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Macro elements in inoculation and co-cultivation medium strongly affect the efficiency of Agrobacterium-mediated transformation in Lilium

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Abstract

A highly efficient Agrobacterium-mediated transformation system for Lilium × formolongi was established by modifying the medium used for inoculation and co-cultivation. Meristematic nodular calli of Lilium were inoculated with an overnight culture of A. tumefaciens strain EHA101 containing the plasmid pIG121-Hm harboring an intron-containing β-glucuronidase (GUS), hygromycin phosphotransferase, and neomycin phosphotransferase II genes. The effects of ten different types of media and carbohydrates (sucrose, d-glucose, and l-arabinose) in both inoculation and co-cultivation media were evaluated. Interestingly, a dramatic increase in the frequency of transformation (25.4%) was observed when Murashige and Skoog (MS) medium containing sucrose and lacking KH2PO4, NH4NO3, KNO3, and CaCl2 was used. Hygromycin-resistant transgenic calli were obtained only in medium supplemented with sucrose. The effects of this modified medium were also investigated for Lilium cultivars ‘Acapulco’, ‘Casa Blanca’, and ‘Red Ruby’. The highest frequency of transformation (23.3%) was obtained for cv. Acapulco. Hygromycin-resistant calli were successfully regenerated into plantlets on plant growth regulator-free MS medium. Transgenic plants were confirmed by GUS histochemical assay, polymerase chain reaction (PCR), and Southern blot analyses.

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Fig. 1a–f
Fig. 2a–c
Fig. 3a, b
Fig. 4

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Abbreviations

AS:

Acetosyringone

CTAB:

Cetyltrimethylammonium bromide

GUS:

β-glucuronidase

Hm:

Hygromycin

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Authors and Affiliations

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

    Pejman Azadi, Dong Poh Chin, Kiyo Kuroda, Raham Sher Khan & Masahiro Mii

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  1. Pejman Azadi
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  2. Dong Poh Chin
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  3. Kiyo Kuroda
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  4. Raham Sher Khan
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  5. Masahiro Mii
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Correspondence to Masahiro Mii.

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Azadi, P., Chin, D.P., Kuroda, K. et al. Macro elements in inoculation and co-cultivation medium strongly affect the efficiency of Agrobacterium-mediated transformation in Lilium . Plant Cell Tiss Organ Cult 101, 201–209 (2010). https://doi.org/10.1007/s11240-010-9677-9

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