For the close-up pictures of the Developmental changes in the Apex, the Release of Pollen at Flowering and of the Whole Wheat Grains, through the Germination, Grain Growth, Grain Filling and Dry Down stages we used digital capture from a video camera mounted on a Leica dissecting microscope.
Photographs of whole plants in the Controlled Environment Rooms, the glasshouses and out in the field were taken with a Fujifilm Finepix A202 digital camera mounted on a tripod. This camera was also used for the studio work and some of the close up photography.
For the micrographs of grain sections we used a Leica DMRB microscope fitted with SLR cameras. Images were recorded on Kodak EPT film. The transparencies were scanned as Bitmap files for incorporation into this website.
Mainstem ears of Mercia wheat were tagged when the anthers first appeared outside the florets of the central spikelets. This established Day 0, the day of fertilisation, for each ear. Developing wheat grains were only taken from the first and second florets of the central five spikelets. Grains were samples at daily intervals from Day 0 until Day 30.
Several steps are involved in the preparation of tissue for microscopy. Firstly the tissue must be 'fixed' in order to preserve its chemical nature and physical structure. Secondly the delicate tissues need to be firmly supported so that they can be cut into very thin slices on a microtome. Thin slices can then be stained to reveal their chemical composition and cellular architecture. The final stage is to examine the stained sections with a microscope and take informative photographs, micrographs, of the cellular structures inside.
The physical characters of the wheat grains changed as they grew and developed and slightly different fixation procedures were needed at different times to ensure good preservation of the tissues. The grains are naturally resistant to liquid penetration; at fertilisation they are covered with many tiny hairs which trap air bubbles and later the cuticle becomes tough and hard. In between times the grain content itself is liquid and the tissue layers are delicate and without strength; they collapse or fly apart if they are treated roughly and at these times slow gentle procedures and great patience were essential. The criterion used to assess successful fixation was whether or not the grains had sunk in the fixative. If the grains persisted in floating then they were rejected because in this case tissue preservation was found to be poor. The best penetration of the fixative was achieved in young grains, from day 0 until about day 13, by either incorporating triton X-100 detergent in the fixative or applying modest controlled vacuum. For older grains from day 11 through to day 30 it was better to assist fixative penetration by making small cuts into the outer pericarp. Slicing grains in half caused damage to the internal structures and the endosperm content was easily lost.
The fixative used throughout was a solution of 3.7% paraformaldehyde in 100mM NaH2PO4, pH7.2.
The fully fixed grains were rinsed briefly in clean phosphate buffer and then very slowly dehydrated through a graded series of ethanol/tertiary butyl alcohol solutions. From pure tertiary butyl alcohol they were gradually transferred to pure paraffin wax (Lambwax; Raymond A Lamb, UK) at 70 degrees Celsius. When cold the wax hardens providing good support for the soft tissues. Longitudinal and transverse sections, 10 microns thick, were cut using a rotary microtome (Reichert-Jung Biocut; Leica, UK). The sections were mounted on glass slides previously treated with Haupt's adhesive (1% gelatine in water with 2% phenol crystals and 15% glycerine).
Several different stains were used to identify tissue structures and cell contents including starch, proteins and lipids. However the images presented were all stained to show cellular architecture.
Wax was removed from the tissue sections with Histoclear (National Diagnostics) and the sections were hydrated. The tissues were then stained with Toluidine Blue, 0.05%, in benzoate buffer (benzoic acid 0.125%, sodium benzoate 0.145%, pH4.4) after Feder and O'Brien, 1968. The stained sections were mounted in Glycerol Jelly (BDH, UK).
The sections were examined at low magnification, in Brightfield and Darkfield illumination, with a Wild Photomacroscope. A Leica DMRB microscope was used for high magnification observations in Brightfield and in polarised light. Images were recorded on Kodak EPT film. The transparencies were scanned as Bitmap files for incorporation into this website.