Electrical fusion for optimal formation of protoplast heterokaryons in Nicotiana |
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Authors: | George W Bates |
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Institution: | (1) Department of Biological Science, Institute of Molecular Biophysics, Florida State University, 32306 Tallahassee, FL, USA |
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Abstract: | The electrical fusion of protoplasts has been studied in order to maximize the formation of heterokaryons for culture. Heterokaryons of Nicotiana tabacum L. mesophyll protoplasts and N. plumbaginifolia Viviani supension-cell protoplasts were identified in fixed and stained as well as living material; a quantitative fusion index was thereby developed. With this index the efficiencies of various electric fields and fusion-chamber designs have been determined. Optimal fusion was obtained with an alternating-current (AC) field of 150 V/cm and direct-current (DC) square-wave pulses of 1000 V/cm. A new, simple-to-use, largescale fusion chamber is described in which batches of up to 5·105 protoplasts (0.5 ml of cells at 106/ml) can be fused in 5–7 min with efficiencies approaching 40%. Half of the fusion products are heterokaryons, thus fusion is random. Of the fusion products, 60% are bi- or trinucleate. Using fusion procedures similar to those described here Bates and C. Hasenkampf (1985, Theor. Appl. Genet., in press) have recovered viable somatic hybrids which have been regenerated.Abbreviations AC
alternating current
- DC
direct current
- PEG
polyethylene glycol |
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Keywords: | Electrical fusion Heterokaryon Nicotiana (protoplast fusion) Protoplast fusion |
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