Abstract
Chimeric β-glucuronidase (GUS) gene expression in an efficientAgrobacterium-mediated transformation system utilising mesophyll cells ofPetunia hybrida synchronized with cell cycle phase-specific inhibitors (mimosine and colchicine) was used to show the absolute requirement of S-phase for transfer and/or integration of the transferred DNA (T-DNA). Flow-cytometric analysis of nuclear DNA content and immunohistological detection of bromodeoxyuridine (BrdUrd) incorporation showed that, prior to phytohormone treatment, most (98%) mesophyll cells were at GO-Gl-phase (quiescent phase) and no cell division was occurring. After 48 h and 72 h of phytohormone treatment, there was a rapid increase in S-G2-M-phase populations (> 75%) and a concomitant decrease (down to 24%) in G0–-G1-phase cells. Assays of GUS showed that maximum transformation (> 95% of explants) also occurred after this period. Our data showed that mimosine and colchicine blocked the mesophyll cells at late Gl-phase and M-phase, respectively. No transformation (= GUS expression) was observed in phytohormone-treated cells inhibited in late G1 by mimosine. However, after removal of mimosine, 82% of the explants were transformed, indicating the non-toxic and reversible effect of the inhibitor. On the other hand, a relatively high transformation frequency (65% of explants) was observed after blocking the cell cycle at M-phase with colchicine. However, only transient, but no stable, gene expression (= kanamycin-resistant callus formation) was observed in colchicine-treated M-phase-arrested cells. Similarly, endoreduplication of nuclear DNA, which occurred during the 48 h of phytohormone treatment in some mesophyll cells and cells located along the minor veins in the leaf explants, resulted in transient GUS expression only. These observations indicate a direct correlation between endoreduplication and transient GUS gene expression. Obviously, for stable GUS gene expression, cell division and proliferation are required, indicating that both DNA duplication (S-phase) and cell division (M-phase) are strongly related to stable transformation. We propose that the present system should facilitate further dissection of the process of T-DNA integration in the host genome and therefore should aid in developing new strategies for transformation of recalcitrant plants.
Similar content being viewed by others
Abbreviations
- BAP:
-
6-benzylaminopurine
- BM:
-
basal medium
- BrdUrd:
-
bromodeoxyuridine
- GUS:
-
β-glucuronidase
- KmR :
-
kanamycin resistant
- T-DNA:
-
transferred DNA
References
Akama K, Shiraishi H, Ohta S, Nakamura K, Okada K, Shimura Y (1992) Efficient transformation ofArabidopsis thaliana: comparison of the efficiencies with various organs, plant ecotypes andAgrobacterium strains. Plant Cell Rep 12: 7–11
An G (1985) High efficiency transformation of cultured tobacco cells. Plant Physiol 79: 568–570
Benediktsson I, Köhler F, Sebieder O (1991) Transient and stable expression of marker genes in cotransformedPetunia protoplasts in relation to X-ray and UV-irradiation. Transgenic Res 1: 38–44
Bergounioux C, Perennes C, Brown SC, Sarda C, Gadal P (1988) Relation between protoplast division cell-cycle stage and nuclear chromatin. Protoplasma 142: 127–136
Carr AM, Hoekstra MF (1995) The cellular responses to DNA damage. Trends Cell Biol 5: 32–39
Citovsky V, Howard E, Winsor B, Zambryski P (1988) Proteins that mediate DNA transfer byAgrobacterium tumefaciens to plant cells: summary and perspectives. In: Staskawicz P, Ahlquist P, Yoder, (eds) UCLA Symp. Mol. Cell. Biol, Ns, vol 101 B. Alan R. Liss, New York
Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 4: 19–21
Ducrocq C, Sangwan RS, Sangwan-Norreel BS (1994) Production ofAgrobacterium-mediated transgenic fertile plants by direct somatic embryogenesis from immature zygotic embryos ofDatura innoxia. Plant Mol Biol 25: 995–1009
Glab N, Labidi B, Qin LX, Trehin C, Bergounioux C, Meijer L (1994) Olomoucine, an inhibitor of the cdc2/cdck2 kinases activity, blocks plant cells at the G1 to S and G2 to M cell cycle transitions. FEBS Lett 353: 207–211
Hooykass PJ, Beijersbergen AGM (1994) The virulence system ofAgrobacterium tumefaciens. Annu Rev Phytopathol 32: 157–179
Janssen BJ, Gardner CR (1989) Localized transient expression of GUS in leaf discs following cocultivation withAgrobacterium tumefaciens. Plant Mol Biol 14: 61–72
Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5: 387–407
Jong T, Mertens MMJ, Rademaker W (1994) Stable expression of the GUS reporter gene inChrysanthemum depends on binary plasmid T-DNA. Plant Cell Rep 14: 59–64
Kartzke S, Saedler H, Meyer P (1990) Molecular analysis of transgenic plants derived from transformations of protoplasts at various stages of the cell cycle. Plant Sci 67: 63–72
Kathen A, de, Jacobsen HJ (1995) Cell competence forAgrobacterium-mediated DNA transfer inPisum sativum L. Transgenic Res 4: 184–191
