Prediction of the effect of chitosan on cell suspension culture of Azadirachta indica by response surface methodology

Key message For the first time, chitosan concentrations and sampling times optimized by response surface methodology for azadirachtin, mevalonic acid and squalene accumulation and production and investigated SQS1 and MOF1 genes expression. Azadirachta indica is used to insects repellent, control diabetes, and combat with cancer. In this study, the effect of different concentrations of chitosan and sampling times on cell suspension culture and SQS1 and MOF1 genes expression were studied and response surface methodology was used to construct the prediction models for azadirachtin, mevalonic acid and squalene accumulation and production. The highest fresh and dry cell weight and azadirachtin accumulation were 726.21 g/L, 20.76 g/L, and 11.74 mg/g DW obtained 6 days after using 50 mg/L chitosan, respectively. The highest azadirachtin production was 165.50 mg/L achieved by using 50 mg/L chitosan for 8 days. Maximum mevalonic acid accumulation (1.87 mg/g DW) and production (22.50 mg/L) were observed 2 and 4 days after the addition of 50 mg/L chitosan and control condition, respectively. The highest amount of squalene accumulation (0.319 mg/g DW) and production (3.56 mg/L) were obtained 10 days after using 50 mg/L chitosan. Also, the prediction results showed the highest azadirachtin accumulation (10.76 mg/g DW) and production (136.83 mg/L), mevalonic acid accumulation (1.869 mg/g DW) and production (20.576 mg/L) and squalene accumulation (0.217 mg/g DW) and production (2.23 mg/L) by using 71.50 mg/L chitosan for 5.24 days, 66.25 mg/L chitosan for 9.1 days, 2.5 mg/L chitosan for 2 days, without chitosan for 2 days, 98.50 mg/L chitosan for 2 days and 80.25 mg/L chitosan for 6.08 days, respectively. The qRT-PCR analysis indicated the maximum relative expression of SQS1 and MOF1 genes by using 25 and 50 mg/L chitosan for 6 days.