Large electro-fused protoplasts ofPopulus alba selected by a micromanipulator: Techniques and some characteristics of cells and their regenerants |
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| Authors: | Hamako Sasamoto Yohichi Wakita Shinso Yokota Nobuo Yoshizawa |
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| Institution: | (1) Cell Manipulation Laboratory, Bio-resources Technology Division, Forestry and Forest Products Research Institute, 305-8687 Ibaraki, Japan;(2) Department of Forest Science, Faculty of Agriculture, Utsunomiya University, 321-8505 Utsunomiya, Japan;(3) Present address: Research Institute of Agricultural Resources, Ishikawa Agricultural College, 921-8836 Nonoichi, Ishikawa, Japan |
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| Abstract: | An improved method for selection of large electro-fused protoplasts ofPopulus alba by a micromanipulator was developed. The conditions for electric cell fusion treatment were optimized. For the best result,
protoplasts with a cell density of 5 × 105/mL were treated with an alternate current (1 MHz, 200 V/cm) and pulsed with a direct current (2 kV/cm) for 100μs in 2.5 mM CaCl2 and 0.55 M mannitol. The electo-fused protoplasts were cultured in NH4NO3-free Murashige and Skoog’s medium containing 0.6 M of mannitol, 0.09 M sucrose, 1μM of 2,4-dichlorophenoxyacetic acid and 0.1μM of benzyladenine, the same medium used for protoplast culture, but at a very low cell density of 5–10 × 102/mL in a well of a 96-well culture plate. When cell aggregates derived from individual fused protoplasts were transferred
to fresh medium with 0, 0.3 or 0.6 M mannitol, large colonies developed. In the shoot differentiation medium, the reaction
of the calluses derived from large fused protoplasts towards the growth regulators differed from the non-fused ones. In medium
containing 1μM each of naphthalene acetic acid andN-(2-chloro-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 (0.1–10μM) was beneficial to shoot regeneration; however, irregularly shaped leaves appeared at high gibberellic acid concentrations.
Shoots regenerated were rooted in Murashige and Skoog’s medium containing 4μM of indole-3-butyric acid. Some plantlets obtained had a varied morphology. Based on the characteristics of growth, some
cells derived from electro-fused protoplasts appear to be physiologically different from the non-fused one. |
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| Keywords: | cell fusion micromanipulation plant hormones Populus alba protoplast |
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