Melia azedarach L. from the Meliaceae, contains a wide range of secondary metabolites used as antibacterial, antiviral, antioxidant and anticancer in traditional medicinal. In the present study, the effect of PGRs and explant type on callus induction and cell suspension culture establishment and growth were investigated. We investigated the biochemical properties, and production and accumulation of secondary metabolites such as rutin, quercetin and kaempferol in M. azedarach L. cell cultures. The results showed that the inflorescence and petiole explants had a high percentage of callus induction compared to the leaf explants, but the highest callus growth were observed in leaf explants in MS+3 mg/L NAA+5 mg/L BAP/3 mg/L Kin and 5 mg/L 2,4-D+5 mg/L Kin (6.307, 5.152 and 3.977 g/explant, respectively). The establishment and growth of cell cultures were significantly influenced by PGRs combination and explant type. The highest cell growth were obtained from the leaf and inflorescence callus transferred into the liquid MS medium supplemented with 1 mg/L 2,4-D+1 mg/L Kin (8.489 g/50 mL suspension) and 1 mg/L 2,4-D+1 mg/L BAP (6.852 g/50 mL suspension), respectively. HPLC analysis showed that the cell cultures derived from inflorescence callus in MS+3 mg/L NAA+1 mg/L BAP showed the highest of rutin (47.536 mg/g FW). However, the highest amount of quercetin (8.570 mg/g FW) and kaempferol (5.420 mg/g FW) were obtained from the petiole cell cultures in the MS+1 mg/L 2,4-D+1 mg/L Kin.
Research Article
Extraction, Identification And Determination of Antiviral And Anticancer Flavonoids By HPLC-DAD In Cell Suspension Culture of Melia Azedarach L.
https://doi.org/10.21203/rs.3.rs-636880/v1
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Melia azedarach L. is a species of deciduous tree belonging to the Meliaceae, also known as Chinaberry tree. M. azedarach L. is considered in the biological programs and medicinal applications. M. azedarach L. contains a wide range of secondary metabolites such as triterpenoids, limonoids, flavonoids, steroids, acids, anthraquinones, alkaloids, saponins, tannins (coria et al., 2008). So that, different parts of M. azedarach L. such as leaf, fruit, bark, seed and roots are used as anti-fungal, anti-malarial, antibacterial, antiviral, hepatoprotective, anti-hyperglycemic, anti-oxidant, anti-fertility, anti-inflammatory, anthelmintic, antipyretic, phytotoxic, anticancer and cytotoxic in pharmaceutical industries (Ervina, 2018) and also has pesticidal activities (Mishra et al. 2013; coria et al., 2008).
Plants produce a great variety of phytochemicals with different biological properties such as antioxidant activity. The antioxidants may inhibit the occurrence of cardiovascular disease, cancer and neurological diseases. Plant antioxidants act as oxygen quenchers, free radical scavengers, peroxide decomposers and enzyme inhibitors (Xiao et al. 2014). Plant growth regulators (PGRs) are one the main important factors in regulating plant growth and developmental processes as well as in modulating plant responses to various biotic and abiotic stresses. Furthermore, oxidative stress may be increased in the cells growing in medium containing high concentrations of PGRs (Sharma et al. 2007), and there is a low study for investigation of how to contribute to the oxidative stress by different PGRs. The sensitivity of cells to oxidative stress is reduced by antioxidant defense mechanisms, including the enzymatic antioxidant systems such as CAT, SOD, GPX and PPO, and non-enzymatic systems such as GSH, α-tocopherol, phenolic compounds (PhOH), flavonoids, alkaloids, and nonprotein amino acids. For conservation of plant products need to the controlling the activity of two enzymes: peroxidase and polyphenol-oxidase (PPO). Peroxidases act as an electron acceptor while polyphenol-oxidase acts as a donor. H2O2 is a reactive oxygen species that normal condition endogenously is produced by the cells and act as a second messenger in cellular signaling pathways in living systems; but the production and accumulation of this molecule in the cells significantly increase under stress conditions, as well as other reactive oxygen species which results in oxidative stress. These oxidative agents attack to proteins, lipids and nucleic acids and interfere with vital processes of the plant cells (Lacey and Walker, 2014). MDA is the end product of lipoperoxidation and is considered to be a reliable indicator of oxidative stress (Bao et al. 2017). So that, the antioxidant defense system maintains play a key role in maintaining cell homeostasis (Oruc-Ozcan and Uner, 2002). Flavonoids are as a main biologically active compound in plants that play significant roles in human health due to their presence in the diet. Flavonoids may promote desirable and non-desirable physiological effects in humans. Earlier studies have been recommended the flavonoids such as rutin, quercetin, and kaempferol as pharmacological compounds with important biological activities such as anti-inflammatory, antioxidant, antithrombotic, hepatoprotective, antiviral, anticancer and antitumor (Havsteen, 2002; Patel and Patel, 2019). Furthermore these secondary metabolites have positive effects on capillary fragility and vascular protection (Trugo et al. 2003). Quercetin inhibits the oxidation and cytotoxicity, and can reduce risk for coronary heart disease or cancer. Also, flavonoids act as free-radical neutralizers and caused to antioxidative characteristics. An in vitro oxidation model showed that quercetin and rutin are being more powerful antioxidants than the traditional vitamins. The quercetin glycosides are predominate compared to kaempferol in vegetables and fruits (Satheeshkumar et al. 2014).
