Transformation of suspension cultures of bromegrass (Bromus inermis) by Agrobacterium tumefaciens |
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Authors: | Toshihide Nakamura Masaya Ishikawa |
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Institution: | (1) Adaptation Systems Laboratory, Genetic Diversity Department, National Institute of Agrobiological Sciences, 305-8602 Tsukuba, Ibaraki, Japan |
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Abstract: | Smooth bromegrass (Bromus inermis Leyss) is an extremely cold hardy perennial grass and its cell culture is an excellent system for studying mechanisms of
cold hardiness induced by low temperature or abscisic acid (ABA). Agrobacterium tumefaciens-mediated transformation of non-embryogenic bromegrass cultures was attempted. Agrobacterium strain EHA105 carrying a binary vector that contained the neomycin phosphotransferase (NPT II), beta-glucuronidase (GUS)
and green fluorescent protein (GFP) genes were co-cultivated for 3 days with bromegrass cells at the late exponential or early
stationary growth phase (7–9 days after subculture). These conditions gave optimal transformation efficiency. Putative transformants
were identified by selection for geneticin resistance and by examining the calluses using fluorescence microscopy. This allows
the elimination of escapes and selection of cells that express the target genes. PCR and Southern blot analyses confirmed
the integration of the GUS and GFP genes into the genome of transformed bromegrass cell lines. Transformants with various
levels of GUS expression were obtained with a high frequency following Agrobacterium-mediated gene transfer and visual selection by GFP. The successful transformation method described allows reverse genetics
approaches for analyzing cold hardiness genes isolated from bromegrass cells. |
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Keywords: | cell growth stage culture growth phase genetic transformation GFP (green fluorescent protein) GUS smooth bromegrass |
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