Fresh and dry cell weight
Fig. 1a-c shows the leaf explant, callus production, and cell suspension culture of A. indica. The obtained results demonstrated that different yeast extract concentrations, sampling time, and thier interactions affected both the fresh and dry cell weight (Table 1). The application of yeast extract decreased the fresh and dry cell weight. The most suitable condition for neem cell suspension growth was control without any concentrations of yeast extract. Under these conditions, the mass of fresh and dry cell weight were maximized, which were 413.41 g/L and 14.47 g/L, respectively. Based on results, by addition 25, 50, 100, 150, and 200 mg/L yeast extract the fresh cell weight reduced 29.61%, 35.61%, 27.73%, 45.52%, and 48.66% and dry cell weight reduced 22.53%, 25.57%, 8.64%, 27.99%, and 33.86% compared to the control, respectively. The sampling time of 6 and 4 days gave the highest fresh cell weight and dry cell weight of 410.69 g/L and 16.23 g/L. The fresh cell weight increased from the 2nd day to the 6th day of sampling time but decreased from the 6th day to the 12th day, but the dry cell weight increased from the 2nd day to the 4th day of sampling time and decreased from the 4th day to the 12th day. Therefore, the fresh cell weight on the 6th day of sampling was 47.00%, 1.66%, 88.52%, 111.52%, and 104.56% higher than 2nd, 4th, 8th, 10th, and 12th days of sampling and dry cell weight on 4th day of sampling was 22.67%, 9.90%, 80.05%, 95.30%, and 99.96% higher than 2nd, 6th, 8th, 10th, and 12th days of sampling. By combination study of the effect of different concentrations of yeast extract and sampling times, it was found the highest fresh and dry cell weight were 580.25 g/L and 21.01 g/L obtained 6 days after addition 100 mg/L yeast extract and 4 days after using 50 mg/L yeast extract. In this treatments, fresh cell weight increased 9.00%, 50.86%, 23.23%, 109.97%, and 146.06% compared to control, 25, 75, and 100 mg/L yeast extract at same sampling time and the dry cell weight increased 24.17%, 34.33%, 21.37%, 40.91%, and 81.43% compared to the control, 25, 75, and 100 mg/L yeast extract at same sampling time (Table S1).
Azaidrachtin accumulation and production
The HPLC chromatogram of azadirachtin showed in Fig. 1d. The analysis demonstrated that the applied concentrations of yeast extract, sampling times, and their interaction significantly stimulated the azadirachtin accumulation and production in treated cells compared to the control (Table 1). Accumulation and production of azadirachtin showed a dose-dependent response to the yeast extract. A more increase in azadirachtin accumulation and production was observed at 25 mg/L yeast extract. Yeast extract at the lower concentration of 25 mg/L showed the highest azadirachtin accumulation and production which was 9.67 mg/g DW and 118.53 mg/L. The azadirachtin accumulation and production from control treatment to 25 mg/L yeast extract increased and then decreased with increasing yeast extract concentration from 25 to 100 mg/L. At the 25 mg/L yeast extract the azadirachtin accumulation was 161.57%, 19.79%, 42.45%, 36.34%, and 59.37% and the azadirachtin production was 119.74%, 33.80%, 31.16%, 51.28%, and 106.81% higher than control, 50, 100, 150, and 200 mg/L (Fig. 2a). In terms of sampling time, the highest azadirachtin accumulation and production were 9.20 mg/g DW and 125.65 mg/L observed at 2 days after treatment. On the 2nd day of sampling, the azadirachtin accumulation increased 17.94%, 42.22%, 75.44%, 58.97%, and 34.02% and azadirachtin production increased 0.53%, 31.53%, 160.65%, 207.37%, and 142.42% compared to the 2nd, 4th, 8th, 10th, and 12th days, respectively (Fig. 2b). The effect of different concentrations of yeast extract along with different sampling times is shown in Table S1. The best conditions for induction of azadirachtin accumulation and production were an application of 25 mg/L for 2 days (16.08 mg/g DW) and 4 days (219.78 mg/L), respectively. Under these conditions, the azadirachtin accumulation increased 462.24%, 42.43%, 66.11%, 82.93%, and 146.62% compared to the control, 50, 100, 150, and 200 mg/L yeast extract the 2nd day of sampling and the azadirachtin production increased 302.16%, 54.09%, 96.23%, 51.01%, and 191.79% compared to the control, 50, 100, 150, and 200 mg/L at same day, respectively (Table S1).
