hygromycin-a and phosphinothricin

Alfalfa (Medicago sativa L.) is a high-quality forage crop widely grown throughout the world. This chapter describes an efficient protocol that allows for the generation of large number of transgenic alfalfa plants by sonication-assisted Agrobacterium-mediated transformation. Binary vectors carrying different selectable marker genes that confer resistance to phosphinothricin (bar), kanamycin (npt II), or hygromycin (hph) were used to generate transgenic alfalfa plants. Intact trifoliates collected from clonally propagated plants in the greenhouse were sterilized with bleach and then inoculated with Agrobacterium strain EHA105. More than 80 % of infected leaf pieces could produce rooted transgenic plants in 4-5 months after Agrobacterium-mediated transformation.

Topics: Agriculture; Aminobutyrates; Cinnamates; Coculture Techniques; Drug Resistance; Genetic Techniques; Genetic Vectors; Hygromycin B; Kanamycin; Medicago sativa; Plant Leaves; Plants, Genetically Modified; Seeds; Sterilization; Transformation, Bacterial

Antibiotic resistance marker genes are powerful selection tools for use in plant transformation processes. However, once transformation is accomplished, the presence of these resistance genes is no longer necessary and can even be undesirable. We herein describe the successful excision of antibiotic resistance genes from transgenic plants via the use of an oxidative stress-inducible FLP gene. FLP encodes a recombinase that can eliminate FLP and hpt selection genes flanked by two FRT sites. During a transformation procedure in tobacco, transformants were obtained by selection on hygromycin media. Regenerants of the initial transformants were screened for selective marker excision in hydrogen peroxide supplemented media and both the FLP and hpt genes were found to have been eliminated. About 13-41% of regenerated shoots on hydrogen peroxide media were marker-free. This auto-excision system, mediated by the oxidative stress-inducible FLP/FRT system to eliminate a selectable marker gene can be very readily adopted and used to efficiently generate marker-free transgenic plants.

Topics: Aminobutyrates; Anti-Bacterial Agents; Cinnamates; Drug Resistance, Bacterial; Genetic Vectors; Hydrogen Peroxide; Hygromycin B; Nicotiana; Oxidative Stress; Plants, Genetically Modified; Promoter Regions, Genetic; Recombinases; Recombination, Genetic

The application of aminoglycoside-3"-adenyltransferase ( aadA) gene-mediated streptomycin resistance for non-lethal selection of transgenic rice resulted in plant regeneration frequencies under selection pressure as high as those in non-transformed controls without selection. Since streptomycin does not kill non-transgenic cells, and allows plant regeneration from them, a selection procedure was developed that made the visual identification of transgenic calli and regenerants possible. For callus-level selection, a vital pH indicator-Chlorophenol Red-was applied together with streptomycin, making use of the phenomenon that fast-growing cell lines lower the pH in the culture medium. Transgenic plants were selected according to their main distinctive features; their green colour (photomixotrophic assimilation), and more intense growth. At the same time, non-transgenic regenerants were bleached (heterotrophic assimilation), and growth was retarded in the presence of streptomycin and sucrose. The final efficiency of genetic transformation based on streptomycin resistance was found to be double that of transformations where the selective agent was l-phosphinothricin, and nearly three times more compared to transformations resulting in hygromycin-resistant regenerants. To the best of our knowledge, this is the first report on producing nuclear transformed rice plants by using a non-lethal selection strategy based on the chimaeric aadA gene.

Topics: Aminobutyrates; Base Sequence; Cinnamates; DNA, Recombinant; Drug Resistance; Genetic Markers; Herbicides; Hygromycin B; Oryza; Plants, Genetically Modified; Selection, Genetic; Streptomycin; Transformation, Genetic

Topics: Agrobacterium tumefaciens; Aminobutyrates; Bleomycin; Cinnamates; DNA, Recombinant; Drug Resistance; Electric Stimulation; Gene Expression; Genetic Markers; Gentamicins; Herbicides; Hygromycin B; Kanamycin; Kanamycin Kinase; Methotrexate; Oryza; Phosphotransferases; Protoplasts; Recombinant Proteins; Transfection; Transformation, Genetic

Chimeric hygromycin phosphotransferase (hph) and phosphinothricin acetyltransferase (bar) genes were introduced, using polyethylene glycol treatment, into protoplasts isolated from embryogenic cell suspension cultures of tall fescue (Festuca arundinacea Schreb.), a graminaceous plant that is an important forage crop in temperate pastures. Colonies resistant to either 200 mg/l hygromycin or 100 mg/l phosphinothricin, respectively, were recovered upon selection using bead-type culture systems. Stable integration of the transgenes in the genomes of plants regenerated from resistant callus clones was shown by Southern hybridization analysis. In situ hybridization of a labeled transgene-probe to metaphase chromosomes is shown for one transgenic primary regenerant. Expression of the transgenes in mature plants was demonstrated by HPH enzyme assay or by phosphinothricin-herbicide spraying.

Topics: Acetyltransferases; Aminobutyrates; Base Sequence; Cells, Cultured; Cinnamates; Drug Resistance; Gene Expression; Hygromycin B; Molecular Sequence Data; Nucleic Acid Hybridization; Phosphotransferases; Phosphotransferases (Alcohol Group Acceptor); Plants; Polyethylene Glycols; Polymerase Chain Reaction; Protoplasts; Transfection