Cinnamomum longepaniculatum (Gamble) N. Chao is an evergreen tree of the genus Cinnamomum of the family Lauraceae. C. longepaniculatum is a precious tree species in the genus Aphididae. The distribution is mainly concentrated in Taiwan, China and Yibin, Sichuan. The oil of C. longepaniculatum can be extracted from the leaves, stems and roots of C. longepaniculatum by steam distillation. At this stage, the oil extracted from C. longepaniculatum in Yibin is mainly essential oil  .
The essential oil of C. longepaniculatum is mainly composed of dozens of different chemicals  , and the main component 1,8-eucalyptus  is widely used in many industries, such as spices, food, medicine, chemical and defense. With the increase of the world’s population and the improvement of people’s material living standards, the demand for medicines, foods, spices, etc. has increased, and the demand for 1,8-eucalyptus has increased rapidly     .
Suspension cell culture refers to establish callus or excised plant cells and transfers to a liquid medium for sterile shaking culture  . The relatively uniform cells obtained by this culture method not only provide a unique experimental system for studying cell proliferation and differentiation, but also have a rapid cell proliferation rate and are suitable for large-scale culture. Therefore, there is a huge application potential in the industrial production of plant products   .
With the rapid development of society and the continuous improvement of people’s quality of life, the demand for natural flavors such as essential oil has doubled, leading to a growing contradiction between supply and demand. Coupled with the plant’s secondary metabolite instability and environmental constraints, the yield and quality of the essential oil cannot meet the demand. Therefore, the study of the influencing factors of essential oil is of great significance for significantly increasing the yield of essential oil, and has become an important content and a new development direction of C. longepaniculatum resources and plant aromatic oil research.
In the process of suspension culture of plant cells, the activity of enzymes in cell metabolism can be activated by adding exogenous inducers  , thus increasing the production of secondary metabolites in oil suspension cells, and sometimes even inducing the new production of secondary metabolites in oil suspension cells   , with the continuous development of plant tissue and cell culture technology, cell suspension culture system is becoming more and more perfect. It has always been a hot spot for researchers to study how to use plant cell suspension culture technology to produce useful secondary metabolites and increase metabolite production   . This technique of using elicitors to increase the production of secondary metabolites in oil suspension cells in the short term is currently a more mature method  . Therefore, finding suitable inducers can effectively increase the secondary metabolites of suspended cells and improve the oil production rate, which has high research value and application prospects.
2. Materials and Methods
All young leaves were sourced from the C. longepaniculatum base of Hongyan Mountain in Yibin. Cut the leaves blade into the appropriate size (should be placed in a sterile container) and rinse with running water for a few minutes to 30 minutes. Then soaked in 70% alcohol for 10 to 30 seconds and washed it 3 to 10 times with sterile water. Inoculated the treated aseptic explants in a 100 ml solid triangular medium.
Main Drugs and Medium
Inducers: CuSO4, FeSO4, Li2SO4, MnSO4, CaSO4, H2O2, tryptophan, SA (salicylic acid), sodium benzoate B5 medium: A large number of elements 1: KNO3 50 g, 1 L (take 50 ml); A large number of elements 2: CaCl2-2H2O 3 g, MgSO4-7H2O 5 g, (NH4)2SO4 2.68 g, NaH2PO4-2H2O 3 g, 1L (take 50 ml); Trace element 1: KI 75 mg, H3BO3 300 mg, MnSO4-H2O 1000 mg, ZnSO4-7H2O 200 mg, Na2MoO4-2H2O 25 mg, 500 ml (take 5 ml); Trace element 2: CuSO4-5H2O 25 mg, CoCl-6H2O 25 mg, 500 ml (take 0.5 ml); Iron salt: FeSO4-7H2O 2780 mg, Na2-EDTA-2H2O 3730 mg, 500 ml (take 5 ml); Organic matter: Inositol 10,000 mg, niacin 100 mg, Pyridyl hydrochloride 100 mg, Thiamine hydrochloride 1000 mg, 500 ml (take 5 ml).
