Abstract
Resistance to bialaphos, a broad-spectrum herbicide, was introduced into Lactuca sativa cv. Evola by Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strains 0310 and 1310, both carrying the bialaphos resistance (bar) and neomycin phosphotransferase (nptII) genes, were used for transformation. Primary transformants were selected on kanamycin sulphate-supplemented shoot regeneration medium. Integration of both transgenes was confirmed by non-radioactive Southern hybridisation. The hypervirulent plasmid ToK47 in A. tumefaciens strain 1310 generated multiple insertions of T-DNA in some transgenic plants; the absence of pToK47 (strain 0310) resulted in single gene inserts in all plants tested. Resistance to glufosinate ammonium was observed in axenic seedlings grown on medium supplemented with the herbicide at 5 mg l−1 and in glasshouse-grown plants sprayed with the compound at 300 mg l−1. Stable expression of the bar gene was observed in R2 generation plants. The kanamycin resistance of R1 seedlings was observed by germinating seeds on medium supplemented with 200 mg l−1 kanamycin sulphate. The presence of NPTII protein and PAT enzyme activity were demonstrated by ELISA and PAT enzyme assay respectively. Transgenes segregated in a Mendelian fashion in some plant lines in the R1 generation; herbicide resistance also segregated in the expected ratio in the R2 generation in most transgenic lines. This study confirmed that an agronomically important transgene can be integrated and stably expressed over several generations in lettuce.
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Akama, K., Puchata, H. and Hohn, B. (1995) Efficient Agrobacterium-mediated transformation of Arabidopsis thaliana using the bar gene as selectable marker. Plant Cell Rep. 14, 450–4.
Anderson, W.P. (1996) Lettuce. In: Richards, L. ed., Weed Science, Principles and Applications, pp. 313–4. Minneapolis, St. Paul, USA: West Publishing Company.
Bedford, D.J., Lewis, C.G. and Buttner, N.J. (1991) Characteriza-tion of a gene conferring bialaphos resistance in Streptomyces coelicolor A3(2). Gene 104, 39–45.
Bradford, M.M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analy. Biochem. 72, 248–54.
Broer, I. (1996) Stress inactivation of foreign genes in transgenic plants. LK 45, 19–25.
Cabrera-Ponce, J.L., Vegas-Garcia, A. and Herrera-Estrella, L. (1995) Herbicide resistant transgenic papaya plants produced by an efficient particle bombardment transformation method. Plant Cel. Rep. 15, 1–7.
Curtis, I.S., Power, J.B., Blackhall, N.W., de Laat, A.M.M. and Davey, M.R. (1994) Genotype-independent transformation of lettuce using Agrobacterium tumefaciens. K. 45, 1441–9.
Curtis, I.S., He, C., Scott, R., Power J.B. and Davey, M.R. (1996a) Genomic male sterility in lettuce, a baseline for the production of F1 hybrids. S 113, 113–9.
Curtis, I.S., He, C., Power, J.B., Mariotti, D., de Laat, A.M.M. and Davey, M.R. (1996b) The effects of Agrobacterium rhizogenes rolAB genes in lettuce. J 115, 123–5.
Dellaporta, S.L., Wood, J. and Hicks, J.B. (1983) A plant DNA minipreparation: version II. KJ. 1, 19–21.
D'Halluin, K., De Block, M., Denecke, J., Janssens, J., Leemans, J., Reynaerts, A. and Botterman, J. (1992) The bar gene as selectable and screenable marker in plant engineering. DH. 216, 415–26.
Finnegan, J. and McElroy, D. (1994) Transgene inactivation: plants fight back! K. 12, 883–8.
Gallo-Meagher, M. and Irivine J.E. (1996) Herbicide resistant transgenic sugarcane plants containing the bar gene. JSDHFK. 36, 1367–74.
Gordon-Kamm, W., Spencer, T.M., Mangano, M.L., Adams, T.R., Daines, R.J., Start, W.G., O'Brien, J.V., Chambers, S.A., Adams, W.R. Jr., Willetts, N.G., Rice, T.B., Mackey, C.J., Krueger, R.W., Kausch, A.P. and Lemaux, P.G. (1990) Transformation of maize cells and regeneration of fertile transgenic plants. The Plant Cell 2, 603–18.
Hoekema, A., Hirsch, P.R., Hooykaas, P.J. and Schilperoort, R.A. (1983) A binary plant vector strategy based on the separation of the vir and T-region of Agrobacteria. JD303, 179–81.
Jin, S., Komari, T., Gordon, M.P. and Nester, E. (1987) Genes responsible for the supervirulence phenotype of Agrobacterium tumefaciens A281. J 169, 4417–25.
Kar, S., Johnson, T.M., Nayak, P. and Sen, S.K. (1996) Efficient transgenic plant regeneration through Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.). Plant Cell Rep. 16, 32–7.
Keller, G., Spatola, L., McCabe, D., Martinell, B., Swain, W. and John, M.E. (1997) Transgenic cotton resistant to herbicide bialaphos. Transgenic Res. 6, 385–92.
Matzke, M.A. and Matzke, A.J.M. (1995) How and why do plants inactivate homologous (trans)genes? Plant Physiol. 107, 679–85.
