Measuring Immune-Induced Inhibition of Seedling Growth

Abstract

Source: Bredow, M. et al., Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana. J. Vis. Exp. (147),(2019).

This video demonstrates the seedling growth inhibition assay in the Arabidopsis thaliana plant. The impact of increased concentration of immune peptide elicitor on seedling growth is determined.

Protocol

1. Seedling Growth Inhibition Assay Sterilize seeds and sow on Murashige and Skoog (MS) plates. Prepare sterile half-strength (0.5x) MS medium (2.16 g/L) containing 0.8% agar [w/v]. Pour media into plates (90 x 15 mm) in a laminar flow hood. Sterilize approximately 100 seeds in a microcentrifuge tube by washing with 1 mL of 70% ethanol for 2 min and remove by aspiration. Add 1 mL of 40% bleach and gently rock for 17 mins at room temperature. Remove bleach by aspiration …

Representative Results

Figure 1. Elicitor-induced seedling growth inhibition assay in Arabidopsis. (A) Sow seedlings on MS agar and grow for 3-4 days under standard short-day conditions. (B) Transplant seedlings to 48-well plates containing MS medium or MS containing different concentrations of elf18. (C) After 8-12 days, visually assess the seedling size and then measure fresh weight using an analytical scale…

Materials

8-Well Sterile Plates with Lid	Sigma-Aldrich	CLS3548
Analytical Scale with Draft Sheid	VWR	VWR-225AC	Any standard analytical scale can be used for growth inibition assays, however, a direct computer output is optimal.
BioHit mLine Mechanical 12 Multichannel Pipette (30-300 uL	Sartorius	725240	Any multichannel pipette can be used, as can a single pipetter if necessary.
elf18 (AcSKEKFERTKPHVNVGTIG)	EZ Biolab	cp7211	Store 10 mM stock peptide at -80 deg C in low protein binding tubes. When thawed, store 100 uM working stock at -20 deg C.
Murisage and Skoog Basal Salts	Cedarlane Labs	MSP09-100LT	Store at 4 deg C.
White Polystyrene 96-Well Plates	Fisher Scientific	07-200-589