B5 medium was used as the basic medium, and the single factor test of different levels of the same inorganic inducer was set up (Table 1), the single factor test of different levels of the same organic inducer (Table 2) and different inducers. Orthogonal test (Table 5), the medium was dispensed into a 150 mL Erlenmeyer flask, repeated for 3 bottles treatment, and sterilized at 115˚C for 20 min in an autoclave. After cooling, callus solid cells were transferred to liquid medium. The callus solid cells were cultured for 28 days in 25˚C in a constant temperature incubator, and its essential oil production was measured every 7 days during its cultivation.
Table 1. Single factor test of inorganic inducer.
Table 2. Single factor test of organic inducer.
Cultivation of suspension cells and establishment of suspension system
The inoculated explants were placed in a culture chamber, and the temperature was controlled at about 23˚C. After the callus induction was completed, it was subcultured twice. The transfer was performed twice, and about 2.0 g of the well-grown and loose pink callus was inoculated into a 150 mL Erlenmeyer flask containing 50 mL of B5 medium at a speed of 110 r/min at 25˚C. Shake culture under shading conditions. 14 days were subcultured once, followed by 2 times.
Inducer Single Factor Test
B5 medium was prepared, and the inorganic inducer was added to the same amount of inducer after each component, and sterilized at 115˚C for 20 min in an autoclave with an inoculation needle; organic inducer for sterilization in a high-pressure steam sterilization pot at 115˚C for 20 min, after cooling, an equal amount of each organic concentration inducer was removed by filtration under a sterile condition using a 0.22 μm water filter, as shown in Table 1 and Table 2. Then, it was placed in a suspension culture at 28˚C, 120 r/min for 28 days, and each concentration was repeated 3 times, and 3 controls were set at the same time.
Different Inducer Orthogonal Test
According to the results of single factor test, after the significance test of spss data processing system, the inducers with significant differences were selected for orthogonal test, and the yield of essential oil was used as the index to carry out the orthogonal test of L16(45) (Tables 3-6), each set of experiments was repeated 3 times.
Extraction and Determination of Essential Oil from Suspension Cells of C. lngepaniculatum
The suspension cells were removed from the culture flask under sterile conditions every 7 days and dried at 55˚C. Accurately weigh 0.2000 g - 0.6000 g of C. longepaniculatum suspension cells, add 4 times (1:4) of cyclohexane or petroleum ether for cold soak overnight, then treat in ultrasonic cleaner at 70˚C for 30 min, and finally centrifuge at 5000 r/min for 4 min. The supernatant was taken and made up to 5 mL with cyclohexane. The liquid was extracted with a syringe, and the filter was filtered into a sample bottle to determine the content of the essential oil, which was analyzed by GC-MS. Chromatographic conditions: column temperature 60˚C; HP-5MS column, 30 m × 0.250 mm; injection volume is 1 μL; programmed temperature: from 60˚C after the temperature increase rate of 10˚C/min to 190˚C and maintained for 2 min, Then, it was raised to 210˚C for 2 min at a temperature increase rate of 5˚C/min, and then increased to 220˚C for 8 min at a temperature increase rate of 10˚C/min, and then injected into a GC-MS to obtain a standard curve, that is, essential oil content response value was obtained. Further, the essential oil content was calculated  (1,8-eucalyptol: Y = 73900X − 299200, R2 = 0.9993).
Table 3. Effects of different inorganic inducers on the accumulation of essential oil in suspension culture cells.
Note: * There is a significant difference in inorganic inducers.
Table 4. Effect of different organic inducers on the accumulation of essential oil in suspension cells.
Note: *There is a significant difference in inorganic inducers.
Table 5. Orthogonal test of different inducers.
Note: A: CaSO4 (mg/L); B: H2O2 (mmol/L); C: SA (mg/L); D: Li2SO4 (mg/L).
Table 6. Analysis of variance of essential oil accumulation in orthogonal experiment of different inducers.
Note: *p < 0.01.
Significance Test of Experimental Data
The experimental data was entered into the SPSS data processing system table and statistical analysis of the single factor test was performed on a completely random design of the test statistics tab. Perform a significant test on the experimental data and create a line graph of the experimental results in an Excel spreadsheet.