McCabe, M.S., Mohapatra, U., Power, J.B., Davey, M.R. and de Laat, A.M.M. (1997a) T-DNA integration and expression of the gus and npt II genes in lettuce (Lactuca sativa L.). J. Exp. Bot. 48(Suppl.) p. 69.
McCabe, M.S., Power, J.B., de Laat A.M.M. and Davey M.R. (1997b) Detection of single-copy genes in DNA from transgenic plants by non-radioactive Southern blot analysis. Mol. Biotechnol. 7, 1–6.
McCabe, M.S., Mohapatra, U., Power, J.B., Davey, M.R., Schepers, F. and van Dun, K. (1998) Delayed senescence in transgenic lettuce using an autoregulated ipt gene. J. Exp. Bot. 49, (Suppl.) p. 49.
McHughen, A. and Holm, F.A. (1995) Development and preliminary field testing of a glufosinate-ammonium tolerant transgenic flax. Can. J. Plant Sci. 75, 117–20.
Meyer, P., Lohuis, M. T., Van Blokland, R., Heidmann, I. and Niedenhof, I. (1996) The role of DNA methylation in transgene silencing in plants. In Grierson, D., Lycett, G.W. and Tucker, G.A. eds., Mechanisms and Applications of Gene Silencing, pp. 43–8. Nottingham: Nottingham University Press.
Michelmore, R.W., Marsh, E., Seely, S. and Landry, B. (1987) Transformation of lettuce (Lactuca sativa) mediated by Agrobacterium tumefaciens. Plant Cell Rep.6, 439–42.
Mohapatra, U., McCabe, M.S., Power, J.B., Davey, M.R. and de Laat, A.M.M. (1997) Herbicide resistance conferred by integration and expression of the bar gene in lettuce (Lactuca sativa L.). J. Exp. Bot. 48 Suppl.) p.68–9.
Mohapatra, U., McCabe, M.S., Power, J.B., Davey, M.R., Schepers, F. and van Dun, K. (1998) Stable inheritance of a herbicide resistance gene driven by a pea plastocyanin promoter in transgenic lettuce. J. Exp. Bot. 49(Suppl.) p. 17.
Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–97.
Ooms G., Hooykaas P.J.J., Moolenaar G., and Schilperoort, R.A. (1981) Crown gall plant tumours of abnormal morphology induced by Agrobacterium tumefaciens carrying mutated octopine Ti plasmids: analysis of T-DNA functions. Gene 14, 33–50.
Pang, S-Z., Jan, F-J., Carney, K., Stout, J., Tricoli, D.M., Quemada., H.D., and Gonsalves, D. (1996) Post-transcriptional transgene silencing and consequent tospovirus resistance in transgenic lettuce are affected by transgene dosage and plant development. The Plant J. 9, 899–909.
Pwee, K.H. and Gray, J.C. (1993) The pea plastocyanin promoter directs cell-specific but not full light-regulated expression in transgenic tobacco plants. The Plant J. 3, 437–49.
Sijmons, P.C., Dekker, B.M.M., Schrammeijer, B., Verwoerd, T.C., Van den Elzen, P.J.M. and Hoekema, A. (1990) Production of correctly processed human serum albumin in transgenic plants. Bio/Technol. 8, 217–21.
Strauch, E., Wohlleben, W. and PuÈhler, A. (1988) Cloning of a phosphinothricin-N-acetyltransferase gene from Streptomyces viridochromogenes Tü494 and its expression in Streptomyces lividans and Escherichia coli. Gene 63, 65–74.
Thompson, C.J., Rao Movva, N., Tizard, R., Crameri, R., Davies, J.E., Lauwereys, M. and Botterman, J. (1987) Characterisation of the herbicide-resistance gene bar from Streptomyces hygroscopicus. EMBO J. 6, 2519–23.
Expression of the bar gene confers herbicide resistance in transgenic lettuce 43 Tinland, B. (1996) The integration of T-DNA into plant genomes. Trends Plant Sci. 1, 178–84.
Wang, M-B. and Waterhouse, P.M. (1997) A rapid and simple method of assaying plants transformed with hygromycin or PPT resistance genes. Plant Mol. Biol. Rep. 15, 209–15.
Wehrmann, A., Van Vliet, A., Opsomer, C., Botterman, J. and Schulz, A. (1996) The similarities of bar and pat gene products make them equally applicable for plant engineers. Nature Biotechnol. 14, 1274–8.
Yamazaki, M., Son, L., Hayashi, T., Morita, N., Asamizu, T., Mourakoshi, I. and Saito, K. (1996) Transgenic fertile Scoparia dulcis L., a folk medicinal plant, conferred with a herbicide-resistant trait using an Ribinary vector. Plant Cell Rep. 15, 317–21.
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Mohapatra, U., McCabe, M.S., Power, J.B. et al. Expression of the Bar Gene Confers Herbicide Resistance in Transgenic Lettuce. Transgenic Res 8, 33–44 (1999). https://doi.org/10.1023/A:1008891216134
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DOI: https://doi.org/10.1023/A:1008891216134