RAPID HIGH-YIELD ISOLATION OF METABOLICALLY INTACT PROTOPLASTS OF LEAVES FROM BETULA-PENDULA ROTH

A short duration isolation method has been developed achieving high yields of protoplasts from leaves of Betula pendula (2-4 x 10(7) protoplasts/g fresh wt.). After optimizing growth conditions of donor plants and the composition of digestion medium, intact protoplasts were isolated within 3 h. The rate of photosynthesis of purified protoplasts is comparable to those of the donor tissue. The maintenance of physiological intactness of that fraction is demonstrated by a diurnal cycle of photosynthetic capacity immediately following the preparation. The homogeneity of the protoplast fractions has been investigated by green and red autofluorescence using a flow fluorescence cytometer. Highest degrees of homogeneity (cytometer record), as highest intactness of plasmalemma (enzyme assay) and highest capacity of photosynthetic activity (oxygen polarography), occurred in the 20-30% Percoll fraction.