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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Planta 165 (1985), S. 205-216 
    ISSN: 1432-2048
    Keywords: Cell division ; Dielectrophoresis ; Electrofusion (electrical parameters) ; Heterokaryon ; Protoplast (mesophyll and suspension) ; Somatic hybridization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The electrical parameters important in the fusion of plant protoplasts aligned dielectrophoretically in high-frequency alternating electric fields have been established. Protoplasts were aligned in an alternating electric field between two relatively distant (1 mm) electrodes, by dielectrophoresis induced by field inhomogeneities caused by the protoplasts themselves. This arrangement allowed ease of manipulations, large throughput and low loss of protoplasts. In analytical experiments, sufficiently large samples could be used to study pulse duration-fusion response relations at different pulse voltages for protoplasts of different species, tissues and size (mesophyll protoplasts of Solanum brevidens, Triticum aestivum, Hordeum vulgare; suspension-culture protoplasts of Nicotiana sylvestris, N. rustica, Datura innoxia and S. brevidens; root-tip protoplasts of Vicia faba, hypocotyl protoplasts of Brassica napus). The percentage of aligned protoplasts that fused increased with increasing pulse parameters (pulse duration; voltage) above a threshold that was dependant on pulse voltage. The maximum fusion values obtained depended on a number of factors including protoplast origin, size and chain length. Leaf mesophyll protoplasts fused much more readily than suspension-culture protoplasts. For both types, there was a correlation of size with fusion yield: large protoplasts tended to fuse more readily than small protoplasts. In short chains (≦five protoplasts), fusion frequency was lower, but the proportion of one-to-one products was greater than in long chains (≧ten protoplasts). In formation by electrofusion of heterokaryons between mesophyll and suspension-culture protoplasts, the fusion-frequency response curves reflected those of homofusion of mesophyll protoplasts rather than suspension-culture protoplasts. There was no apparent limitation to the fusion of the smallest mesophyll protoplast with the largest suspension-culture protoplasts. Based on these observations, it is possible to direct fusion towards a higher frequency of one-to-one (mesophyll/suspension) products by incorporating low densities of mesophyll protoplasts in high densities of suspensionculture protoplasts and by using a short fusion pulse. The viability of fusion products, assessed by staining with fluorescein diacetate, was not impaired by standard fusion conditions. On a preparative scale, heterokaryons (S. brevidens mesophyll-N. sylvestris or D. innoxia suspension-culture) were produced by electrofusion and cultured in liquid or embedded in agar, and were capable of wall formation, division and growth. It is concluded that the electrode arrangement described is more suitable for carrying out directed fusions of plant protoplasts than that employing closer electrodes.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-2242
    Keywords: Amino acid analogue-resistance ; Electrofusion ; Potato ; Solanum tuberosum L. ; Somatic hybridization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Intraspecific somatic hybridization between amino acid analogue-resistant cell lines of potato (Solanum tuberosum L.) has been carried out following electrofusion of protoplasts. In initial analytical electrofusion experiments (1 mm electrode separation) optimal fusion conditions were determined by changing the fusion medium (addition of Ca and/or spermine) and the electrical parameters. Subsequently, in large scale experiments, cell suspension protoplasts of aec-1, a variant resistant to AEC, were fused with the same type of protoplasts of 5mt-26 or 5mt-27, both variants resistant to 5MT and cross-resistant to 3 FT. After an extensive selection procedure only somatic hybrid lines of aec-1 + 5mt-26 were obtained. The resistance traits of aec-1 and 5mt-26 were expressed fully, indicating that the variant characters involved are transmitted dominantly. Quantitative examination of the free amino acid content revealed characteristics of both the parental cell lines in most of the somatic hybrids. However, initially selected double resistant colonies from fusions of aec-1 + 5mt-27 lines appeared not to be somatic hybrids.
    Type of Medium: Electronic Resource
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