The effect of chemical facilitators on the frequency of electrofusion of tobacco mesophyll protoplast

Trypsin, pronase, protease, dispase, spermine, spermidine, and DMSO were characterized for their effect on the frequency of electrofusion of tobacco mesophyll protoplasts. Protease (Boehringer Mannheim) and Sigma pronase (1.26 mg/ml; 15 min incubation) increased the fusion frequency from 7% (control) to 20.7% (2.9X increase). Following protease and pronase were trypsin (2.8X), spermine (2.4X), dispase (2.1X), DMSO (2.0X), and spermidine (1.4X). BM Protease and polyamines caused the least amount of damage, followed by DMSO and trypsin (26% and 24% decrease in viability respectively), pronase (41%) and dispase (57%). Callus formed from all but dispase-treated protoplasts. Shoots regenerated from calli of all but trypsin-treated protoplasts.Abbreviations BA 6-Benzylaminopurine - BM Boehringer Mannheim - DMSO dimethyl sulfoxide - FDA fluorescein diacetate - FM protoplast fusion medium - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, sodium salt - MES 2-(N-morpholino)ethanesulfonic acid - MS Murashige and Skoog (1962) - NAA -Naphthaleneacetic acid - PEG polyethylene glycol - SE standard error of the mean