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A simple shoot multiplication procedure using internode explants, and its application for particle bombardment and Agrobacterium-mediated transformation in watercress

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Abstract

A shoot multiplication system derived from internode explants was investigated with the aim of improving genetic characteristics of watercress (Nasturtium officinale R. Br.). Internodes of ca. 1 cm excised from in vitro stock shoot culture were placed on half-strength Murashige and Skoog (MS) medium supplemented with 3 μM 2,4-dichlorophenoxyacetic acid as a pre-treatment. Laser scanning microscopy indicated clearly that the first sign of meristematic cell division could be seen after 1–2 days of pre-culture, and meristematic tissues multiplied along the vascular cambium of the internode segment during 7 days of culture. Multiple shoots could be obtained from more than 90% of the pre-treated explants when they were subsequently transferred to MS medium supplemented with 1 μM thidiazuron for 3 weeks. These findings indicate that pre-treatment of the internodes for 7 days promoted their capacity for organogenesis. Using this pre-treatment, frequent generation of transgenic watercress plants was achieved by adapting particle bombardment and Agrobacterium-mediated transformation techniques with a construct expressing a synthetic green florescent protein gene.

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Abbreviations

BA:

6-Benzyladenine

2,4-D:

2,4-Dichlorophenoxyacetic acid

MS medium:

Murashige and Skoog medium

PPM:

Plant preservation mixture

TDZ:

Thidiazuron

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Acknowledgments

The authors thank Dr. Edward C. Yeung of Calgary University for critical reading of the manuscript. The authors also thank Misses Ayumi Kurata, Minori Kuroda, and Rie Yamamura, and Messrs Tatsuya Miura, and Ryusaku Shigemi of Toyama Prefectural University for technical assistance. This research was supported in part by a Grant-in-Aid for scientific research, C (20580082) from the Japan Society for the Promotion of Science.

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Authors and Affiliations

  1. Laboratory of Plant and Cell Engineering, Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama, 939-0398, Japan

    Shinjiro Ogita, Miki Usui, Nanae Shibutani & Yasuo Kato

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  1. Shinjiro Ogita
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  2. Miki Usui
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  3. Nanae Shibutani
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  4. Yasuo Kato
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Correspondence to Shinjiro Ogita.

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Ogita, S., Usui, M., Shibutani, N. et al. A simple shoot multiplication procedure using internode explants, and its application for particle bombardment and Agrobacterium-mediated transformation in watercress. J Plant Res 122, 455–463 (2009). https://doi.org/10.1007/s10265-009-0226-8

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