Evaluating <em>Arabidopsis</em> Primary Root Growth in Response to Osmotic Stress Using an In Vitro Osmotic Gradient Experimental System

The root meristem navigates the highly variable soil environment where water availability limits water absorption, slowing or halting growth. Traditional studies use uniform high osmotic potentials, poorly representing natural conditions where roots gradually encounter increasing osmotic potentials. Uniform high osmotic potentials reduce root growth by inhibiting cell division and shortening mature cell length. This protocol describes a simple and effective in vitro system using a gradient mixer that generates a vertical gradient in an agar gel based on the principle of communicating vessels, exploiting gravity to generate a continuous mannitol concentration gradient (from 0 to 400 mM mannitol) reaching osmotic potentials of -1,2 MPa. It enables long-term <i>Arabidopsis</i> root growth analysis under progressive water deficit, improving phenotyping and molecular studies in soil-like conditions. Key features • Novel approach: Unique method to evaluate primary root growth in <i>Arabidopsis</i> under increasing osmotic potentials. • Osmotic gradient system: Simulating a gradual osmotic gradient in the root growth zone while maintaining aerial tissues under control conditions. • Sustained growth: <i>Arabidopsis</i> Col-0 and <i>ttl1</i> mutant seedlings maintain proper root growth for 25 days, even at osmotic potentials as low as -1.2 MPa. • Enhanced growth rates: Roots grown in the osmotic gradient exhibit higher growth rates than those in homogeneous high osmotic potential conditions. • Phenotypic observation: <i>ttl1</i> seedlings grown in the osmotic gradient do not show the typical swelling phenotype observed at extreme osmotic potentials (-1.2 MPa).