Physiological,
Biochemical and Chemical Studies on Desiccation Tolerance; Primarily in
Developing Wheat Seeds
PhD Dissertation. The University of Adelaide. South Australia.
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This thesis is the result of her investigations into the phenomenon of desiccation tolerance in developing seeds of sweet corn and wheat. Methods employed include the HPLC of sugar, predominantly sucrose and raffinose, thin layer chromatography together with diffuse reflectance using infrared dispersion spectra (DRUIDS), confocal microscopy of lipids, solid state 13C NMR CP/MAS spectroscopy and embryo culture.
The major results obtained were:
1) Neither sugar concentration nor the mole%ratio of raffinose/sucrose were found to be good predicters of desiccation tolerance. Sugar increases in embryos take place due to an alteration of atmospheric conditions affecting of detached seeds.
2) Diffuse reflectance Using Infrared Dispersion Spectra (DRUIDS) and thin layer chromatography (TLC) have established a substantial rise of ester carbonyl of triacylglycerols from a low amount at the desiccation-sensitive stage (15 DAA) to a high amount at the desiccation-tolerant stage (20 DAA). Subsequent desiccation leads to corresponding diminution in the ester carbonyl signal and the appearance of free fatty acids as indicated by a carboxyl peak at 1710 cm-1 and an associated loss of the TAG ester carbonyl at 1743cm-1.
3) 13C NMR spectra are predominantly the signals corresponding to carbohydrates and has enabled the comparison of the spectra from desiccated embryos and fresh embryos of the same corresponding age. After subtraction of one spectrum from the other, there were clearly large difference in the substracted spectra for the desiccation-tolerant stage when compared to the substracted spectra for the desiccation-sensitive stage.
4) A confocal microscope study of the neutral lipids attained with Nile red established that although there were virtually a complete loss of TAGs as measured by infrared ester carbonyl diminution, there was no corresponding decrease in Nile red fluorescence suggesting that ester bond cleavage did not lead to complete loss of Nile red staining. This was interpreted as most un-ionized residual free fatty acid.
The literature on desiccation tolerance has been reviewed and the results have been discussed in the context of this literature. She concluded that both the carbohydrate and lipid content of embryos exhibit major changes during desiccation. The independent methods and novel observations from this thesis suggest that future workers should carefully examine the role of triacylglycerols in desiccation tolerance and the nature and role of the carbohydrate species characterized in the 13C NMR of the difference spectra of embryos at the desiccation-tolerant stage.