ISHS Acta Horticulturae 479: IX International Asparagus Symposium
TRANSIENT β-GLUCURONIDASE EXPRESSION IN ASPARAGUS PROTOPLASTS FOLLOWING ELECTROPORATION |
| Authors: | C. Guangyu, A.J. Conner, A.G. Fautrier, R.J. Field |
| Keywords: | Asparagus officinalis, electroporation efficiency, energy level, genetic engineering, survival rate, transformation |
| Abstract:
Electroporation was performed with asparagus protoplasts using an exponential machine. Electrical parameters used for protoplasts of other species were not successful in achieving transient -glucuronidase (GUS) expression using chimeric GUS genes with various promoters. Therefore, a series of experiments was conducted on the effect of energy and other related components on electroporation efficiency (EE). These experiments demonstrated that the energy dissipated in the process of electroporation is the major factor which controls the EE. The traditional concepts of voltage and capacitance are only parameters determining electrical energy. The relationship of these parameters was theoretically determined to be = 0.5CV02, which allows the level of total energy dissipation (  ) to be calculated for different combinations of capacitance (C) and initial voltage (V0). Both EE and survival rate of protoplasts were determined by, and in proportion to, the energy dissipation levels.
A good linear relationship exists between the energy dissipation per unit volume (EDV) and both the EE and survival rate of protoplasts.
The time constant, which characterizes the waveform of energy dissipation, also influences the EE. When energy is used as the key component, the optimization of electrical parameters for efficient electroporation is simple and accurate.
The application of this new principle to transformation of asparagus protoplasts using a GUS gene was successful in achieving transient GUS expression.
This principle should be generally applicable to electroporation of other species, and is not machine dependent.
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