Relationship between protoplast size and critical field strength in protoplast electropulsing and application to reliable DNA uptake in Brassica

Summary Protoplast diameter, a physical parameter controlling the susceptibility to electropermeation, was observed to vary between different batches of Brassica napus and Brassica campestris protoplasts. In order to control the permeation field strength associated with the protoplast size, we investigated the mathematical relationship between permeant field strength and protoplast size by measuring electroloading of the calcein dye under conditions that did not alter protoplast survival. A roughly linear relationship was obtained when the permeant field strength was plotted against the reciprocal of the radius. The plot characteristics were found to be tissue specific (mesophyll, hypocotyl and cotyledon), species specific (turnip, rapeseed) and modulated by the pulse duration. These plots were used as reference curves to determine accurate electrical conditions for DNA transfer whatever the size distributions of different protoplast batches.Abbreviations GUS -glucuronidase - CAT chloramphenicol acetyltransferase - MOPS 3-(N-morpholino)propanesulfonic acid - TRIS tris(hydroxymethyl)aminomelhane - BSA bovine serum albumin - ATP adenosine 5-triphosphate