Winged-bean protoplasts: Isolation and culture to callus |
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Authors: | A E Cuddihy P J Bottino |
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Institution: | (1) Department of Botany, University of Maryland, 20742 College Park, MD, USA |
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Abstract: | A system was developed for protoplast isolation and culture from suspension cultured cells of winged bean,Psophocarpus tetragonolobus. Cells from a three-day-old suspension were incubated in an enzyme mixture containing 6% cullulysin, 1% Macerase, 1% desalted
Rhozyme, 0.4M sorbitol, and 0.1M CaCl2 at pH 5.5. Average yields of protoplasts were 6.5 × 106 per gram fresh weight of cells. Protoplasts were cultured in modified B5 medium containing 68.4 g/l glucose, 250 mg/l xylose,
0.1 mg/l 2,4-D, 0.5 mg/l BAP, 250 mg/l N-Z amine type AS, and 20 ml/l coconut water.
After 24 h of culture, the protoplasts had synthesized a new wall, and in three days had begun division. The optimum plating
density was 1–2 × 103 protoplasts/ml. The division frequency ranged between 40%–60% for most experiments with a high of 72% in one experiment.
After three weeks, cell colonies could be transferred to solid MS medium containing N-Z amine and coconut water where callus
developed. This protoplast system is technically comparable to soybean for experiments concerned with genetic manipulation
involving legumes. |
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Keywords: | protoplasts suspension cells callus formation winged bean Psophocarpus tetragonolobus |
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