BACKGROUND: Phenotypic characterization of transgenic cell lines, frequently used in plant biology studies, is complicated because transgene expression in individual cells is often heterogeneous and unstable. To identify the sources and to reduce this heterogeneity, we transformed tobacco (Nicotiana tabacum L.) BY-2 cells with a gene encoding green fluorescent protein (GFP) using Agrobacterium tumefaciens, and then introduced a simple cloning procedure to generate cell lines derived from the individual transformed cells. Expression of the transgene was monitored by analysing GFP fluorescence in the cloned lines and also in lines obtained directly after transformation. RESULTS: The majority ( approximately 90%) of suspension culture lines derived from calli that were obtained directly from transformation consisted of cells with various levels of GFP fluorescence. In contrast, nearly 50% of lines generated by cloning cells from the primary heterogeneous suspensions consisted of cells with homogenous GFP fluorescence. The rest of the lines exhibited "permanent heterogeneity" that could not be resolved by cloning. The extent of fluorescence heterogeneity often varied, even among genetically identical clones derived from the primary transformed lines. In contrast, the offspring of subsequent cloning of the cloned lines was uniform, showing GFP fluorescence intensity and heterogeneity that corresponded to the original clone. CONCLUSION: The results demonstrate that, besides genetic heterogeneity detected in some lines, the primary lines often contained a mixture of epigenetically different cells that could be separated by cloning. This indicates that a single integration event frequently results in various heritable expression patterns, which are probably accidental and become stabilized in the offspring of the primary transformed cells early after the integration event. Because heterogeneity in transgene expression has proven to be a serious problem, it is highly advisable to use transgenes tagged with a visual marker for BY-2 transformation. The cloning procedure can be used not only for efficient reduction of expression heterogeneity of such transgenes, but also as a useful tool for studies of transgene expression and other purposes.

Charles University Prague Faculty of Science Department of Plant Physiology Prague 2 Czech Republic

Zobrazit více v PubMed

Nagata T. When I encountered tobacco BY-2 cells! In: Nagata T, Hasezawa S, Inzé D, editor. Tobacco BY-2 Cells Biotechnology in Agriculture and Forestry. Vol. 53. Berlin Heidelberg New York: Springer; 2004. pp. 1–5.

Harashima H, Shinmyo A, Sekine M. Phosphorylation of threonine 161 in plant cyclin-dependent kinase A is required for cell division by activation of its associated kinase. Plant J. 2007;52:435–448. PubMed

Kuthanova A, Fischer L, Nick P, Opatrny Z. Cell cycle phase-specific death response of tobacco BY-2cell line to cadmium treatment. Plant Cell Environ. 2008;31:1634–1643. PubMed

Klein TM, Harper EC, Svab Z, Sanford JC, Fromm ME, Maliga P. Stable genetic-transformation of intact Nicotiana cells by the particle bombardment process. Proc Natl Acad Sci U S A. 1988;85:8502–8505. PubMed PMC

An GH. High-efficiency transformation of cultured tobacco cells. Plant Physiol. 1985;79:568–570. PubMed PMC

Higaki T, Kutsuna N, Sano T, Hasezawa S. Quantitative analysis of changes in actin microfilament contribution to cell plate development in plant cytokinesis. BMC Plant Biol. 2008;8:80. PubMed PMC

Joubes J, Inzé D, Geelen D. Improvements of the molecular toolbox for cell cycle studies in tobacco BY-2 cells. In: Nagata T, Hasezawa S, Inzé D, editor. Tobacco BY-2 Cells Biotechnology in Agriculture and Forestry. Vol. 53. Berlin Heidelberg New York: Springer; 2004. pp. 7–20.

Pröls F, Meyer P. The methylation patterns of chromosomal integration regions influence gene activity of transferred DNA in Petunia hybrida. Plant J. 1992;2:465–475. PubMed

Iglesias VA, Moscone EA, Papp I, Neuhuber F, Michalowski S, Phelan T, et al. Molecular and cytogenetic analyses of stably and unstably expressed transgene loci in tobacco. Plant Cell. 1997;9:1251–1264. PubMed PMC

van Leeuwen W, Ruttink T, Borst-Vrenssen AWM, Plas LHW van der, Krol AR van der. Characterization of position-induced spatial and temporal regulation of transgene promoter activity in plants. J Exp Bot. 2001;52:949–959. PubMed

Schubert D, Lechtenberg B, Forsbach A, Gils M, Bahadur S, Schmidt R. Silencing in Arabidopsis T-DNA transformants: The predominant role of a gene-specific RNA sensing mechanism versus position effects. Plant Cell. 2004;16:2561–2572. PubMed PMC

Francis KE, Spiker S. Identification of Arabidopsis thaliana transformants without selection reveals a high occurrence of silenced T-DNA integrations. Plant J. 2005;41:464–477. PubMed

Depicker A, Sanders M, Meyer P. Transgene silencing. In: Meyer P, editor. Plant Epigenetics. Vol. 19. Blackwell Publishing; 2005. pp. 1–31.