Model for predicting of azadirachtin accumulation and production
The sampling times of 2, 4, 6, 8, and 10 days chose to RSM analysis and prediction of azadirachtin accumulation and production. The combined effects of different concentrations of yeast extract and sampling times were investigated by the response surface methodology using a central composite design (CCD). The specific interaction of different concentrations of yeast extract and sampling times with the measured and predicted response values of azadirachtin accumulation and production are shown in Table 2. In this study, experiment No. 2 in 100 mg/L yeast extract and sampling on the 2nd day had the highest amount of azadirachtin accumulation (9.89 mg/g DW) and experiment No. 13 in 150 mg/L yeast extract and sampling on the 8th day had the lowest azadirachtin accumulation (4.29 mg/g DW). Also, experiment No. 2 with an application of 100 mg/L yeast extract and sampling time of 2 days had the highest azadirachtin production (170.37 mg/L) and experiment No. 3 with application of 50 mg/L yeast extract and sampling time of 8 days had the lowest azadirachtin production (32.44 mg/L).
Analysis of variance (ANOVA) of the results of the response surface model was presented in Table 3. In the analysis of variance of CCD, the coefficient of determination of the model for azadirachtin accumulation and production were 95.76% and 94.08%, respectively; which indicates that 95.76% and 94.08% of the actual levels can correspond to the predicted levels. Also, the p-value of the models were significant and the proposed models were appropriate. Therefore, the following formulas were obtained to predict the azadirachtin accumulation and production using yeast extract and sampling times:
Azadirachtin accumulation (mg/g DW) = 6.46 + 0.2452 A – 1.36 B – 1.09 AB – 0.4178 A2 + 0.2269 B2
Azadirachtin production (mg/L) = 121.32 + 1.97 A – 38.14 B + 11.21 AB – 17.73 A2 – 3.25 B2
Optimization of the response surface of azadriachtin accumulation and production
The interaction between different yeast extract concentrations and different sampling times is shown in Fig. 2. Increasing the concentration of yeast extract along with increasing the exposure time to 6 days had a significant effect on the azadirachtin accumulation. The best yeast extract concentrations and sampling times for maximum azadirachtin accumulation were between 100-200 mg/L and 2-4 days. The highest amount of azadirachtin accumulation in this analysis was obtained with application of 100 mg/L yeast extract and sampling time was 2 days. However, the optimal conditions for maximizing the azadirachtin accumulation (13.607 mg/g DW) predicted two days after application of 245 mg/L yeast extract (Fig. 2c and d). Also, the results indicated that azadirachtin production depends on sampling time. According to Fig. 2e and f, at all concentrations of yeast extract, azadirachtin production gradually decreases with increasing sampling time. The highest azadirachtin production is achieved by applying 0-150 mg/L of yeast extract and sampling time of 2-6 days. In this analysis, the highest azadirachtin production was 2 days after application of 100 mg/L yeast extract, but it is predicted that the highest azadirachtin production with 71 mg/L is obtained by culturing of cell for 2 days at medium containing 190.50 mg/L yeast extract.
