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Deviation of protoplast media from isotonic concentrations causes the protoplasts to shrink or swell. This process may influence membrane physiology and long-term cell survival. On close examination of standard protoplast procedures, we found that considerable protoplast volume changes can occur during routine manipulation. Techniques for balancing isolation and culture media to cell isotonic concentration were evaluated for two plant genera ( Betula and Solanum ). Two methods for determining cell sap concentration were examined. Plasmometric methods revealed that different cell types within a single leaf vary markedly in isotonic concentrations. The accuracy of cell sap extraction methods was influenced by pretreatment, handling and selection of plant material. The influence of salts, sugars and other media components on osmotic strength was also investigated. It is suggested that low success rates for protoplast culture of certain plants may in part be due to use of osmotically imbalanced media.

Anatomical and physiological comparisons were made between tissues of Betula platyphylla var. szechuanica (Schneid.) Rehd. (Asian white birch) obtained directly from microcultured shoots, from seedlings grown in a micro-culture-like environment, or from micropropagated plants grown in a greenhouse. The diminished stature of microcultured shoots resulted primarily from reduced cell division, although their leaves had reduced cell size as compared to shoots from greenhouse-grown plants. The area occupied by vascular tissue in midrib veins and petioles was significantly reduced in microcultured shoots as was the extent of the palisade layer, indicating that cell differentiation had also been altered. The photosynthetic capacity of leaves from microcultured shoots was less than half that of leaves from greenhouse-grown plants. However, this capacity was restored after non-in vitro rooting of the microcultured shoots. Unlike greenhouse-grown plants, photosynthetic rates of leaves from microcultured shoots were not influenced by decreasing light intensities from 1200 to 200 μmol·s−1·m−2 nor was the respiration rate strongly inhibited by cyanide. Seedlings from a high-humidity, microculture-like environment generally had characteristics intermediate between those of microcultured and greenhouse-grown tissues, indicating that the environment and not the juvenile state of the tissue was probably of overriding importance in influencing these trends.

Summary Greenhouse-grown and shoot-cultured stock plants from 2 woody plant genera (Betula and Rhododendron) were compared as source material for protoplast isolation. Shoot cultures provided higher yields/gram source tissue and enhanced viability of isolated protoplasts. Successful isolation depended on stock culture age and culture environment, with best results from young (20-30-day-old) shoot cultures. Additional advantages of the use of shoot cultures for protoplast research, especially on woody species, are discussed.