian fashion in the T1 generation. The establishment of this selection system in rice provides an efficient way for producing transgenic plants without using antibiotics or herbicides with a transformation frequency of up to 41%.

Agrobacterium-mediated transformation Mannose selection Phosphomannose isomerase (PMI) Transgenic rice 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Ayres N.M. and Park W.D. 1994. Genetic transformation of rice. Crit. Rev. Plant Sci. 13: 219–239.Google Scholar
  2. 2.
    Bliffeld M., Mundy J., Potrykus I. and Futterer J. 1999. Genetic engineering of wheat for increased resistance to powdery mildew disease. Theor. Appl. Genet. 98: 1079–1086.Google Scholar
  3. 3.
    Burkhardt P.K., Beyer P., Wünn J., Kloti A., Armstrong G.A., Schledz M., von Lintig J. and Potrykus I. 1997. Transgenic rice (Oryza sativa) endosperm expressing daffodil (Narcissus pseudonarcissus) phytoene synthase accumulates phytoene, a key intermediate of provitamin A biosynthesis. Plant J. 11: 1071–1078.Google Scholar
  4. 4.
    Christou P. 1997. Rice transformation: bombardment. Plant Mol. Biol. 35: 197–203.Google Scholar
  5. 5.
    Dale P.J. 1992. Spread of engineered genes to wild relatives. Plant Physiol. 100: 13–15.Google Scholar
  6. 6.
    Daniell H. 1999. Environmentally friendly approaches to genetic engineering. In Vitro Cell Dev. Biol. Plant 35: 361–368.Google Scholar
  7. 7.
    Ferguson J.D., Street B.E. and David S.B. 1958. The carbohydrate nutrition of tomato roots. IV. The inhibition of excised root growth by galactose and mannose and its reversal by dextrose and xylose. Ann. Bot. 22: 523–538.Google Scholar
  8. 8.
    Haensch R., Mendel R. and Schulze J. 1998. A rapid and sensitive method to evaluate genotype specific tolerance to phosphinothricin-based selective agents in cereal transformation. J. Plant. Physiol. 152: 145–150.Google Scholar
  9. 9.
    Haldrup A., Petersen S.G. and Okkels F.T. 1998a. Positive selection: a plant selection principle based on xylose isomerase, an enzyme used in the food industry. Plant Cell Rep. 18: 76–81.Google Scholar
  10. 10.
    Haldrup A., Petersen S.G. and Okkels F.T. 1998b. The xylose isomerase gene from Thermoanaerobacterium thermosulfurogenes allows effective selection of transgenic plant cells using D-xylose as the selection agent. Plant Mol. Biol. 37: 287–296.Google Scholar
  11. 11.
    Hiei Y., Komari T. and Kubo T. 1997. Transformation of rice mediated by Agrobacterium tumefaciens. Plant Mol. Biol. 35: 205–218.Google Scholar
  12. 12.
    Hiei Y., Ohta S., Komari T. and Kumashiro T. 1994. 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.Google Scholar
  13. 13.
    Hodal L., Bochardt A., Nielsen J.E., Mattsson O. and Okkels F.T. 1992. Detection, expression and specific elimination of endogenous β-glucuronidase activity in transgenic and nontransgenic plants. Plant Sci. 87: 115–122.Google Scholar
  14. 14.
    Hoekema A., Roelvink P.W., Hooykaas P.J.J. and Schilperoort R.A. 1984. Delivery of T-DNA from the Agrobacterium tumefaciens chromosome into plant cells. EMBO J. 3: 2485–2490.Google Scholar
  15. 15.
    Joersbo M., Donaldson I., Kreiberg J., Petersen S.G., Brunstedt J. and Okkels F.T. 1998. Analysis of mannose selection used for transformation of sugar beet. Mol breed 4: 111–117.Google Scholar
  16. 16.
    Joersbo M., Petersen S.G. and Okkels F.T. 1999. Parameters interacting with mannose selection employed for the production of transgenic sugar beet. Physiol. Plant. 105: 109–115.Google Scholar
