Abstract
Variation in the tissue structure of short rotation coppice (SRC) willow is a principle factor driving differences in lignocellulosic sugar yield yet much of the physiology and development of this tissue is unknown. Traditional sectioning can be both difficult and destructive in woody tissue; however, technology such as three dimensional X-ray micro-computational tomography (μCT) scanning can be used to move biological researchers beyond traditional two dimensional assessment of tissue variation without having to destructively cut cells. This technology does not replace classical microscopic techniques but rather can be carefully integrated with traditional methods to improve exploration of the world of plant biology in three dimensions. The procedures below outline preparation of willow for 3D X-ray μCT and associated xylem staining and visualisation techniques, in particular secondary xylem programmed-cell-death (PCD) delay during gelatinous fibre (g-fibre) development. Many of the staining techniques here are transferable to other woody species such as poplar and Eucalyptus.
Keywords: Micro-computed tomography, Tension wood, Cell wall staining, Xylem visualisation, Plant histology
Materials and Reagents
Equipment
Procedure
X-ray μCT can also potentially be used for a broader range of crops, and a more diverse set of biological questions. In most cases, care should be taken to air-dry material prior to scanning; water saturation will reduce cell wall contrast while forced drying beyond fibre saturation point runs the risk of inducing cell collapse, altering the plant’s tissue architecture. An example of this wider potential for the technology in plant research is provided in Figure 1 and Video 1. Figure 1. Miscanthus internode 3D render. X-ray 3D Computational tomography example images of mature Miscantus x giganteus stem.
Acknowledgments
The author was financially supported by BioFuelNet Canada. X-ray μCT scanning was performed in collaboration with Farah Ahmed and Dan Sykes at the Natural History Museum with funding from BBSRC Sustainable Bioenergy Centre (BSBEC), working within the BSBEC BioMASS (http://www.bsbec-biomass.org.uk/) Programme. In situ staining techniques were developed in collaboration with Dr. Michael Jasmine Ray.
References
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