Table 2 Microscopic, biochemical, and molecular analysis methods used with in vitro tracheary elements

Microscopic analysis

Bright field microscopy

TE observation under light and polarised light

TE morphological analysis

Pesquet and Tuominen ([2011]); Pillai et al. ([2011])

TEs secondary wall fragments generated by mild sonication

Internal organisation of TEs

Lacayo et al. ([2010])

Cell counting under light and polarised light

Quantitative analysis of TEs

Kärkönen et al. ([2011]); Pesquet and Tuominen ([2011])

Evans blue staining

Localisation of non-viable cells

Groover et al. ([1997])

Phloroglucinol-HCl, safranin, O-toluidine blue staining

Lignin localisation

McCann ([1997]); Oda et al. ([2005]); Pillai et al. ([2011]);

Sato et al. ([2011])

Atomic force microscopy

Topography of TE cell walls

Lacayo et al. ([2010])

Time-lapse microscopy and analysis

Monitoring of TE PCD and cell wall synthesis

Groover et al. ([1997]); Pesquet et al. ([2010])

Fluorescence microscopy

Calcofluor white staining

Cellulosic secondary cell walls observation

Pesquet and Tuominen ([2011])

DAPI, FDA viability staining

Localisation of living (non-lignified) cells

Groover et al. ([1997]); Pesquet and Tuominen ([2011])

Lignin autofluorescence

Visualisation of TEs lignified cell walls

Pesquet and Tuominen ([2011])

Green fluorescent protein specific for crystalline cellulose

Cellulose accessibility and binding in TEs

Lacayo et al. ([2010])

Flow cytometry cell sorting

Separation of TEs from mesophyll cells

Ito et al. ([2004]); Weir et al. ([2005])

Biochemical analysis

2D-NMR spectroscopy

Lignin qualitative analysis

Wagner et al. ([2007])

AcBr lignin assay

Lignin quantitative analysis

Wagner et al. ([2007])

Alkaline-nitrobenzene oxidation

Monomeric composition of lignin

Sato et al. ([2011])

Pyrolysis-GC-MS and FTIR spectroscopy

Lignin qualitative analysis

Pillai et al. ([2011])

FTIR spectromicroscopy

Polysaccharides qualitative analysis

Lacayo et al. ([2010])

Klason lignin

Lignin qualitative analysis

Möller et al. ([2005b])

Lignothioglycolic acid assay

Lignin content in differentiated cell wall

Pillai et al. ([2011])

NMR spectroscopy

Polysaccharides qualitative analysis

Ramsden and Northcote ([1987])

TFA hydrolysis and GC/GC-MS

Monosaccharide content in differentiated cell wall

Pillai et al. ([2011]); Möller et al. ([2003])

Thioacidolysis

Lignin qualitative analysis

Möller et al. ([2005a])

Thioglycolic acid lignin assay

Lignin quantitative analysis

Möller et al. ([2003])

Molecular investigation

RT-PCR expression analysis

Gene expression analysis

Pesquet et al. ([2010], [2013]); Wagner et al. ([2009]); Milioni et al. ([2002])

RNA-dependent gene overexpression or silencing (RNA interference)

Gene function analysis

Pesquet et al. ([2010]); Endo et al. ([2008]); Endo et al. ([2009]); Möller et al. ([2005b]); Wagner et al. ([2013]); Wagner et al. ([2009]); Wagner et al. ([2007])