Summary
We investigated whether the efficiency of transformation of plant cells by Agrobacterium tumefaciens during cocultivation is limited by the properties of the plant cells or by the infecting bacteria.
Therefore, tobacco protoplasts were infected by cocultivation with two different agrobacteria strains carrying Ti plasmids with distinguishable T-DNAs. These T-DNAs cotransform plant cells at a frequency equal to the product of their independent transformation frequencies, which indicates that all plant cells are equally competent. On the other hand, when these T-DNAs are located on the same Ti plasmid vector within one bacterial strain, the cotransformation frequency is significantly higher than the product of the single transformation frequencies. We interpret these results to indicate that transformation is limited more by the establishment of effective bacteria/plant cell interaction than by (i) the process of DNA integration and (ii) by the number of plant cells capable of being transformed by Agrobacterium. We found that most plant cells are transformed by only one or a few agrobacteria. Analysis of the number of T-DNA copies in these clonally transformed lines indicates amplification of the original, infecting T-region copy.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Aerts M, Jacobs M, Hernalsteens J-P, Van Montagu M, Schell J (1979) Induction and in vitro culture of Arabidopsis thaliana crown gall tumours. Plant Sci Lett 17:43–50
An G, Watson BD, Stachel S, Gordon MP, Nester EW (1985) New cloning vehicles for transformation of higher plants. EMBO J 4:277–284
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl Acids Res 7:1513–1523
Caplan AB, Van Montagu M, Schell J (1985) Genetic analysis of integration mediated by single T-DNA borders. J Bacteriol 161:655–664
Deblaere R, Bytebier B, De Greve H, Deboeck F, Schell J, Van Montagu M, Leemans J (1985) Efficient octopine Ti plasmid-derived vectors for Agrobacterium-mediated gene transfer to plants. Nucl Acids Res 13:4777–4788
De Block M, Herrera-Estrella L, Van Montagu M, Schell J, Zambryski P (1984) Expression of foreign genes in regenerated plants and their progeny. EMBO J 3:1681–1689
De Greve H, Dhaese P, Seurinck J, Lemmers M, Van Montagu M, Schell J (1982) Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octopine synthase gene. J Mol Appl Genet 1:499–512
Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1:19–21
Dhaese P, De Greve H, Decraemer H, Schell J, Van Montagu M (1979) Rapid mapping of transposon insertion and deletion mutations in the large Ti-plasmids of Agrobacterium tumefaciens. Nucl Acids Res 7:1837–1849
Douglas CJ, Halperin W, Nester EW (1982) Agrobacterium tumefaciens mutants affected in attachment to plant cells. J Bacteriol 152:1265–1275
Douglas CJ, Staneloni RJ, Rubin RA, Nester EW (1985) Identification and genetic analysis of an Agrobacterium tumefaciens chromosomal virulence region. J Bacteriol 161:850–860
Gheysen G, Dhaese P, Van Montagu M, Schell J (1985) DNA flux across genetic barriers: the crown gall phenomenon. In: Hohn B, Dennis ES (eds) Genetic flux in plants. Advances in plant gene research, vol 2. Springer, Wien New York, pp 11–47
Herrera-Estrella L, Depicker A, Van Montagu M, Schell J (1983a) Expression of chimaeric genes transferred into plant cells using a Ti-plasmid-derived vector. Nature (London) 303:209–213
Herrera-Estrella L, De Block M, Messens E, Hernalsteens J-P, Van Montagu M, Schell J (1983b) Chimeric genes as dominant selectable markers in plant cells. EMBO J 2:987–995