  17. 17.
    Ki C.S., Soo C.Y., Jin P.S., Sheop S.J., Ji K.H. and Ho K.K. 1998. Efficient transformation of Korean rice cultivars (Oryza sativa L.) mediated by Agrobacterium tumefaciens. J. Plant Biol. 41: 262–268.Google Scholar
  18. 18.
    Komari T., Hiei Y., Saito Y., Murai N. and Kumashiro T. 1996. Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J. 10: 165–174.Google Scholar
  19. 19.
    Kramer C., Dimaio J., Carfswell G.K. and Shillito R.D. 1993. Selection of transformed protoplast-derived Zea mays colonies with phosphinothricin and a novel assay using the pH indicator chlorophenol red. Planta 190: 454–458.Google Scholar
  20. 20.
    Lindsey K. and Gallois P. 1990. Transformation of sugar beet (Beta vulgaris) by Agrobacterium tumefaciens. J. Exp. Bot. 41: 529–536.Google Scholar
  21. 21.
    Malca I., Endo R.M. and Long M.R. 1967. Mechanism of glucose counteraction of inhibition of root elongation by galactose, mannose and glucosamine. Phytopathology 57: 272–278.Google Scholar
  22. 22.
    Malik V.S. and Saroha M.K. 1999. Marker gene controversy in transgenic plants. J. Plant Biochem. Biotechnol. 8: 1–13.Google Scholar
  23. 23.
    McGookin R. 1994. RNA extraction by the guanidine thiocyanate procedure. In: Walker J.M. (ed.) Methods in Molecular Biology, Vol. 2, Humana Press, Totawa, NJ, pp. 113–116.Google Scholar
  24. 24.
    Miles J.S. and Guest J.R. 1984. Nucleotide sequence and transcriptional start point of the phosphomannose isomerase gene (mana) of Escherichia coli. Gene 32: 41–48.Google Scholar
  25. 25.
    Murashige T. and Skoog F. 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15: 473–497.Google Scholar
  26. 26.
    Nap J.P., Bijvoet J. and Stiekema W.J. 1992. Biosafety of kanamycin-resistant transgenic plants. Transgenic Res. 1: 239–249.Google Scholar
  27. 27.
    Negrotto D., Jolley M., Beer S., Wenck A.R. and Hansen G. 2000. The use of phosphomannose-isomerase as a selectable marker to recover transgenic maize plants (Zea mays L.) via Agrobacterium transformation. Plant Cell Rep. 19: 798–803.Google Scholar
  28. 28.
    Potrykus I. and Spangenberg G. 1995. Gene transfer to plants. Springer-Verlag, Berlin.Google Scholar
  29. 29.
    Sambrook J., Fritsch E.F. and Maniatis T. 1989. Molecular Cloning. A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  30. 30.
    WHO 1993. Health aspects of marker genes in genetically modified plants. Report of a WHO workshop of the Food Safety Unit.Google Scholar
  31. 31.
    Wright M.S., Launis K., Bowman C., Hill M., DiMaio J., Kramer C. and Shillito R.D. 1996. A rapid visual method to identify transformed plants. In Vitro Cell Dev. Biol. Plant 32: 11–13.Google Scholar
  32. 32.
    Ye X., Al-Babili S., Klöti A., Zhang J., Lucca P., Beyer P. and Potrykus I. 2000. Engineering the provitamin A (β-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287: 303–305.Google Scholar
  33. 33.
    Yoder J.I. and Goldsbrough A.P. 1994. Transformation systems for generating marker-free transgenic plants. Bio/technology 12: 263–267.Google Scholar

Copyright information

© Kluwer Academic Publishers 2001

Authors and Affiliations

  • Paola Lucca
    • 1
  • Xudong Ye
    • 2
  • Ingo Potrykus
    • 1
    • 2
  1. 1.Institute for Plant Science, Swiss Federal Institute of Technology (ETHZ)Universitätsstrasse 2ZürichSwitzerland
  2. 2.Agracetus Campus, Monsanto CompanyUniversity GreenMiddletonU.S.A

Personalised recommendations