Mevalonic acid accumulation and production
The HPLC chromatogram of azadirachtin showed in Fig. 1e. The mevalonic acid accumulation and production were significantly changed under different yeast extract concentrations, sampling times, and their interactions (Table 1). Among the studied concentrations of yeast extract, 50 mg/L increased the accumulation of mevalonic acid compared to the control, 25, 100, 150, and 200 mg/L decreased it compared to the control. With increasing the concentration of yeast extract in the culture medium from 0 to 25 mg/L, the accumulation of mevalonic acid decreased, and then with increasing the concentration of yeast extract to 50 mg/L increased and with increasing the concentration of yeast extract form 50 mg/L to 200 mg/L, its amount decreased. The highest amount of mevalonic acid accumulation was 0.63 mg/g DW obtained at 50 mg/L yeast extract, which was 17.31% higher than the control and 48.71%, 381.59%, 265.15%, and 353.86% higher than the 25, 100, 150, and 200 mg/L yeast extract, respectively. Therefore, higher concentrations of yeast extract in the culture medium prevented the mevalonic acid accumulation. With increasing the yeast extract concentration from 0 to 25 mg/L the production of malonic acid decreased and with increasing the concentration of yeast extract from 25 to 50 mg/L increased; however, the difference between control and application of 50 mg/L yeast extract was not statistically significant. The highest mevalonic acid production (8.45 mg/L) was obtained at 50 mg/L yeast extract. By adding 50 mg/L yeast extract to culture medium, the mevalonic acid production increased 8.94%, 47.06%, 381.84%, 302.82%, and 424.19% compared to the control and 25, 100, 150, and 200 mg/L yeast extract, respectively (Fig. 3a). Therefore, the application of moderate concentrations of yeast extract had a positive effect on the accumulation and production of mevalonic acid. By investigating the effect of sampling times, we founded that prolonged exposure of neem cell suspension culture with yeast extract reduces the mevalonic acid accumulation. The highest amount of mevalonic acid accumulation with an average of 0.78 mg/g DW was observed on the second day of sampling. In general, two days after treatment the amount of mevalonic acid accumulation was 55.88%, 79.76%, 213.19%, 915.48%, and 5525.94% higher than the 4th, 6th, 8th, 10th, and 12th days, respectively. Also, between different sampling times, the highest mevalonic acid production (9.95 mg/L) was observed on the 2nd day of sampling. By culturing of neem cells for 2 days, the mevalonic acid production was increased 23.63%, 50.01%, 359.17%, 1724.97%, and 11214.56% compared to days 4, 6, 8, 10, and 12, respectively (Fig. 3b). The interactions of yeast extract concentrations and sampling times showed that the highest mevalonic acid accumulation (1.75 mg/g DW) and production (23.77 mg/L) were obtained two days after application of 50 mg/L yeast extract. In this conditions, mevalonic acid accumulation was 8.92%, 212.90%, 1954.11%, 334.27%, and 2716.13% higher then the control, 25, 100, 150, and 200 mg/L yeast extract and mevalonic acid production was 29.82%, 216.93%, 1801.60%, 370.69%, and 525.53% higher than the 25, 50, 75, and 100 mg/L yeast extract on same day (Table S1).
Model for predicting of mevalonic acid accumulation and production
Based on the results, the sampling times of 2, 4, 6, 8, and 10 days were selected for RSM analysis and prediction of mevalonic acid accumulation and production. The specific interaction of different concentrations of yeast extract and sampling times with the measured and predicted response values of mevalonic acid accumulation and production is shown in Table 2. Experiment No. 10 (six days after the addition of 200 mg/L yeast extract) had the highest amount of mevalonic acid accumulation (0.51 mg/g DW) and experiment No. 2 (two days after application of 100 mg/L yeast extract) had the lowest of mevalonic acid accumulation. Also, experiment No. 12 with the application of 100 mg/L yeast extract for 6 days had the highest amount of mevalonic acid production (6.97 mg/g DW) and experiment No. 2 with using 100 mg/L yeast extract for two days produced the lowest of mevalonic acid production. Analysis of variance results showed that the coefficient of determination (R2) of the model for mevalonic acid accumulation and production were 91.67% and 87.53%, respectively; which indicates that 91.67% and 87.53% of the actual levels can correspond to the predicted levels. Also, the p-value of the models were significant and the proposed models were appropriate (Table 3). Therefore, the following formulas were obtained to predict the mevalonic acid accumulation and production using yeast extract and sampling times:
