Analyses of several seed viability markers in individual recalcitrant seeds of Eugenia stipitata McVaugh with totipotent germination.

The use of biochemical seed viability markers is often compromised by the unknown partitioning of analytes in bulk seed lots consisting of inseparable populations of viable and nonviable seeds. We took advantage of an unusual morphological syndrome found in the recalcitrant, undifferentiated seeds of Eugenia stipitata: one seed can be cut into several parts, each of which can germinate and develop into seedlings. We used four seed parts from one individual seed to analyse seed moisture content (MC), seed viability and the antioxidant glutathione (γ-glutamyl-cysteinyl-glycine; GSH), glutathione disulphide (GSSG) and intermediates of glutathione synthesis and breakdown. Seeds were exposed to different environmental MC to induce various levels of desiccation stress. Upon storage at high seed MC, seed viability was maintained, while GSH concentration increased and the glutathione half-cell reduction potential (EGSSG/2GSH ) was less negative than -215 mV, indicating GSH production and highly reducing conditions. Storage at low seed MC led to loss of GSH, resulting in a shift in EGSSG/2GSH , and seed death. In contrast, the cyst(e)ine half-cell reduction potential (ECySS/2CYS ) could not distinguish between the viability categories. Previous studies on seed populations revealed that the probability for a seed being alive is 50% at EGSSG/2GSH values between -180 and -160 mV. The single seed approach revealed that the window in which seed viability was lost could be slightly shifted towards more negative values. We discuss the contribution of cellular pH to EGSSG/2GSH and recommend E. stipitata as a recalcitrant seed model to study stress response on a single seed basis.