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Production of bialaphos-resistant Nierembergia repens by electroporation

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Abstract

Transgenic plants with the herbicide-resistance gene (bar gene) were obtained via organogenesis from isolated mesophyll protoplasts of Nierembergia repens after applying electroporation. Transient β-glucuronidase (GUS) activity of electroporated protoplasts assayed 2 days after applying an electric pulse showed that optimum condition (transient GUS activity 319 pmol 4 MU/mg per min and plating efficiency 2.43%) for electroporation was 0.5 kV/cm in field strength and 100 μF in capacitance. The protoplasts electroporated with the bar gene at this condition initiated formation of microcolonies on medium after 2 weeks. After 4 weeks of culture, equal volume of fresh 1/2-strength Murashige and Skoog (MS) medium containing 0.2 mg/l bialaphos was added for selection of transformed colonies. After 6 weeks of culture, growing colonies were transferred onto regeneration medium containing 1.0 mg/l bialaphos, on which they formed adventitious shoots 1–2 months after electroporation. The adventitious shoots rooted easily after transfer onto MS medium with bialaphos lacking plant-growth regulators. Transformation of these regenerants with the bar gene was confirmed by Southern analysis. Some of the transformants showed strong resistance to the application of bialaphos solution at 10.0 mg/l.

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Abbreviations

BA:

Benzyladenine

MS:

Murashige and Skoog

1/2 MS:

Half-strength MS

NAA:

α-Naphthaleneacetic acid

PGR:

Plant-growth regulator

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Authors and Affiliations

  1. Graduate School of Horticulture, Chiba University, Matsudo, Chiba, 271-8510, Japan

    Yoshiaki Shizukawa & Masahiro Mii

  2. Kyoto Prefectural Institute of Agricultural Biotechnology, 74 Ooji, Kitaine-yatsuma, Seika, Souraku, Kyoto, 619-0244, Japan

    Yoshiaki Shizukawa

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  1. Yoshiaki Shizukawa
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  2. Masahiro Mii
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Correspondence to Yoshiaki Shizukawa.

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Shizukawa, Y., Mii, M. Production of bialaphos-resistant Nierembergia repens by electroporation. Plant Biotechnol Rep 2, 219–226 (2008). https://doi.org/10.1007/s11816-008-0065-4

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  • DOI: https://doi.org/10.1007/s11816-008-0065-4

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