Arabidopsis, Tomato, and Barley Seed Sterilization

Arabidopsis thaliana (L. Heynh), ecotype Columbia (Col-0) reporter lines, transformants DR5rev:GFP and DR5:GUS^{54 (link)}, WOX5:GFP^{29 (link)}, PIN1:PIN1-GFP^{55 (link)}, YUC2:GUS^{56 (link)}, mutants npr1–1^{57 (link)}, coi1–40^{58 (link)} and the triple mutant myc2/3/4^{59 (link)}, were tested with chitosan. Arabidopsis seeds were surface-sterilised using 1% NaClO for 2 min and then washed 3 times with sterile distilled water. Surface sterilised seed were stratified at 4 °C for 48 h and then grown on MS (Murashige and Skoog medium; Sigma) plates as in Ripoll et al.^{60 (link)}.
Tomato (Solanum lycopersicum Mill cv. Marglobe) and barley (Hordeum vulgare L. var. Disticum) seeds were also surface-sterilised using 50 ml 4% sodium hypochlorite with 3 drops of Tween-20 (Sigma-Aldrich) for 20 min and 1 h, respectively. They were then washed three times (5 min each) with sterile distilled water. Surface-sterilised seeds were plated on 9 cm petri dishes with a germinating medium (1.2% agar supplemented with glucose (10 g l⁻¹), peptone (0.1 g l⁻¹) and yeast extract (0.1 g l⁻¹)^{61 (link)}. Seeds were stratified for two days at 4 °C and then incubated at 25 °C in the dark for 5 days and finally 4 days under a 16 h/8 h (light/darkness) photoperiod.