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Towards Universally Applicable Seed Viability Stains

Positive tetrazolium staining of viable embryo in seed of Najas marina (Najadaceae)

This project is one of a series in the theme 'Diagnosis of Viability and Germination'.

Viability tests are routinely used to differentiate between dead and dormant seeds and are particularly useful for rapidly assessing seed lot viability, especially for slow or difficult to germinate species. Tetrazolium chloride (TZ) is routinely used as a viability test for agricultural and horticultural species and there are precise international guidelines published by, inter alia, the International Seed Testing Association on the application and interpretation of staining patterns for different taxa. In addition, the fluorescent stain fluorescein diacetate (FDA) is widely used for micro-seeds, e.g. Orchidaceae.

TZ has been widely used within the Seed Conservation Department over many years. Whilst a single simple methodology has been applied across a broad range of germplasm, test validation and wider acceptance has been precluded by the use of small seed numbers and variable repeatability. As a consequence, we have followed two approaches to improving tetrazolium staining. Firstly we have analysed historical viability test data from the Seed Bank Database for 171 species. Results from this analysis indicate that for species for which there are no international guidelines, the test often under-estimated seed viability, presumably as a result of poor stain permeation into seed tissues. Therefore without improvement, the usefulness of the test on a wide range of germplasm may be limited. Secondly, we have developed a technique for improving stain permeation into seed tissues by vacuum degassing seeds in the TZ solution. Application of this method to 17 Pinus species has shown that degassing substantially shortens the period required for stain development and also decreases the incidence of unclear, patchy staining patterns. We are applying this technique to other seed material e.g. Asteraceae to assess its wider applicability.

FDA has been used as a viability test for a range of UK orchids, however it has not been used on orchids from a wider range of habitats or on seed lots of the same species differing in viability level. Consequently we have assessed the effectiveness of FDA on orchids from Kenya which has revealed that stain permeation into the Kenyan species was problematic as a result of a thicker seed coat compared to UK species. We have also compared the effectiveness of FDA on multiple seed lots, differing in viability, of a range of UK species, which has further demonstrated that, at least for UK species, FDA provides a fast and reliable assessment of viability. Further testing is anticipated as Australia and Botswana enhance their orchid collecting.

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