Importance of co-cultivation medium pH for successful Agrobacterium-mediated transformation of Lilium x formolongi

• Published in: Plant cell reports Vol. 27; no. 4; pp. 699 - 705
• Main Authors: Ogaki, M, Furuichi, Y, Kuroda, K, Chin, D. P, Ogawa, Y, Mii, M
• Format: Journal Article
• Language: English
• Published: Germany Berlin/Heidelberg : Springer-Verlag 01.04.2008
• Subjects: Agrobacterium; Culture Media; Genetic Vectors; Hydrogen-Ion Concentration; Lilium; Lilium - genetics; Lilium - growth & development; Lilium - microbiology; Meristem - growth & development; Meristem - microbiology; Meristem - physiology; Plants, Genetically Modified - growth & development; Plants, Genetically Modified - microbiology; Plants, Genetically Modified - physiology; Rhizobium - metabolism
• ISSN: 0721-7714; 1432-203X
• DOI: 10.1007/s00299-007-0481-x

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 β-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 μM 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.