Isolation of protoplasts from developing xylem of <i>Pinus</i><i>banksiana</i> and <i>Pinus</i><i>strobus</i>

Protoplasts were isolated from developing xylem of Pinusbanksiana Lamb, and Pinusstrobus L. by incubating freshly harvested tissue in a cellulose–pectinase mixture having mannitol as osmoticum. Protoplasts were then purified using a discontinuous sucrose–mannitol gradient. More than 70% of the isolated protoplasts were of small diameter (12–27 μm) and had dense cytoplasm and many small vacuoles, suggesting that they originated from ray cells. Larger protoplasts constituted about 25% of the protoplast population; these contained single large vacuoles and only parietal cytoplasm, suggesting that they originated from fusiform cells. Using combined gas chromatography–mass spectrometry, coniferin was confirmed to be present in protoplast preparations. By high-performance liquid chromatography (258 nm UV detection), coniferin was readily detected in protoplasts and in extracts of developing xylem from both species. On a fresh-weight basis, coniferin occurred at 1.0–1.6 mM in protoplasts. In late June, coniferin in developing xylem could be accounted for totally by protoplast coniferin content. In late July, protoplasts contained 93 and 61% of the coniferin content in developing xylem of P. strobus and P. banksiana, respectively.