Abstract
A very simple leaf assay is described that rapidly and reliably identifies transgenic plants expressing the hygromycin resistance gene, hph or the phosphinothricin resistance gene, bar. Leaf tips were cut from plants propagated either in the glasshouse or in tissue culture and the cut surface embedded in solid medium containing the appropriate selective agent. Non-transgenic barley or rice leaf tips had noticeable symptoms of either bleaching or necrosis after three days on the medium and were completely bleached or necrotic after one week. Transgenic leaf tips remained green and healthy over this period. This gave unambiguous discrimination between transgenic and non-transgenic plants. The leaf assay was also effective for dicot plants tested (tobacco and peas).
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References
Datta, S.K., K. Datta, N. Soltanifar, G. Donn and I. Potrykus. 1992. Herbicide–resistant Indica rice plants from IRRI breeding line IR72 after PEG–mediated transformation of protoplasts. Plant Mol. Biol. 20:619–629.
Ellis, J.G., D.J. Llewellyn, E.S. Dennis and W.J. Peacock. 1987. Maize Adh1 promoter sequences control anaerobic regulation: addition of upstream promoter elements from constitutive genes is necessary for expression in tobacco. EMBO J. 6:11–16.
Gritz, L. and J. Davies. 1983. Plasmid–encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae. Gene 25:179–188.
Hiei, Y., S. Ohta, T. Komari and T. Kumashiro. 1994. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T–DNA. Plant J. 6:271–282.
Komari, T., Y. Hiei, Y. Saito, N. Murai and T. Kumashiro. 1996. Vectors carrying two separate T–DNAs for co–transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J. 10:165–174.
Linn, F., I. Heidmann, H. Saedler and P. Meyer. 1990. Epigenetic changes in the expression of the maize A1 gene in Petunia hybrida: a role of numbers of integrated gene copies and state of methylation. Mol. Gen. Genet. 222:329–336.
Murakami, T., H. Anzai, S. Imai, A. Satoh, K. Nagaoka and C.J. Thompson. 1986. The bialaphos biosynthetic genes of Streptomyces hygroscopicus: Molecular cloning and characterization of the gene cluster. Mol. Gen. Genet. 205:42–50.
Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473–497.
Schroeder, H.E., A.H. Schotz, T. Wardley–Richardson, D. Spencer and T.J.V. Higgins. 1993. Transformation and regeneration of two cultivars of pea (Pisum sativum L.). Plant Physiol. 101:751–757.
Tingay, S., D. McElroy, R. Kalla, S. Fieg, M. Wang, S. Thornton and R. Brettell. 1997. Agrobacterium tumefaciens–mediated barley transformation. Plant J. 11:1369–1376.
Wang, M.–B., N.M. Upadhyaya, R.I.S. Brettell and P.M. Waterhouse. 1997. Intronmediated improvement of a selectable marker gene for plant transformation using Agrobacterium tumefaciens. J. Genet. Breed. (in press).
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Wang, MB., Waterhouse, P.M. A Rapid and Simple Method of Assaying Plants Transformed with Hygromycin or PPT Resistance Genes. Plant Molecular Biology Reporter 15, 209–215 (1997). https://doi.org/10.1023/A:1007446721394
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DOI: https://doi.org/10.1023/A:1007446721394