The Effect of Single Factor of Inorganic Inducer on the Yield of Essential Oil from Oil Cell Suspension Cells
B5 medium was prepared, and the essential oil production was measured on the 7th, 14th, 21st and 28th day of suspension cell culture. The effect of single factor of each inorganic inducer on the accumulation of essential oil in the suspension cells of C. longepaniculatum was examined. The results are shown in Table 3. The results shown in the table are the average of 3 trials.
Table 3 shows that the oily suspension cells treated with each inorganic inducer showed an increase in the yield of most of the essential oil in the range of 21 d, and reached the maximum at 21 d. When the culture time was greater than 21 d, the essential oil content in the suspension cells gradually decreased; as the concentration of the inorganic inducer increased, the essential oil production increased first and then decreased, but the cells may be aging, and some cells reached the maximum in the second cycle. The overall trend of the increase was higher than that of the no-inducer group (CK). The effects of essential oil accumulation on the C. longepaniculatum cell suspension cells treated with inorganic inducers were basically consistent. Among them, at the 21st day, the induced concentrations of CuSO4, FeSO4, Li2SO4, MnSO4, CaSO4, H2O2 were 0.02 mmol/L, 0.05 mmol/L, 1.0 g/L, 3 mmol/L, 1.5 mg/L, 9 mmol/L, respectively. The essential oil yield was the largest under the treatment of this inorganic inducer, 0.0530 mg/L, 0.0635 mg/L, 0.0792 mg/L, 0.0632 mg/L, 0.0824 mg/L, 0.0692 mmol /L, respectively. The yield increased by 3.52%, 24.02%, 54.69%, 23.44%, 60.94%, 35.16%, respectively. According to the significance test, the induced concentrations of CuSO4, FeSO4, Li2SO4, MnSO4, CaSO4 and H2O2 were respectively 0.02 mmol/L, 0.05 mmol/L, 1.0 mg/L, 3 mmol/L, 1.5 mg/L, 9 mmol/L at 21 d, there was a significant difference between the yield of 1,8-eucalyptus oil and other concentrations under the inducer at this time (P < 0.05). According to the SPSS significance test, CaSO4 had the most significant difference compared with other inorganic inducers on the 21st day.
Effects of Organic Inducer Single Factor on the Yield of Essential Oil from Suspension Cells
B5 medium was prepared, and the same concentration of organic inducer was added after sterilization and cooling. The essential oil production was measured on the 7th, 14th, 21st and 28th day of suspension culture, and the single factor of each organic inducer was detected. The results are shown in Table 4, the results shown in the table are the average of 3 trials.
Table 4 shows that the essential oil production increased in the range of 21 d when the cultured cells were treated with organic inducers, and reached the maximum at 21 d. When the culture time was greater than 21 d, the content of essential oil in suspended cells decreased gradually. With the increase of organic inducer concentration, the yield of essential oil increased first and then decreased. The yield was higher than that of the no-inducer group (CK). The effects of essential oil accumulation on the suspension cells of C. longepaniculatum treated by organic inducers were basically consistent. Among them, on the 21st day, when the concentrations of tryptophan, SA and sodium benzoate were 0.148 g/L, 15 mg/L and 10 mg/L, respectively, the essential oil yield was the highest under the treatment of organic inducer, which was 0.0597 mg/L, 0.0700 mmol/L, 0.0674 mmol/L, the yield increased by 16.60%, 36.72%, 31.64% compared with CK; according to significant test analysis, on the 21st, tryptophan, SA, sodium benzoate induced concentration of 0.148 g/L, 15 mg/L and 10 mg/L, the yield of 1,8-eucalyptus oil was significantly different from that of other concentrations under the inducer at this time (P < 0.05).
Different inducer orthogonal test
Four factors including CaSO4, H2O2, Li2SO4 and SA were selected by orthogonal test, and L16(45) was used as the index of essential oil production of orthogonal test (Table 5). The results of the variance analysis of the essential oil production in the inducer induction test are shown in Table 6. The variance analysis of essential oil production by orthogonal test of different inducers showed that the inducer for the significant difference in essential oil production was CaSO4, and the other three inducers did not reach a significant level for the essential oil production. Through the orthogonal test, the influence of the R value of the four factors in Table 5 on the essential oil yield is: CaSO4 > Li2SO4 > SA > H2O2.
