Germination and seedling establishment assays

Seeds were raised from plants grown in long day conditions (16 h/8 h at 22 °C) as described in;²¹ all genotypes to be compared were raised in the same cabinet. ABA sensitivity of germination was determined using 0.5 X MS medium containing 0–10 μM ABA. Seeds were sterilised as described in⁶³ and stratified for 2 d at 4 °C before transfer to 22 °C under continuous light. Germination is defined as emergence of the radicle (embryonic root) from the seed coat. A linear mixed model was fitted using REML to the logit transformation of percentage germination, using an offset of 0.5. A minimum of three seed batches (50 seeds per batch, except for prt6 abi3–6; 25 seeds per batch) was used for each line, with the same batch used for all ABA concentrations. Thus, seed batch was included as a random term. Fixed effects were assessed using approximate F-statistics⁶⁴ and included the 2 × 2 factorial structure to assess whether the abi and prt6 mutations acted independently across the ABA concentrations. Sugar sensitivity was assessed by germinating seeds as above on 0.5 X MS medium containing 0.5% or 4% sucrose. Following 5 d growth in long day conditions, establishment was scored as the development of photosynthetic competence (green cotyledons). Experiments were carried out in triplicate, using a minimum of 25 seeds per replicate. A non-parametric one-way Kruskal-Wallis ANOVA was applied to the set of different genotypes for each sugar condition. The associated test statistics (calculated after an adjustment for ties) were evaluated against a chi-squared distribution.