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Plant Physiology 80:117-121 (1986)
© 1986 American Society of Plant Biologists

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Articles

Behavior and Viability of Tobacco Protoplasts in Response to Electrofusion Parameters

James A. Saunders, Lee Ann Roskos, Sue Mischke1, Mohammed A. M. Aly2 and Lowell D. Owens

United States Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, Tobacco Laboratory, Beltsville, Maryland 20705, Tissue Culture and Molecular Biology Laboratory, Beltsville, Maryland 20705

This investigation examines responses of protoplasts in a systematic and quantitative way to the various electrical treatments used to achieve electrofusion and their individual and cumulative effect on protoplast viability. Mesophyll and cell suspension protoplasts from two species of the same genera, Nicotiana tabacum and N. rustica var brasilia were used in these experiments. Optimal frequencies for alignment of tobacco protoplasts were between 500 kilohertz and 2 megahertz at 100 volts per centimeter. Variations in frequency and voltage of the alternating current (AC) field caused predictable movements of protoplasts within an electrofusion chamber. AC frequencies below 10 hertz or above 5 megahertz significantly decreased the viability of protoplasts in the fusion chamber as estimated by fluorescein diacetate staining 1 hour after treatment. Although the direct current (DC) pulse appeared to have a slight detrimental effect on protoplast viability, this effect was not significantly different from untreated control preparations.

Protoplasts from both leaf mesophyll cells and suspension cells were induced to fuse with one or more 10 to 30 microseconds DC square wave pulses of approximately 1 kilovolt per centimeter after the protoplasts had been closely appressed with an AC field.

1 Present address: United States Department of Agriculture, Agricultural Research Service, Soilborne Diseases Laboratory, Beltsville, MD 20705.

2 A portion of this research was submitted by M. A. M. A. to the University of Maryland, Baltimore County to fulfill part of the requirement for the Ph.D. degree.






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Copyright © 1986 by the American Society of Plant Biologists