Naked A. sativa L. 'Moyencourt' seeds (i.e. seeds without glumellae) were used for this study. In order to maintain the initial primary dormancy, freshly harvested seeds were stored at -20°C until experiments started. Dormancy was broken by storing the seeds dry for at least 6 months at 20°C in open air (Corbineau et al., 1986). Thermodormancy was induced by incubating primary dormant seeds at 30°C for 42 h (Corbineau et al., 1993).
Germination assays were performed with 100 naked seeds placed in Petri dishes 10 cm in diameter (i.e. four replicates of 25 seeds per dish) on a layer of cotton wool wetted with deionized water or various solutions of polyethylene glycol-8000 (PEG-8000) or NaN3. The water potential of various solutions was adjusted by the addition of PEG-8000 and calculated according to Michel and Kaufmann (1973). A seed was considered to have germinated as soon as the radicle protruded from the grain envelopes (i.e. pericarp and seed coat).
Adenosine phosphate and non-adenylic triphosphate nucleotide determinations
ATP, adenylic diphosphate nucleotide (ADP), adenylic monophosphate nucleotide (AMP) and NTP were extracted from embryos isolated from primary dormant, non-dormant and thermodormant seeds incubated for various durations on water or PEG-8000 solutions at 20°C or 30°C, according to the method described by Lecat et al. (1992). They were also extracted after a subsequent 3 h incubation of the seeds in the presence of 10 mM adenosine. Adenylic nucleotide and NTP levels were determined using a bioluminescence method (Gendraud, 1977; Lecat et al., 1992) with a pico-ATP biophotometer (Jobin et Yvon, France). The results obtained are expressed as picomol ATP, ADP, AMP or NTP per milligram dry weight (DW), and are the means of values obtained with five extracts from two embryos each ± standard deviation (sd). The ATP/ADP and NTP/ATP ratios and the EC, expressed as (ATP + 0.5 ADP)/(ATP + ADP + AMP), were also calculated.
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