Transformation of haploid Datura innoxia protoplasts and analysis of the plasmid integration pattern in regenerated transgenic plants |
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Authors: | Thilo Schmidt-Rogge Martin Meixner Vibha Srivastava Sipra Guha-Mukherjee Otto Schieder |
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Institution: | (1) Institute for Applied Genetics, Free University of Berlin, Albrecht-Thaer-Weg 6, 14195 Berlin, Germany;(2) School of Life Sciences, Jawaharlal Nehru University, New Mehrauli Road, 11067 New Dehli, India |
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Abstract: | We developed a highly efficient transformation protocol for the PEG-mediated direct transfer of plasmid DNA into protoplasts of haploid Datura innoxia. Vectors harbouring a neomycin phosphotransferase II gene or a hygromycin B phosphotransferase gene under the control of different promoters were used in the transformation experiments. Various amounts of plasmid DNA were applied without any carrier DNA to show the direct influence of the plasmid DNA concentration on the transformation efficiency. Approximately 95% of the selected calli were regenerated to plants; 20% of them remained haploid. Total DNA of different transgenic plants was analysed with regard to the integration pattern of the plasmid DNA. Plants carrying only one or two copies of the vector DNA were observed as well as individuals with multi-copy integration (up to ten or more copies).Abbreviations ATF/RTF
absolute/relative transformation frequency
- BAP
6-benzylaminopurine
- CaMV
cauliflower mosaic virus
- CTAB
N-cetyl-N,N,N-trimethyl-ammonium bromide
- HPT
hygromycin B phosphotransferase gene
- PEG
polyethyleneglycol
- MES
2-(N-morpholino) ethanesulfonic acid
- NPT II
neomycin phosphotransferase II gene |
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Keywords: | Datura haploids direct gene transfer |
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