Mevalonic acid accumulation (mg/g DW) = 0.2513 + 0.0569 A + 0.0199 B – 0.0289 AB + 0.0313 A2 – 0.0364 B2
Mevalonic acid production (mg/L) = 5.26 – 0.0101 A + 0.0210 B – 0.2599 AB + 0.0543 A2 – 1.02 B2
Optimization of the response surface of mevalonic acid accumulation and production
The interaction between different yeast extract concentrations and different sampling times is shown in Fig. 3. The results showed that yeast extract concentration is the most important factor. Accordingly, gradual increases in the concentration of yeast extract lead to a decrease in the accumulation of mevalonic acid in the cell suspension culture of neem. Therefore, in this analysis, the highest amount of accumulation of mevalonic acid was observed at their highest level. Depending on the concentration of yeast extract, sampling time increased or decreased the accumulation of mevalonic acid. According to Fig. 3c and d, the highest amount of mevalonic acid accumulation was obtained 6 days after applying 200 mg/L of yeast extract, but it is predicted that the highest amount of mevalonic acid accumulation with the amount of 0.50 mg/g DW was obtained by culture of the cells for 4.96 days in medium containing 200 mg/L yeast extract. Also, according to the results of this analysis, the production of mevalonic acid depends on the sampling time. At different concentrations of yeast extract, increasing the duration of cell suspension culture exposure to yeast extract increased the amount of mevalonic production from 2 days to 6 days and then decreased. The highest mevalonic acid production was obtained by exposing the cell suspension with 100 mg/L yeast extract for 6 days. The optimal condition for maximizing the mevalonic acid production is predicted at cell suspension culture without yeast extract after 6.54 days, which can produce 5.57 mg/L mevalonic acid.
Squalene accumulation and production
The HPLC chromatogram of azadirachtin showed in Fig. 1f. The results showed that different concentrations of yeast extract, sampling times, and their interactions had a significant effect on squalene accumulation and production (Table 1). The study of squalene accumulation in cell suspension culture in the presence of yeast extract showed that yeast extract inhibited squalene accumulation. Increasing the concentration of yeast extract in the culture medium reduced the accumulation of squalene. With increasing the yeast extract concentration from 25 to 50 mg/L, the squalene accumulation increased and then decreased. The highest squalene accumulation between different concentrations of yeast extract was obtained (0.07 mg/g DW) at the control condition. With using 25, 50, 100, 150, and 200 mg/L yeast extract squalene accumulation decreased 59.70%, 5.97%, 35.82%, 42.27%, and 64.17% than control, respectively. Application of 50 mg/L yeast extract increased squalene production compared to the control and application of 25, 100, 150, and 200 mg/L decreased it. Among different concentrations of yeast extract, the highest production of squalene (1.13 mg/L) was obtained at 50 mg/L, which was 14.85%, 227.71%, 66.62%, 123.72%, and 398.77% higher compared to the control and 25, 100, 150, and 200 mg/L, respectively (Fig. 4a). Between different sampling times, the highest amount of squalene accumulation was 0.10 mg/g DW obtained on the 4th day of sampling. In general, 4 days after treatment the amount of squalene accumulation compared to 2, 6, 8, 10, and 12 days was 547.87%, 44.95%, 363.76%, 149.67%, and 361.09% higher, respectively. Investigation of different sampling times showed that during the first 4 days, the production of squalene in the cell suspension culture of neem increased and then decreased. The highest amount of squalene production (1.74 mg/L) was observed on the 4th day of sampling. So, on the 4th day of sampling the amount of squalene production compared to the 2nd, 6th, 8th, 10th, and 12th days was 732.07%, 68.63%, 748.53%, 343.38%, and 474.63% higher, respectively (Fig. 4b). Further study of squalene accumulation and production was performed using the simultaneous examination of different concentrations of yeast extract and sampling times. The results showed that 4 days after application of 50 mg/L yeast extract the highest amount of squalene accumulation (0.22 mg/g DW) and production (4.53 mg/L) obtained, which were 283.93%, 1333.3%, 69.29%, 41.45%, and 923.81% and 377.43%, 1817.79%, 105.54%, 99.47%, and 1739.84% higher compared to the control, 25, 100, 150, and 200 mg/L, respectively (Table S1).
