Computational protocol: Arabidopsis ICK/KRP cyclin-dependent kinase inhibitors function to ensure the formation of one megaspore mother cell and one functional megaspore per ovule

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[…] For seedling biomass analysis, seeds of WT, ick12567 and septuple mutants were planted in soil in the plant growth room. For each line, three pots each having five plants were used in the analysis. At 21 days after planting, seedlings were collected from each pot and weighed immediately for the fresh weight. Experiments were repeated at four different locations in the growth room. The relative fresh weight for each mutant (fresh weight of mutant/fresh weight of WT) was obtained.For morphological phenotyping, the 1st, 2nd, 3rd and 4th pairs of true leaves from seedlings at the indicated plant ages were separated, placed on a flat surface and photographed with a digital camera. The leaf sizes, leaf length and width were measured from the obtained images using ImageJ software (http://rsbweb.nih.gov/ij/index.html). The leaf length/width ratio for each line was obtained from its length and width data. For seed weight analysis, mutants and control plants were grown side by side to minimize the variation in growth conditions. Seeds were collected on the basis of one pot each having four plants, and three seed lots (each from the four plants in one pot) were used, with 1000 seeds from each seed lot counted and weighed. Three different batches of seeds from plants grown at different locations of the plant growth room were analyzed. For the analyses of silique and seed development, ten fully extended siliques (6th -15th from the bottom) from four plants per line were measured. Six measured siliques per plant were opened using a pairs of sharp tweezers, and counted for the number of the seeds and aborted ovules under a Zeiss dissecting microscope. For statistical analysis, one-way ANOVA and post-hoc Tukey test (using the software SPSS) were performed to determine the significance between lines. […]

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