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Electrical fusion for optimal formation of protoplast heterokaryons in Nicotiana.

Authors
Type
Published Article
Journal
Planta
Publication Date
Volume
165
Issue
2
Pages
217–224
Identifiers
DOI: 10.1007/BF00395044
PMID: 24241046
Source
Medline

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 5·10(5) protoplasts (0.5 ml of cells at 10(6)/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.

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