Kim SI, Veena , Gelvin SB. Genome-wide analysis of Agrobacterium T-DNA integration sites in the Arabidopsis genome generated under non-selective conditions. Plant J. 2007;51:779–791. PubMed

Fischer U, Kuhlmann M, Pecinka A, Schmidt R, Mette MF. Local DNA features affect RNA-directed transcriptional gene silencing and DNA methylation. Plant J. 2008;53:1–10. PubMed

Müller E, Lorz H, Lutticke S. Variability of transgene expression in clonal cell lines of wheat. Plant Sci. 1996;114:71–82.

Down RE, Ford L, Bedford SJ, Gatehouse LN, Newell C, Gatehouse JA, et al. Influence of plant development and environment on transgene expression in potato and consequences for insect resistance. Transgenic Res. 2001;10:223–236. PubMed

Kain SR, Adams M, Kondepudi A, Yang TT, Ward WW, Kitts P. Green fluorescent protein as a reporter of gene-expression and protein localization. Biotechniques. 1995;19:650–655. PubMed

Ruiz MT, Voinnet O, Baulcombe DC. Initiation and maintenance of virus-induced gene silencing. Plant Cell. 1998;10:937–946. PubMed PMC

Kalantidis K, Tsagris M, Tabler M. Spontaneous short-range silencing of a GFP transgene in Nicotiana benthamiana is possibly mediated by small quantities of siRNA that do not trigger systemic silencing. Plant J. 2006;45:1006–1016. PubMed

Mirabella R, Franken C, Krogt GNM van der, Bisseling T, Geurts R. Use of the fluorescent timer DsRED-E5 as reporter to monitor dynamics of gene activity in plants. Plant Physiol. 2004;135:1879–1887. PubMed PMC

Henderson IR, Jacobsen SE. Epigenetic inheritance in plants. Nature. 2007;447:418–424. PubMed

Lunerova-Bedrichova J, Bleys A, Fojtova M, Khaitova L, Depicker A, Kovarik A. Trans-generation inheritance of methylation patterns in a tobacco transgene following a post-transcriptional silencing event. Plant J. 2008 PubMed

Fojtova M, Van Houdt H, Depicker A, Kovarik A. Epigenetic switch from posttranscriptional to transcriptional silencing is correlated with promoter hypermethylation. Plant Physiol. 2003;133:1240–1250. PubMed PMC

Gelvin SB, Kim SI. Effect of chromatin upon Agrobacterium T-DNA integration and transgene expression. Biochim Biophys Acta. 2007;1769:410–421. PubMed

Neuhuber F, Park YD, Matzke AJM, Matzke MA. Susceptibility of transgene loci to homology-dependent gene silencing. Mol Gen Genet. 1994;244:230–241. PubMed

Brodersen P, Voinnet O. The diversity of RNA silencing pathways in plants. Trends Genet. 2006;22:268–280. PubMed

Santi DV, Norment A, Garrett CE. Covalent bond formation between a DNA-cytosine methyltransferase and DNA containing 5-azacytosine. Proc Natl Acad Sci U S A. 1984;81:6993–6997. PubMed PMC

Koukalova B, Fojtova M, Lim KY, Fulnecek J, Leitch AR, Kovarik A. Dedifferentiation of tobacco cells is associated with ribosomal RNA gene hypomethylation, increased transcription, and chromatin alterations. Plant Physiol. 2005;139:275–286. PubMed PMC

Muir WH, Hildebrandt AC, Riker AJ. The preparation, isolation, and growth in culture of single cells from higher plants. Am J Bot. 1958;45:589–597.

Bergmann L. New technique for isolating and cloning cells of higher plants. Nature. 1959;184:648–649.

Pischke MS, Huttlin EL, Hegeman AD, Sussman MR. A transcriptome-based characterization of habituation in plant tissue culture. Plant Physiol. 2006;140:1255–1278. PubMed PMC

Nagata T, Nemoto Y, Hasezawa S. Tobacco BY-2 cell-line as the Hela-cell in the cell biology of higher-plants. Int Rev Cytol. 1992;132:1–30.

Murashige T, Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plantarum. 1962;15:473.

Deblaere R, Bytebier B, Degreve H, Deboeck F, Schell J, Vanmontagu M, et al. Efficient octopine Ti plasmid-derived vectors for Agrobacterium-mediated gene-transfer to plants. Nucleic Acids Res. 1985;13:4777–4788. PubMed PMC

Bolte S, Brown S, Satiat-Jeunemaitre B. The N-myristoylated Rab-GTPase m-Rab(mc) is involved in post-Golgi trafficking events to the lytic vacuole in plant cells. J Cell Sci. 2004;117:943–954. PubMed

Davis SJ, Vierstra RD. Soluble, highly fluorescent variants of green fluorescent protein (GFP) for use in higher plants. Plant Mol Biol. 1998;36:521–528. PubMed

Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 1989.

Sulfadiazine and phosphinothricin selection systems optimised for the transformation of tobacco BY-2 cells

Detailed insight into the dynamics of the initial phases of de novo RNA-directed DNA methylation in plant cells

Correlation of Autophagosome Formation with Degradation and Endocytosis Arabidopsis Regulator of G-Protein Signaling (RGS1) through ATG8a

Pervasive read-through transcription of T-DNAs is frequent in tobacco BY-2 cells and can effectively induce silencing

Hybrid proline-rich proteins: novel players in plant cell elongation?

Successive silencing of tandem reporter genes in potato (Solanum tuberosum) over 5 years of vegetative propagation