Köhler F, Cardon G, Pöhlman M, Gill R, Schieder O (1989) Enhancement of transformation rates in higher plants by lowdose irradiation: Are DNA repair systems involved in the incorporation of exogenous DNA into the plant genome? Plant Mol Biol 12: 189–199
Koncz C, Olsson O, Langridge WHR, Schell J, Szalay AA (1987) Expression and functional assembly of bacterial luciferase in plants. Proc Natl Acad Sci USA 84: 131–135
Li XQ, Liu CN, Ritchie SW, Peng JY, Gelvin SB, Hodges TK (1992) Factors influencingAgrobacterium-mediated transient expression of GUS A in rice. Plant Mol Biol 20: 1037–1048
Lida A, Yamashita T, Yamada Y, Morikawa H (1991) Efficiency of particle-bombardement-mediated transformation is influenced by cell cycle stage in synchronized cultured cells of tobacco. Plant Physiol 97: 1585–1587
Lowe BA, Krul WR (1991) Physical, chemical, developmental, and genetic factors that modulate theAgrobacterium-vitis interaction. Plant Physiol 96: 121–129
Marie D, Brown SC (1993) A cytometric exercise in plant DNA histograms with 2C values for 70 species. Biol Cell 78: 41–51
Mayerhofer R, Koncz-Kalman Z, Nawrath C, Bakkeren G, Crameri A, Angelis K, Redei GP, Schell J, Hohn B, Koncz C (1991) T-DNA integration: a mode of illegitimate recombination in plants. EMBO J 10: 697–704
Mozo T, Hooykaas PJJ (1992) Factors affecting the rate of T-DNA transfer fromAgrobacterium tumefaciens toNicotiana glauca plant cells. Plant Mol Biol 19: 1019–1030
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 437–497
Nagata Z, Okada K, Takebe I (1986) Strong dependency of the transformation of plant protoplast on cell cycle. In: Kado C (ed) Fallen Leaf Lake conference onAgrobacterium and crown gall. University of California, Davis, USA, pp 9
Nagl W (1972) Selective inhibition of cell cycle stages in theAllium root meristem by colchicine and growth regulators. Am J Bot 59: 346–359
Perennes C, Qin LX, Glab N, Bergounioux C (1993)Petunia p32 cdc2 protein kinase activity in G2/M cells obtained with a reversible cell cycle inhibitor, mimosine. FEBS Lett 333: 141–145
Rossi L, Hohn B, Tinland B (1993) The VirD2 protein ofAgrobacterium tumefaciens carries nuclear localization signals important for transfer of T-DNA to plants. Mol Gen Genet 239: 345–535
Sangwan RS, Harada H (1975) Chemical factors contolling morphogenesis ofPetunia cell cultured in vitro. Biochem Physiol Pflanz 170: 77–84
Sangwan RS, Bourgeois Y, Sangwan-Norreel B (1991) Genetic transformation ofArabidopsis thaliana zygotic embryos and identification of critical parameters influencing transformation efficiency. Mol Gen Genet 230: 475–485
Sangwan RS, Bourgeois Y, Brown S, Vasseur G, Sangwan-Norreel B (1992) Characterization of competent cells and early events ofAgrobacterium-mediated genetic transformation inArabidopsis thaliana. Planta 188: 439–456
Sangwan RS, Dubois F, Ducrocq C, Bourgeois Y, Vilcot B, Pawlicki N, Sangwan-Norreel BS (1995) The embryo as a tool for genetic engineering in higher plants. In: Teeri M, Cella R, Falavigna A (eds) Current issues in plant molecular and cellular biology. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 271–277
Shivji MKK, Kenny MK, Wood RD (1992) Proliferating cell nuclear antigen is required for DNA excision repair. Cell 69: 367–374
Sonti RV, Chiurazzi M, Wong D, Davis CS, Harlow GR, Mount DW, Sigmer ER (1995)Arabidopsis mutants deficient in TDNA integration. Proc Natl Acad Sci USA 92: 11786–11790
Teeri TH, Lehaväslaiko H, Franck M, Uotila J, Heino P, Palva ET, Van Montagu M, Herrera-Estrella L (1989) Gene fusion to lacZ reveals new expression patterns of chimeric genes in transgenic plants. EMBO 18: 343–350
Valvekens D, Van Montagu M, Van Lijsbettens M (1988)Agrobacterium tumefaciens mediated transformation ofArabidopsis thaliana root explants by using kanamycin selection. Procd Natl Acad Sci USA 85: 5536–5540
Vancanneyt G, Schmidt R, O'Connor-Sanchez A, Willmitzer R, Rocha-Sosa M (1990) Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events inAgrobacterium-mediated plant transformation. Mol Gen Genet 220: 245–250
Watson PA, Hanauske-Abel HH, Flint A, Lalande M (1991) Mimosine reversibly arrests cell cycle progression at the G1-S phase border. Cytometry 12: 242–246
Zambryski P (1992) Chronicles from theAgrobacterium plant cell DNA transfer story. Annu Rev Plant Physiol Plant Mol Biol 43: 465–490
Zupan JR, Zambryski P (1995) Transfer of T-DNA fromAgrobacterium to the plant cell. Plant Physiol 107: 1041–1047
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Villemont, E., Dubois, F., Sangwan, R.S. et al. Role of the host cell cycle in theAgrobacterium-mediated genetic transformation ofPetunia: Evidence of an S-phase control mechanism for T-DNA transfer. Planta 201, 160–172 (1997). https://doi.org/10.1007/BF01007700
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01007700