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

  • Genetic Transformation and Hybridization
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Abstract

An efficient system for Agrobacterium-mediated transformation of Lilium × 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.

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Abbreviations

AS:

Acetosyringone

CTAB:

Cetyltrimethylammonium bromide

GUS:

β-Glucuronidase

Hm:

Hygromycin

MES:

2-morpholinoethane-sulfonic acid

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Author notes

  1. Y. Ogawa

    Present address: Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba, 292-0818, Japan

Authors and Affiliations

  1. Laboratory of Plant Cell Technology, Graduate School of Horticulture, Chiba University, 648 Matsudo, Matsudo, Chiba, 271-8510, Japan

    M. Ogaki, Y. Furuichi, K. Kuroda, D. P. Chin, Y. Ogawa & M. Mii

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  1. M. Ogaki
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  2. Y. Furuichi
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  3. K. Kuroda
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  4. D. P. Chin
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  5. Y. Ogawa
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  6. M. Mii
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Correspondence to M. Mii.

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Communicated by K. Kamo.

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Ogaki, M., Furuichi, Y., Kuroda, K. et al. Importance of co-cultivation medium pH for successful Agrobacterium-mediated transformation of Lilium × formolongi . Plant Cell Rep 27, 699–705 (2008). https://doi.org/10.1007/s00299-007-0481-x

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  • DOI: https://doi.org/10.1007/s00299-007-0481-x

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