Establishing the Use of Metabolomics / Genomics to Identify Diagnostic Markers of Seed Quality
High-Performance Liquid Chromatogram (HPLC) used in seed metabolomics studies
This project is one of a series in the theme ‘Genetics and Chemical Traits’
Conventional germination tests are convenient to assess seed viability in species of which certain seed traits are known. In particular, it is helpful to know whether or not a seed displays dormancy and if it is orthodox or recalcitrant. Such knowledge is available for crops, but not for the majority of the species stored in the Millennium Seed Bank, where we are challenged with a biodiversity of thousands of wild species. Some species only germinate after many months, which makes germination testing an unsuitable technique of viability assessment. Therefore, we endeavour to use methods of biochemistry and molecular biology to find markers of viability and germination that allow for a rapid assessment of the basic seed traits of a plant species.
Before such markers of viability and germination can be developed, the underlying physiological processes of ageing and dormancy must be understood (dealt with in other sectors / projects). However, the analysis of certain compounds is often a major challenge in itself. The chemical composition of plant seeds is dominated by their reserves, carbohydrates, lipids and proteins. However, when co-extracted with, for example nucleic acids, these compounds can interfere with conventional assays. In addition, many seeds are rich in (poly)phenols, which can be oxidized to quinones during extraction and then covalently bind to the analyte, for example, antioxidants, leading to erroneous results. Modifying existing methods, finding adequate combinations of existing methods to deal with several interferents simultaneously, or developing new assays for reproducible and reliable measurement of a certain group of chemical compounds is a challenging and often very time-consuming task for the analytical chemist. Here we summarise recent advances in the analysis of chemical compounds in seeds.
Species in the families Brassicaceae, Fabaceae, Fagaceae, Poaceae and Sapindaceae have been studied in more detail. So far (2003-2005), the following techniques have been applied to seeds in our labs.
· Reversed-phase liquid chromatography in combination with fluorescence or diode array detection: 1) low-molecular-weight thiols, 2) pigments and ABA precursors (chlorophylls, carotenoids, xanthophylls) and 3) tocopherols.
· Ion-pairing liquid chromatography in combination with diode array detection: dehydroascorbate / ascorbate.
· Liquid chromatography / pulsed amperometry: anions (fluoride, chloride, nitrite, nitrate, phosphate and sulphate).
· Gas Chromatography-Mass Spectroscopy of 1) volatile hydrocarbons and 2) FAMES (fatty acid methyl esters).
· A method to isolate high-quality RNA from challenging materials was developed that simultaneously eliminates all problems posed by potential interferents in seeds, carbohydrates, proteins, lipid and phenols.
· ‘DNA laddering’ was developed for the use in project ‘Programmed cell death in seeds’ to study DNA degradation.
See Annex 1 for papers from this project.
Project Team
Project Leader: Kranner, Ilse
Seed Conservation Department
Simona Birtic, Ilse Kranner, Charlotte Seal
Funders
UK
MSBP
Annex Material
Annex 1: Information outputs (Word document)