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A technique for bulk production of cytoplasts and miniprotoplasts from suspension culture-derived protoplasts

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Protoplasts isolated from suspension cultures of atrazine resistant black nightshade (Solanum nigrum L.) a weed biotype, were enucleated by centrifugation through a stepwise mannitol/sucrose gradient. Two cytoplast, enucleated subprotoplast, bands were routinely formed: one, a minor band at the 6.4%/18.2% mannitol border containing highly vacuolate cytoplasts with 95%+ enucleation; secondly a major cytoplast band at the 18.2% mannitol/33% sucrose border containing 90%+ enucleated protoplasts in quantities up to 4 million per 50 ml gradient tube. Efficient production of cytoplasts depended on the subculture procedures used for the cell suspensions. Optimal cytoplast yield (44%) occurred for protoplasts isolated three days after subculture. The vigor of the donor suspension cultures as visually monitored had to be controlled in order to obtain consistently high enucleation percentages.

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Cell and Protoplast Wash Solution


2,4-dichlorophenoxyacetic acid




Fluorescein diacetate


Murashige and Skoog medium (1962)


Uchimiya and Murashige medium (1976)


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Author information

Correspondence to M. S. Lesney.

Additional information

Communicated by J.M. Widholm

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Lesney, M.S., Callow, P.W. & Sink, K.C. A technique for bulk production of cytoplasts and miniprotoplasts from suspension culture-derived protoplasts. Plant Cell Reports 5, 115–118 (1986). https://doi.org/10.1007/BF00269248

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  • Sucrose
  • Cell Suspension
  • Mannitol
  • Suspension Culture
  • Atrazine