Model for predicting of squalene accumulation and production
The sampling times of 4, 6, 8, 10, and 12 days were selected for RSM analysis and prediction of squalene accumulation and production. The experimental and predicted values of yeast extract-induced squalene accumulation are shown in Table 4. Experiment No. 6 (six days after application of 50 mg/L yeast extract) had the highest squalene accumulation (0.14 mg/g DW) and experiment No. 10 (150 mg/L yeast extract after 10 days) had the lowest squalene production (0.01 mg/g DW). Experiment No. 3 with an application of 100 mg /L yeast extract and sampling time of 4 days had maximum squalene production (2.21 mg/L) and experiment No. 13 with an application of 50 mg/L yeast extract and sample time of 10 days had the lowest squalene production. The results of the analysis of variance of central composite design showed that the coefficient of determination (R2) of the model is 94.20% for squalene accumulation and 94.48% for squalene production. This indicates that 94.20% and 94.48% of the actual levels of squalene accumulation and production correspond to the predicted levels. Also, the p-value of the models were significant for squalene accumulation and production, which indicates the suitability of the models (Table 3). Therefore, mathematical models were obtained to predict the squalene accumulation and production under yeast extract elicitation:
Squalene accumulation (mg/g DW) = 0.0199 – 0.0169 A – 0.0304 B + 0.0274 AB + 0.0058 A2 + 0.0135 B2
Squalene production (mg/L) = 0.2150 – 0.2509 A – 0.5438 B + 0.4834 AB + 0.0725 A2 + 0.2466 B2
Optimization of the response surface of squalene accumulation and production
Fig. 4 shows the interaction between different yeast extract concentrations and different sampling times. According to the results of the response surface methodology, the squalene accumulation depends on the concentration of yeast extract and the sampling time. The yeast extract concentration is the most important parameter and with increasing that the squalene accumulation has reached a minimum. In the different concentrations of yeast extract except for 200 mg/L, the accumulation of squalene decreased over time. At 200 mg/L yeast extract, the squalene accumulation decreased till the tenth day of sampling and then increased. Therefore, depending on the concentration of yeast extract, sampling time can have a positive or negative effect on squalene accumulation. Based on this analysis results, the highest amount of squalene accumulation was obtained 6 days after application of 50 mg/L yeast extract, but the prediction results showed that the highest squalene accumulation was obtained at free-yeast extract medium after 4 days, which can accumulate 0.30 mg/g DW of squalene. Also, increasing the yeast extract concentration during low exposure periods reduced squalene production, but increasing yeast extract concentration at high exposure times enhanced squalene production. According to Fig. 4e and f, it can be said that using 0-100 mg/L yeast extract for 4-8 days can produce an acceptable amount of squalene. It is predicted that the highest amount of squalene production is achieved by long-term exposure to high concentrations of yeast extract.
qRT-PCR analysis of SQS1 and MOF1 genes expression
The qRT-PCR analysis showed relative gene expression of SQS1 was significantly increased 68.96% at 4 days application of 150 mg/L yeast extract compared with the control cells after 12 days. But, after the addition of 25 mg/L yeast extract for 2 days, 50 mg/L yeast extract for 2 days, 100 mg/L yeast extract for 2 days, and 200 mg/L yeast extract for 12 days, the relative expression of SQS1 gene decreased 73.26%, 74.49%, 38.29%, and 73.53% compared to the control after 12 days. Also, the application of yeast extract significantly up-regulated MOF1 gene. The highest relative expression of the MOF1 gene obtained by addition 25 mg/L yeast extract for 2 days, which was 52.41%, 14.46%, 26.02%, 22.51%, and 39.90% higher than control at 12 days, 50 mg/L yeast extract at 2 days, 100 mg/L yeast extract at 2 days, 150 mg/L yeast extract at 4 days, and 200 mg/L yeast extract at 12 days (Fig. 5).