Developing a model system in vitro to understand tracheary element development in douglas-fir (Pseudostuga Mensziesii)

Abstract

Callus cells were initiated on cambial strips obtained from 4 to 8 y old Douglas-fir (Pseudostuga menziesii) trees, cultured on solidified Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyaceticacid (2,4-D) and benzylaminopurine (BA). The cultures could be maintained by sub-culturing on fresh medium every four weeks. When the callus cells were subsequently transferred to liquid MS medium supplemented with different phytohormones, suspension cultures could be initiated and maintained by periodic sub-culture. Approximately 65% of the callus cells cultured on liquid MS medium supplemented with 2,4-D, when maintained for 6-7 weeks without sub-culture, differentiated to tracheary element (TE) like cells. The formation of TE like cells was confirmed histochemically by staining with phloroglucinol-HCl. Secondary thickening of the cell walls were confirmed by polarized light microscopy, which showed strong birefringence of the cell wall due the presence of crystalline cellulose. The presence of lignin was determined by pyrolysis-GC-MS and FTIR spectroscopy. The lignin content in differentiated cell wall samples was quantified at 21% by the lignothioglycolic acid assay. Analysis of monosaccharide composition of cell wall samples after acid hydrolysis showed that the percentage of glucose, xylose and mannose had increased in the differentiated cell walls. These increases correspond to the formation of cellulose, glucomannan and xylan, primarily associated with secondary cell walls.