The yields of essential oil and the K value of each group in different orthogonal test tables were analyzed. The optimal combination of 4 groups was selected, namely A4B4C1D4, A4B4C4D4, A4B4C1D1, A4B4C4D1 4 groups conduct verification tests. The results showed that when CaSO4 2.0 mg/L, H2O2 9 mmol/L, SA 15 mg/L, Li2SO4 0.5 mg/L were added to the suspension medium, the yield was the largest. It was 0.1768 mg/L, and compared with the yield of CK which have no inducer was increased by 245.31%.
Studies have found that the addition of inducers can alter the pathway of secondary metabolite synthesis; it can also promote or inhibit the activity of key rate-limiting enzymes in the bio-metabolite synthesis pathway  ; this experiment shows that there are differences between different inducers against C. longepaniculatum suspension cells and the effects of essential oil production, indicating that different inducers can affect the accumulation of essential oil in suspension cells to varying degrees.
In the single factor experiment, the suspension cells were treated with inorganic inducers CaSO4, H2O2, Li2SO4, and the essential oil yield was compared with other inducers. There are significant difference (P < 0.05), and CaSO4 was more significant than the other two, the yield was larger, reaching 0.0824 mg/L, which was 60.94% higher than CK. This is related to calcium ion Activator-related, it activates certain secondary metabolic pathways to increase metabolite production; H2O2 regulates gene expression and signaling as a signaling molecule to regulate secondary metabolic pathways  . The yield of essential oil treated by CuSO4, FeSO4 and MnSO4 is higher than that of CK, but the difference is not significant, which promotes the accumulation of essential oil, but it is not significant, and the specific reasons need to be further carried out and explored. In summary, the promotion of the inorganic inducer on the accumulation of essential oil in the suspension cells of C. longepaniculatum is CaSO4 > Li2SO4 > H2O2 > FeSO4 > MnSO4 > CuSO4.
Treatment of C. longepaniculatum suspension cells with organic inducer SA can significantly promote the yield of essential oil from C. longepaniculatum suspension cells, reaching 0.0700 mmol/L, which is 36.72% higher than CK. It is found that SA acts as a signal molecule involved in signal transduction, activation and inhibition. The corresponding transcription factors affect the expression of genes and regulate the synthesis of secondary metabolites  . In summary, the promoting effect of organic inducers on the accumulation of essential oil in oil suspension cells was: SA > sodium benzoate > tryptophan. Therefore, based on the difference in the accumulation of essential oil, the dominant inducer can be found, thereby optimizing the way to induce the accumulation of essential oil in the suspension of cells.
In the orthogonal experiment, CaSO4 had significant differences in the yield of essential oil from the C. longepaniculatum cell suspension compared with H2O2, Li2SO4, SA. The three inducers had no yield for essential oil. To achieve the level of significance, the effect of promoting essential oil production in orthogonal test was: CaSO4 > Li2SO4 > SA > H2O2. Orthogonal test showed that when the callus suspension cells were added CaSO4 2.0 mg/L, H2O2 9.0 mmol/L, SA 15 mg/L, Li2 SO4 0.5 mg/l. The yield of essential oil was the highest at 0.1768 mg/L, which was 245.31% higher than that without inducer. This indicated that the orthogonal combination of different inducers had the best effect on the yield of essential oil in suspension cells and the inducers in several different pathways added simultaneously have synergistic effects  .
In this experiment, the effects of nine inducers and their partial combinations on the accumulation of essential oil in the suspension cells of C. longepaniculatum were studied, which promoted the accumulation of essential oil to some extent, but also encountered some problems in the experiment: such as the freshness of the cells and the effect of the length of the suspension cell culture cycle on the accumulation of essential oil, which needs further study. In the process of suspension culture of callus in the future, we can also optimize the culture conditions of its growth stage and metabolite synthesis stage, which lays a foundation for artificial regulation and optimization of the accumulation of secondary metabolites in induced callus.
The first and corresponding author acknowledges that this work was supported by Key Lab of Aromatic Plant Resources Exploitation and Utilization in Sichuan Higher Education (grant no. 2015XLY002).
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