Importance of co-cultivation medium pH for successful Agrobacterium-mediated transformation of Lilium x formolongi.
An efficient system for Agrobacterium-mediated transformation of Lilium x formolongi was established by preventing the drastic drop of pH in the co-cultivation medium with MES. Meristematic nodular calli were inoculated with an overnight culture of A. tumefaciens strain EHA101 containing the plasmid pIG121-Hm which harbored intron-containing beta-glucuronidase (GUS), hygromycin phosphotransferase (HPT), and neomycin phosphotransfease II (NPTII) genes. After three days of co-cultivation on 2 g/l gellan gum-solidified MS medium containing 100 microM acetosyringone, 30 g/l sucrose, 1 mg/l picloram and different concentrations of MES, they were cultured on the same medium containing 12.5 mg/l meropenem to eliminate Agrobacterium for 2 weeks and then transferred onto medium containing the same concentration of meropenem and 25 mg/l hygromycin for selecting putative transgenic calli. Transient GUS expression was only observed by adding MES to co-cultivation medium. Hygromycin-resistant transgenic calli were obtained only when MES was added to the co-cultivation medium especially at 10 mM. The hygromycin-resistant calli were successfully regenerated into plantlets after transferring onto picloram-free medium. Transformation of plants was confirmed by histochemical GUS assay, PCR analysis and Southern blot analysis.
Graduate School of Horticulture, Chiba University,Matsudo, Chiba, 271-8510, Japan. ogaki-mmk@msb.biglobe.ne.jp
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Ogaki
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Furuichi
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Kuroda
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Chin
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Ogawa
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Mii
D003470:Culture Media; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D027762:Lilium / Q000235:genetics* / Q000254:growth & development / Q000382:microbiology; D018519:Meristem / Q000254:growth & development / Q000382:microbiology / Q000502:physiology; D030821:Plants, Genetically Modified / Q000254:growth & development / Q000382:microbiology / Q000502:physiology*; D012231:Rhizobium / Q000378:metabolism*
D003470:Culture Media