Aurelia Brezeanu

Stimulation of tobacco shoot regeneration by alternating weak electric field

Nicotiana tabacum morphogenetic callus was used to study the stimulative effects of external alternating electric currents of low level and low frequency on the cytodifferentiation processes. The N. tabacum calli were grown on standard medium for shoot regeneration and subjected to electric currents (50 Hz, 0.1–50 μA) during the whole period of treatment (30 days). The number of shoots, total mass, DNA and protein content were measured for each callus sample at the end of the experiment. The number of shoots increased by up to 300% for the samples stimulated with 50 μA although no significant changes were noted in total mass, DNA and protein content in the stimulated samples compared to control. We suggest that in the presence of the external electric current, the callus cells become more sensitive to chemical signals (hormones and/or ions) in the culture medium. A series of changes in the activity of membrane components, as a consequence of the modulation of membrane potential by the external electric field, could support our hypothesis.

Control of Nicotiana Tabacum Callus Growth by Alternating and Pulsed Electric Field

Tobacco callus was subjected to alternating and pulsed electric current of different intensities. The electrically treated callus showed a significant stimulation of growth (50% mass growth percentage) for both wave shapes in the 0.3–1.0 μA current intensity range. Stimulated growth of the treated callus was observed in a given intensity window of the electric current. These results allow us to consider the callus culture as a sensitive system to external weak electric field. We propose that the interaction of the applied electric field with the biological processes at the membrane level (transduction of signals, transport phenomena) determines the stimulation of some metabolic processes.

GENE TRANSFER BY ELECTROPORATION INTO INTACT TOBACCO PETIOLE TISSUE

Electroporation has been investigated as a potential transformation method for Nicotiana tabacum cv.xanthi intact petiole tissue. Field strength values from 0.25 to 2 kV/cm, one and five exponential pulses, 20 ms width, were used. The experiments were done with a pJIT 79 plasmid, containing P-glucuronidase (gus) and neomycin phosphotransferase (npt 11) genes, conferring GUS activity and kanamycin (Km) resistance. Resistant explants were selected in the presence of maximum 100μg/ml Km. Maximal transformation efficiency of about 50% was observed for one pulse up to 1 kV/cm. A histochemical GUS assay was performed 8 weeks after electroporation and revealed the activity of the introduced gene for different electrical pulse conditions.

Short High-Voltage Pulses Promote Adventitious Shoot Differentiation from Intact Tobacco Seedlings

A modified electroporation protocol was used for studying the influence of short-duration, high-voltage pulses on the differentiation of adventitious shoots. Intact tobacco seedlings with two expanded leaves were subjected to 78-ms rectangular pulses, with field strengths varying between 50 and 400 V/cm. After electric treatment, the seedlings were explanted onto shoot differentiation medium. Inoculum final mass and regeneration capacities were significantly enhanced by electric treatment, showing a “window response” that was dependent on the field strength and had a maximum at 300 V/cm. Shoot regeneration dynamics were also accelerated in treated seedlings compared with controls. Our results revealed that short high-voltage pulses stimulate in vitro morphogenetic processes in plant systems more complex than protoplasts.