Isolation of protoplasts from developing xylem of Pinus banksiana and Pinus strobus
1993
Leinhos, V. | Savidge, R.A.
Protoplasts were isolated from developing xylem of Pinus banksiana Lamb. and Pinus strobus L. by incubating freshly harvested tissue in a cellulase-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 micrometers) 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.
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