Holsters M, Silva B, Van Vliet F, Genetello C, De Block M, Dhaese P, Depicker A, Inzé D, Engler G, Villarroel R, Van Montagu M, Schell J (1980) The functional organization of the nopaline A. tumefaciens plasmid pTiC58. Plasmid 3:212–230
Koukolíková-Nicola Z, Shillito RD, Hohn B, Wang K, Van Montagu M, Zambryski P (1985) Involvement of circular intermediates in the transfer of T-DNA from Agrobacterium tumefaciens to plant cells. Nature (London) 313:191–196
Leemans J, Langenakens J, De Greve H, Deblaere R, Van Montagu M, Schell J (1982) Broad-host-range cloning vectors derived from the W-plasmid Sa. Gene 19:361–364
Lemmers M, De Beuckeleer M, Holsters M, Zambryski P, Depicker A, Hernalsteens J-P, Van Montagu M, Schell J (1980) Internal organization, boundaries and integration of Ti-plasmid DNA in nopaline crown gall tumors. J Mol Biol 144:353–376
Maliga P, Sz.-Breznovits A, Márton L, Joo F (1975) Non-Mendelian streptomycin-resistant tobacco mutant with altered chloroplasts and mitochondria. Nature (London) 255:401–402
Márton L, Wullems GJ, Molendijk L, Schilperoort RA (1979) In vitro transformation of cultured cells from Nicotiana tabacum by Agrobacterium tumefaciens. Nature (London) 277:129–130
Miller JH (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, New York, pp 466
Nagy JI, Maliga P (1976) Callus induction and plant regeneration from mesophyll protoplasts of Nicotiana sylvestris. Z Pflanzenphysiol 78:453–455
Neff NT, Binns AN (1985) Agrobacterium tumefaciens interaction with suspension-cultured tomato cells. Plant Physiol 77:35–42
Okker RJH, Spaink H, Hille J, van Brussel TAN, Lugtenberg B, Schilperoort RA (1984) Plant-inducible virulence promoter of the Agrobacterium tumefaciens Ti plasmid. Nature (London) 312:564–566
Otten LA, Schilperoort RA (1978) A rapid microscale method for the detection of lysopine and nopaline dehydrogenase activities. Biochim Biophys Acta 527:497–500
Shillito RD, Paszkowski J, Potrykus I (1983) Agarose plating and a bead type culture technique enable and stimulate development of protoplast-derived colonies in a number of plant species. Plant Cell Rep 2:244–247
Stachel SE, An G, Flores C, Nester EW (1985) A Tn3 lacZ transposon for the random generation of β-galactosidase gene fusions: applications to the analysis of gene expression in Agrobacterium. EMBO J 4:891–898
Van Haute E, Joos H, Maes M, Warren G, Van Montagu M, Schell J (1983) Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of Ti plasmids of Agrobacterium tumefaciens. EMBO J 2:411–418
Van Lijsebettens M, Inzé D, Schell J, Van Montagu M (1985) Transformed cell clones as a tool to study T-DNA integration mediated by Agrobacterium tumefaciens. J Mol Biol, in press
Villarroel R, Hedges RW, Maenhaut R, Leemans J, Engler G, Van Montagu M, Schell J (1983) Heteroduplex analysis of P-plasmid evolution: the role of insertion and deletion of transposable elements. Mol Gen Genet 189:390–399
Wang K, Herrera-Estrella L, Van Montagu M, Zambryski P (1984) Right 25-bp terminus sequences of the nopaline T-DNA is essential for and determines direction of DNA transfer from Agrobacterium to the plant genome. Cell 38:455–462
Zambryski P, Depicker A, Kruger K, Goodman H (1982) Tumor induction of Agrobacterium tumefaciens: analysis of the boundaries of T-DNA. J Mol Appl Genet 1:361–370
Author information
Authors and Affiliations
Additional information
Communicated by H. Saedler
Rights and permissions
About this article
Cite this article
Depicker, A., Herman, L., Jacobs, A. et al. Frequencies of simultaneous transformation with different T-DNAs and their relevance to the Agrobacterium/plant cell interaction. Molec Gen Genet 201, 477–484 (1985). https://doi.org/10.1007/BF00331342
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00331342