Seeds of the M. truncatula genotype Jemalong R108 were used in this study. The della1 (NF12399), della2 (NF4302) and della3 (NF10539) mutant lines are described in Fonouni-Farde et al.31 (link). Seeds were scarified and sterilized as described in Gonzalez-Rizzo et al. (2006)48 .
For in vitro pharmacological treatments, germinated R108 seeds were grown vertically on a growth paper (Mega International;http://www.megainternational.com/index.htm ) on a “i” medium49 supplemented with 1.5% Bacto-Agar (Gibco), in growth chambers at 24 °C under long-day conditions (16 h light at 150μE light intensity/8 h dark). After two days, the growth paper carrying the plants was transferred on a fresh “i” medium with or without GA3 (0,1 µM, Sigma-Aldrich) or paclobutrazol (PAC, 0,01 µM, Sigma-Aldrich), defined as the minimal concentrations leading to significant effects on root development. Root length was measured using the ImageJ software and emerged LRs were quantified two weeks post-germination.
For hormonal treatments, seedlings grown on a grid in a Magenta box containing a liquid “i” medium were pre-treated for 3 h with 10−6 M GA3 (Sigma-Aldrich) and then treated for 6 h with 10−7 M BAP (Sigma-Aldrich). Control experiments performed in parallel consisted in mock-treated samples. Roots were collected and immediately frozen in liquid nitrogen.
For in vitro pharmacological treatments, germinated R108 seeds were grown vertically on a growth paper (Mega International;
For hormonal treatments, seedlings grown on a grid in a Magenta box containing a liquid “i” medium were pre-treated for 3 h with 10−6 M GA3 (Sigma-Aldrich) and then treated for 6 h with 10−7 M BAP (Sigma-Aldrich). Control experiments performed in parallel consisted in mock-treated samples. Roots were collected and immediately frozen in liquid nitrogen.
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