Physiological tests
For all tests, three replicates of 30 seeds each for M.
truncatula or three replicates of 150-200 seeds each of Arabidopsis
were used unless mentioned otherwise. To test for germination, M.truncatula seeds were scarified with a sandpaper and imbibed in
the dark at 20°C (Chatelain et al. 2012). Seeds of A.
thaliana were imbibed at 20°C and a photoperiod of 16 hours light. For
both species, germinated seeds were scored at regular time intervals by
counting seeds having an emerged radicle with length ≥ 1 mm. To release
physiological dormancy prior to germination tests, scarified seeds ofM. truncatula were imbibed at 20 °C for 4 h in Petri
dishes onto filter papers then transferred at 4 °C for three days in the
dark. For A. thaliana, seeds were imbibed for 3 days directly at
4°C in the dark. Water contents were assessed gravimetrically for
triplicate samples of 5 seeds by determination of the fresh weight and
subsequent dry weight after 2 d in an oven at 96°C. Water contents are
expressed on a dry weight basis. Seed weight of the genotypes was
measured on five replicates of 50 seeds after oven drying at 96°C for
2d.
For accelerated aging assays, scarified seeds of M. truncatula or
mature seeds of Arabidopsis were placed over a saturated solution of
NaCl (75% RH) at 35°C in hermetically sealed boxes and viability was
evaluated by germination at regular time intervals. Longevity was
expressed as P50, defined as the time (days) at which the stored seed
lost 50% of viability during storage. Controlled deterioration assays
were carried out by equilibration of seeds at 85% RH (saturated KCl) or
incubation in water vapor (100%) and 35°C or 40°C.
The effect of ABA inhibition on the T50, the time of imbibition needed
to reach 50% of germination, was determined by imbibition of mature
seeds of M. truncatula in a 10 µM fluridone solution freshly
prepared from a 1 mM stock solution where fluridone was dissolved in
ethanol and Tween 20 solution (2/1 v/v).
ABA sensitivity was determined by imbibition of mature, 4 months after
ripened seeds on filter paper on a range of ABA concentrations (mixed
isomers, Sigma, St Louis, MO, USA) at 20°C, 16h light or in the dark.
ABA was dissolved in methanol prior to dilution in water. Control seeds
were imbibed in the MeOH concentration corresponding to the highest ABA
concentration (0.05% MeOH). Germination was scored after 14 days.
To evaluate dormancy model of the Mthsfa9 mutants is imposed by
the embryo or by the surrounding tissues (testa, endosperm) or
combinations of these tissues, three replicates of 30 seeds were imbibed
for 6 hours at 20 ° C. Using a magnifying glass, the endosperm and seed
coat were carefully removed, taking care of not damaging the embryos.
Scarified intact seeds and naked embryos were imbibed in water at 25 ° C
in the dark. Germination (for intact seeds) or embryo growth were
recorded over time.