Large Electro-fused Protoplasts of Populus alba Selected by a Micromanipulator : Techniques and Some Characteristics of Cells and Their Regenerants
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概要
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An improved method for selection of large electro-fused protoplasts of Populus alba by a micromanipulator was developed. Theconditions for electric cell fusion treatment were optimized. For the best result, protoplasts with a cell density of 5 x 10^5/mL weretreated with an alternate current (l MHz, 200V/cm) and pulsed with a direct current (2 kV/cm) for 100 μs in 2.5 mM CaCl_2 and0.55 M mannitol. The electo-fused protoplasts were cultured in NH_4NO_3-free Murashige and Skoog's medium containing 0.6Mof mannitol, 0.09 M sucrose, 1 μM of 2,4-dichlorophenoxyacetic acid and 0.1 μM of benzyladenine, the same medium used for protoplast culture, but at a very low cell density of 5-10 X 10^2/mL in a well of a 96-well culture plate. When cell aggregates derived fromindividual fused protoplasts were transferred to fresh medium with 0, 0.3 or 0.6 M mannitol, large colonies developed. In the shootdifferentiation medium, the reaction of the calluses derived from large fused protoplasts towards the growth regulators differed fromthe non-fused ones. In medium containing 1 μM each of naphthalene acetic acid and N-(2-chlor0-4-pyridyl)-N'-phenylurea,growth of callus from electro-fused ones was not reduced by much compared to the control, but shoot differentiation was inhibited. Gibberellic acid (0.1-10 μM) was beneficial to shoot regeneration; however, irregularly shaped leaves appeared at high gibberellicacid concentrations. Shoots regenerated were rooted in Murashige and Skoog's medium containing 4 μM of indole-3-butyric acid.Some plantlets obtained had a varied morphology. Based on the characteristics of growth. some cells derived from eiectro-fused protoplasts appear to be physiologically different from the non-fused one.
- 一般社団法人日本森林学会の論文
- 2000-11-16
著者
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Wakita Y
Research Institute Of Agricultural Resources Ishikawa Agricultural College
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Sasamoto Hamako
Cell Manipulation Laboratory, Bio-resources Technology Division, Forestry and Forest Products Resear
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Wakita Y
Cell Manipulation Laboratory Bio-resources Technology Division Forestry And Forest Products Research
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Wakita Yohichi
Research Institute Of Agricultural Resources Ishikawa Agricultural College
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YOKOTA Shinso
Department of Forest Science, Faculty of Agriculture, Utsunomiya University
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YOSHIZAWA Nobuo
Department of Forest Science, Faculty of Agriculture, Utsunomiya University
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Yokota Shinso
Department Of Forest Science Faculty Of Agriculture Utsunomiya University
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Wakita Yohichi
Cell Manipulation Laboratory Bio-resources Technology Division Forestry And Forest Products Research
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Sasamoto Hamako
Cell Manipulation Laboratory Bio-resources Technology Division Forestry And Forest Products Research
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Yoshizawa Nobuo
Department Of Dentistry And Oral Surgery Yamagata University School Of Medicine
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