A reproducible procedure for plant regeneration from seedling hypocotyl protoplasts of Vigna sublobata L. |
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Authors: | S. K. Bhadra N. Hammatt J. B. Power M. R. Davey |
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Affiliation: | (1) Plant Genetic Manipulation Group, Department of Life Science, University of Nottingham, NG7 2RD Nottingham, UK;(2) Department of Botany, University of Chittagong, 4331 Chittagong, Bangladesh;(3) Horticulture Research International, East Mailing, West Mailing, ME19 6BJ Kent, UK |
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Abstract: | Viable protoplasts of Vigna sublobata L. were isolated enzymatically from hypocotyls of axenic seedlings. Protoplast yields were dependent upon seedling age, with maximum yields (2.25 ± 0.35 × 106 g fwt–1) from seedlings aged 6 d. Protoplasts regenerated cell walls and underwent sustained divisions when cultured in either agarose-solidified or liquid K8P medium. The plating density affected the division frequency and plating efficiency; the division frequency (68 ±0 6.0%) was maximum at 4.0 × 104 ml–1 while plating efficiency was maximum (1.3 ± 0.1%) at 5.0 × 104 ml–1. Dividing protoplasts developed into microcalli, which produced glossy green compact nodular calli on transfer to 8.0 gl–1 w/v agar-solidified medium containing MS salts, B5 organic components, 30 g l–1 sucrose, NAA (0.2–0.5 mg l–1), zeatin riboside (0.5–2.0 mg l–1) and GA3 (0.5–1.0 mg l–1). These calli, after sub-culture on the same medium, produced shoot buds which underwent elongation following transfer of tissues to 6.0 g l–1 agar-solidified B5 medium containing 30g l–1 sucrose, IBA (0.01 mg l–1) and BAP (1.0 mg l–1). Elongated shoots developed roots after transfer to 8.0g l–1 agar-solidified, hormone-free MS medium with 30 g l–1 sucrose.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - BAP 6-benzyladenine or benzylaminopurine - B5 medium after Gamborg et al (1968) - 2,4-D 2,4-dichlorophenoxyacetic acid - GA3 gibberellic acid - 2,i-P 6-(--dimethylallylamino) purine - MS medium after Murashige and Skoog (1962) - NAA 1-naphthaleneacetic acid |
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Keywords: | Vigna sublobata protoplasts microcalli shoot bud formation plant regeneration |
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