Grow seedlings on square (12×12 cm) vertical plates, 8-12 seedlings per plate spaced ~1 cm apart along a horizontal sowing line, with genotype/treatment randomized across plate positions and plate locations within the chamber to control for position effects. Use ≥3 biological replicate plates per condition and ≥15-25 seedlings per condition total. Image daily from day 4 to day 10 post-germination (or a single endpoint at day 7-10). Include all genotypes/treatments on interleaved plates to avoid confounding plate-to-plate variation.
BSL-1. Auxins (IAA, NAA) and ABA are handled in ethanol/DMSO stocks — wear gloves and avoid skin contact. Bleach and ethanol are corrosive/flammable; use in a ventilated hood and never mix bleach with acids. NaCl and MS salts are low-hazard. Autoclave used plates and any transgenic seedling material before disposal. Standard PPE: gloves, lab coat, eye protection. No biohazard beyond plant material; dispose of plant waste per institutional rules.
Wild-type Col-0 grown on the same plates as the internal genotype control. Mock/vehicle control for chemical treatments (equal volume of ethanol or DMSO carrier without the active compound). A positive-control treatment with a known root-length effect (e.g., 100 nM IAA shortens primary roots; 100 mM NaCl reduces growth) to confirm assay sensitivity. Empty-position 'no-seed' lanes to verify no contamination. Plate-orientation control by interleaving genotypes within each plate so plate-effects are not confounded with genotype.
Wild-type Col-0 primary root reaches ~3-5 cm by day 7 and ~5-7 cm by day 10, growing ~0.5-0.7 cm/day, with lateral root density of ~3-6 laterals/cm in the mature region. A growth-inhibiting treatment (e.g., 100 mM NaCl) reduces primary root length by 30-60%. Auxin at 100 nM shortens primary roots and can increase lateral root density. Clean data show tight intra-genotype distributions (CV typically <20%) and clearly separated genotype/treatment means when effects are real.
To measure primary root length, root growth rate, lateral root number/density, and gravitropic set-point in Arabidopsis thaliana seedlings grown on vertically oriented agar plates, enabling rigorous comparison of root-system architecture between genotypes (e.g., mutant vs. Col-0) or across treatments (e.g., ±auxin, ±NaCl, varied phosphate). The assay provides a reproducible, non-destructive, time-resolved readout of root development.
Independent variables: genotype, treatment compound and concentration, time post-germination. Dependent variables: primary root length (mm), root growth rate (mm/day), number of emerged lateral roots, lateral root density (laterals per cm of primary root), gravitropic angle. Controlled variables: medium composition (0.5× MS, 1% sucrose, 1% agar), pH 5.7, photoperiod, light intensity, temperature, plate angle, seed sterilization protocol, stratification time, sowing density, image acquisition distance/resolution.
A genotype or treatment affecting root development (e.g., an auxin-signaling mutant, or addition of 100 nM IAA, or 75 mM NaCl) will produce a statistically detectable change in primary root length and/or lateral root density relative to wild-type/mock controls, measurable within 7-10 days of vertical growth.
Calibrate each image to its embedded scale bar in Fiji. Trace primary roots with the segmented-line tool or SmartRoot to extract length; compute growth rate as the slope of length vs. day. Count laterals manually or with SmartRoot; compute lateral root density = laterals ÷ branched primary root length. Export per-seedling values to a spreadsheet/R; treat each seedling as a technical unit nested within plate (biological replicate). Normalize treatment values to the within-experiment wild-type/mock mean (% of control) when combining experiments.
Roots growing into the agar instead of along the surface: agar too soft or plates not vertical — increase agar to 1.0-1.2% and ensure a steady ~80° angle. Uneven germination: incomplete stratification or old seed — stratify 3 days and use fresh, sterilized seed. Fungal/bacterial contamination: improve sterilization (extend bleach to 10 min) and use micropore tape, not parafilm over the whole plate. Roots waving/skewing: a known agar-surface artifact — keep medium batch and angle constant and quantify a skewing index if relevant. Hard-to-distinguish laterals: image at higher resolution (≥600 dpi scan) and count emerged laterals (>0.5 mm) under a stereomicroscope.
For two groups, use a two-tailed Student's t-test (or Welch's t-test for unequal variances); for multiple genotypes/treatments use one-way or two-way ANOVA with Tukey's HSD post-hoc correction; for dose-response or time-course use mixed-effects models with plate as a random effect to account for non-independence within plates. Use n ≥ 15-25 seedlings per condition across ≥3 plates; set α=0.05. Report means ± SEM and effect sizes; verify normality (Shapiro-Wilk) and homoscedasticity (Levene's), using non-parametric Mann-Whitney/Kruskal-Wallis if assumptions fail.