ELECTROFUSION BETWEEN MESOPHYLL PROTOPLASTS OF Lycopersicon esculentum AND SHOOT TIP PROTOPLASTS OF LYCOPERSICON HIRSUTUM

Mesophyll protoplasts and shoot tip protoplasts of Lycopersicon esculentum and L. hirsutum were enzymatieally isolated, and the electrofusion experiments with mesophyll protoplasts of L. esculentum and shoot tip protoplasts of L. hirsutum were carried out.Applying a high-frequency alternating electrical field (sine wave, 1mHz, 200V/cm)generated from a self-made cell fusion apparatus, protoplasts were aligned between two parallel electrodes (200μm in distance)by dieleetrophorsis. This chain arrangement of the proteplasts could he manipulated by regulating the electrical parameters and protoplast density. After supplement with a single square wave pulse, the reversible breakdown of plasma membranes was induced and then protoplast fusion was triggered. The fusion frequency of the aligned protoplasts increased with the increase of pulse voltage between 1000-3000V/cm, and the pulse duration of 40μs induced higher fusion frequency than that of 20μs. The maximal fusion frequency which depended on a number of factors including protoplast origin, size and chain arrangement was 57% in this experiment, compared with 11% in the PEG fusion experiment here. Mesophyll protoplasts tended to fused more easily than shoot tip protoplasts, but no obvious limitation for fusion of protoplasts different greatly in size was observed. Fluorescein diaeetate staining showed that both PEG induced fusion and electrofusion had no apparent effects on protoplast viability.The electrofusion-treated protoplasts were capable of cell wall formation and cell division when cultured in liquid medium, but no constant cell growth.