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.

Formation of plant tracheary elements in vitro – a review

“…Its short generation cycle and very small genome size enables a wide range of molecular and genetic approaches. Arabidopsis thaliana is one of the best genetically characterised plants with a genome size of 1.25 × 10 8 base pairs (The Arabidopsis Genome Initiative 2000) (Pillai et al 2011). In comparison, the size of Pinus radiata genome is much larger (2.6 × 10 10 base pairs) (Ahuja and Neale 2005).…”

“…The major advantages of using Populus spp. as model systems are their economic importance as a forestry species, their relatively small genomic size of 5.5 × 10 8 base pairs (Pillai et al 2011), and their ability to be genetically transformed. Ohlsson et al (2006) established two cell suspension cultures to investigate the function of enzymes involved in cell wall biosynthesis in the hybrid aspen, Populus tremula x P. tremuloides.…”

“…In some cases, similar methods are applicable to the analysis of cell-wall polysaccharides, e.g. nuclear magnetic resonance spectroscopy (Ramsden and Northcote 1987), or using trifluoroacetic acid hydrolysis followed either by gas chromatography (Möller et al 2003) or by gas chromatography coupled mass spectroscopy (Pillai et al 2011). Lacayo et al (2010) generated an architectural model of Zinnia elegans TEs using biochemical extraction and three imaging platforms (atomic force microscopy, synchrotron radiation-based Fouriertransform infrared and fluorescence microscopy).…”

“…These include: a thioglycolic acid lignin assay (Möller et al 2003); Klason lignin analysis (Möller et al 2005b); thioacidolysis (Möller et al 2005a(Möller et al , 2005bWagner et al 2013); two dimensional nuclear magnetic resonance (Wagner et al 2007); an acetyl bromide lignin assay (Wagner et al 2007); alkalinenitrobenzene oxidation (Sato et al 2011); pyrolysis-gas chromatography coupled mass spectroscopy (Pillai et al 2011); and Fourier-transform infrared (FTIR) spectroscopy (Pillai et al 2011). In some cases, similar methods are applicable to the analysis of cell-wall polysaccharides, e.g.…”

“…Clumps of cells are usually observed when callus is used as in vitro source of xylogenic cells. Pillai et al (2011) segregated clumps of cells using a ground glass grinder followed by filtration on a nylon mesh prior to microscopic observations of Pseudostuga menziesii (Mirb.) Franco TEs.…”