Alpha 1 4 ld plus freeze drier

Approximately 5 g of chitin or chitosan powder was weighed in a 500‐mL Erlenmeyer flask and added with 50 mL of 85% phosphoric acid. Then, another 50 mL of 85% phosphoric acid was slowly added with continuous stirring. The mixture was incubated at 4°C overnight. Pre‐cooled 500 mL of ethanol was added to dilute the colloid and incubated at 4°C overnight again. The mixture was filtered through a double layer of Whatman No 1 filter paper using a Buchner funnel and then washed with water (approximately 3 L) until pH approached neutral (approximately 5–6). The retentate of colloidal chitin or chitosan was collected in a 50‐mL conical tube and frozen at −80°C overnight. Lyophilization was carried out using an Alpha 1‐4 LD plus freeze‐drier (Christ, Germany). Dried colloidal chitin and chitosan were kept at room temperature. Before use, chitin and chitosan were resuspended in a Hoagland solution in final concentrations of 0.1%, 0.2% and 0.5% w/v. The mixture was sonicated for 20 min to allow dispersion before applying to the plant.

Colloidal chitosan was prepared according to the previous method [73 (link)] with a slight modification. Five g of chitosan powder (medium molecular weight; Sigma, US) was first mixed with 50 ml of 85% phosphoric acid, followed by slowly adding another 50 ml of the acid with continuous stirring. The mixture was left at 4 °C overnight to form a colloidal suspension. Pre-cooled 500 ml of 50% ethanol was added to dilute the mixture, then left at 4 °C overnight again. The suspension was filtered through Whatman Grade 1 filter paper (Whatman plc, UK), aided by vacuum filtration. Colloidal chitosan was retained in the funnel and then washed with distilled water until pH above 5. The retentate was transferred to 50-ml conical tubes and then lyophilized in an Alpha 1–4 LD plus freeze-drier (Christ, Germany). Before use, dried chitosan was resuspended to 1% w/v in Hoagland solution.