Plant Cell, Tissue and Organ Culture

, Volume 29, Issue 2, pp 101–108 | Cite as

Agrobacterium-mediated transformation and plant regeneration of buckwheat (Fagopyrum esculentum Moench.)

  • Jovanka Miljuš-Djukić
  • Mirjana Nešković
  • Slavica Ninković
  • Radomir Crkvenjakov


Genetic transformation of buckwheat (Fagopyrum esculentum Moench.) and regeneration of transgenic plants were obtained by using Agrobacterium tumefaciens strains as vectors. Buckwheat cotyledons were excised from imbibed seeds, co-cultivated with A. tumefaciens and subjected to previously reported protocols for callus and shoot regeneration. The transformation with oncogenic strains was confirmed by opine and DNA analyses of tumour tissue extracts. Plants were regenerated on cotyledon fragments incubated with strain A281, harboring pGA472, which carries the neomycin phosphotransferase II gene for kanamycin resistance. The transformation of resistant shoot clones was confirmed by NPTII enzyme assay and DNA hybridization. A large number of transformed shoots were rooted and fertile plantlets were raised in the greenhouse. Transgenic plants comprised pin and thrum clones, which were allowed to cross-pollinate. In about 180 R2 seeds tested for kanamycin resistance, the ratio of resistant to sensitive seedlings was roughly 3:1.

Key words

Agrobacterium tumefaciens buckwheat Fagopyrum esculentum NPTII gene transgenic plants 





dichloro-phenoxyacetic acid


6-(γ, γ,-dimethylallyl-amino)-purine


indole-3-butyric acid


indole-3-acetic acid




neomycin phosphotransferase II


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  1. Adachi T, Yamaguchi A, Miike Y & Hoffmann F (1989) Plant regeneration from protoplasts of common buckwheat (Fagopyrum esculentum). Plant Cell Rep. 8: 247–250Google Scholar
  2. An G, Watson BD & Chiang CC (1986) Transformation of tobacco, tomato, potato, and Arabidopsis thaliana using a binary Ti vector system. Plant Physiol. 81: 301–305Google Scholar
  3. An G, Watson BD, Stachel S, Gordon MP & Nester EW (1985) New cloning vehicles for transformation of higher plants. EMBO J. 4: 277–284Google Scholar
  4. Bohanec B (1987) Improvements in buckwheat micro-propagation procedures. Fagopyrum 7: 13–15Google Scholar
  5. David C, Chilton MD & Tempe J (1984) Conservation of T-DNA in plants regenerated from hairy root cultures. Bio/Technol. 2: 73–76Google Scholar
  6. De Jong H (1972) Buckwheat. Field Crop Abstracts 25: 389–395Google Scholar
  7. Delaporta SL, Wood J & Hicks JB (1985) Maize DNA Miniprep. In: Malmberg R, Messing J & Sussex I (Eds) Molecular Biology of Plants: A Laboratory Course Manual (pp 36–37). Cold Spring Harbor, New YorkGoogle Scholar
  8. De Vos G, De Beuckeleer M, Van Montagu M & Schell J (1981) Restriction endonuclease mapping of the octopine tumor-inducing plasmid pTiAch5 of Agrobacterium tumefaciens, Plasmid. 6: 249–253Google Scholar
  9. Gamborg OL, Miller RA & Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50: 151–158Google Scholar
  10. Garfinkel DJ, Simpson RB, Ream LW, White FF, Gordon MP & Nester EW (1981) Genetic analysis of crown gall: Fine structure map of the T-DNA by site-directed mutagenesis. Cell 27: 143–153Google Scholar
  11. Herrman B, Bućan M, Meins PE, Frischhauf AM, Silver LM & Lechrach H (1986) Genomic Southern blots. Cell 44: 469–476Google Scholar
  12. Horsch RB, Fry JE, Hoffmann NL, Eichholz D, Rogers SG & Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227: 1229–1231Google Scholar
  13. Lichtenstein C & Draper J (1985) Genetic Engineering of Plants. In: Glover DM (Ed) DNA Cloning, Vol II (pp 67–121). IRL Press, OxfordGoogle Scholar
  14. Maniatis T, Fritsch FF & Sambrook J (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New YorkGoogle Scholar
  15. Montoya AL, Chilton MD, Gordon MP, Sciaky D & Nester EW (1977) J. Octopine and nopaline metabolism in Agrobacterium tumefaciens and crown gall tumors. J. Bacteriol. 129: 101–107Google Scholar
  16. Nešković M, Vinterhalter B, Miljuš-Djukić J, Ninković S, Vinterhalter D, Jovanović V & Knežević J (1990) Susceptibility of buckwheat (Fagopyrum esculentum Moench.) to Agrobacterium tumefaciens and A. rhizogenes. Fagopyrum 10: 57–61Google Scholar
  17. Nešković M, Vujičić R & Budimir S (1987) Somatic embryogenesis and bud formation from immature embryos of buckwheat (Fagopyrum esculentum Moench.) Plant Cell Rep. 6: 423–426Google Scholar
  18. Nešković M, Vujičić R & Srejović V (1985) Differential responses of buckwheat leaf cells to growth substances stimulating cell division. Ann. Bot. 56: 755–760Google Scholar
  19. Pomeranz Y & Robbins GS (1972) Amino acid composition of buckwheat. J. Agric. Food Chem. 20: 270–274Google Scholar
  20. Radke SE, Andrews BM, Moloney MM, Crouch ML, Kridl JC & Knauf VC (1988) Transformation of Brassica napus L. using Agrobacterium tumefaciens: developmentally regulated expression of a reintroduced napin gene. Theor. Appl. Genet. 75: 685–694Google Scholar
  21. Rogers SG, Horsch RB & Fraley RT (1986) Gene transfer in plants: Production of transformed plants using Ti plasmid vectors. Meth. Enzymol. 118: 627–640Google Scholar
  22. Srejović V & Nešković M (1981) Regeneration of plants from cotyledon fragments of buckwheat (Fagopyrum esculentum Moench.). Z. Pflanzenphysiol. 104: 37–42Google Scholar
  23. Van Larebeke N, Genetello Ch, Hernalsteens JP, De Picker A, Zaenen I, Messens E, Van Montagu M & Schell J (1977) Transfer of Ti plasmids between Agrobacterium strains by mobilization with the conjugative plasmid RP4. Molec. Gen. Genet. 152: 119–124Google Scholar
  24. Yamane Y (1974) Induced differentiation of buckwheat plants from subcultured calluses in vitro. Jap. J. Genet. 49: 139–146Google Scholar
  25. Zimmer EA & Newton KJ (1982) A simple method for the isolation of high molecular weight DNA from individual maize seedlings and tissues. In: Sheridan WF (Ed) Maize for Biological Research (pp 165–168). Plant Molec. Biol. Assoc. VirginiaGoogle Scholar

Copyright information

© Kluwer Academic Publishers 1992

Authors and Affiliations

  • Jovanka Miljuš-Djukić
    • 1
  • Mirjana Nešković
    • 1
    • 2
  • Slavica Ninković
    • 1
  • Radomir Crkvenjakov
    • 3
  1. 1.Institute for Biological Research ‘S. Stanković’University of BelgradeBelgradeYugoslavia
  2. 2.Institute of Botany, Faculty of ScienceUniversity of BelgradeBelgradeYugoslavia
  3. 3.Institute for Molecular Biology and Genetic EngineeringUniversity of BelgradeBelgradeYugoslavia

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