The cell suspension culture, as a biotechnological approach represents an efficient system for producing, isolating or handling of secondary metabolites because of their fast growth rate and easy (Kaviani, 2014). Rutin, quercetin and kaempferol are the most important phenolic compounds of M. azedarach L. (Ahmed et al. 2013). Till now the reports are limited for estimation of these flavonoids in the plant extract of M. azedarach L. Also, no reports are available on the cell suspension culture, antioxidant properties, and secondary metabolites production in cell suspension cultures of M. azedarach L. For estimates total phenolic, flavonoid and anthocyanin production in plants, and increase their potential use as nutraceuticals in medicinal and agricultural applications or functional foods, the production and accumulation of these metabolites in cell suspension cultures will be very useful. So, the aims of the current study were the investigation of the effects of different concentration and combination of PGRs on the callus induction, cell suspension culture establishment and evaluation antioxidant properties of the cell cultures of M. azedarach L. Furthermore, the production and accumulation of antiviral and anticancer secondary metabolites in M. azedarach L. cells were measured using HPLC.
2.1. Plant material and callus culture
The young twigs of Chinaberry (M. azedarach L.) were collected from naturally growing trees the Qazvin province (36°19'15.1"N 50°00'42.8"E and 1287m HSL), Iran. The young leaves, inflorescences and petioles of M. azedarach L. were cleaned with commercial detergent, washed under running tap water for 30 min and then rinsed with sterile distilled water (SDW). Explants were surface sterilized with 70% ethanol for 20 s and washed with SDW followed by 0.5% NaOCl for 20 min. Finally, explants were rinsed with SDW for three times and were cultured on MS medium supplemented with 1, 3 and 5 mg/L NAA or 2,4-D along with 1, 3 and 5 mg/L Kin or BAP and 3% sucrose, that were solidified with 0.8% plant agar. The cultures were maintained in a growth chamber under 24 ± 1°C and 16 h photoperiod. The percentage of callus induction was recorded one month after culture, and callus yield (mg/single explants) were measured in the three month after culture.
2.2. Cell suspension cultures and growth measurement
Cell suspension cultures were established from one-month-old callus cultures. One single callus was transferred to flasks (250 mL) containing 20 mL liquid MS medium supplemented with abovementioned PGRs. The cultures were maintained on a rotatory shaker at 110 rpm and 24 ± 1 ºC, with 16 h photoperiod. The cells were subcultured every 22 days with an initial cell density of 100×104 cells/mL and 50 mL/suspension liquid MS. Finally, the fresh cells were filtered and were lyophilized for next measuring of biochemical and secondary metabolite analysis.
2.3. Evaluation of the cell growth and growth efficiency
The cells of medium supplemented with 1 mg/L 2,4-D + 1 mg/L Kin/BAP from leaf and inflorescence, were collected at 2-day intervals during a 24-day period; and packed cell volume (PCV), settled cell volume (SCV) and cell density (Godoy-Hernández and Vázquez-Flota,, 2012) of cultures were determined. Each point of the growth curve is represented by the mean of six independent measurements. Cell growth efficiency was evaluated using growth index (GI), specific growth rate (SGR) and doubling time (Dt) indice according to Bhatia et al. (2015):
where, Fm and Im are the final and initial cell density, respectively.
2.4. Determination of H2O2 content and lipid peroxidation level (MDA)
The cells (100 mg FW of the cell suspension) were pulverized in liquid nitrogen and were homogenized in ice bath with 1 mL 0.1% (w:v) TCA. The samples were centrifuged at 12000 rpm for 20 min and supernatant transferred to a clean vial and used for H2O2 and MDA measurement.
The content of Hydrogen peroxide was measured according to Zaragoza-Martinez et al. (2016). Briefly, 0.5 mL of the supernatant was added to 0.5 mL 10 mM potassium phosphate buffer (pH 7.0) and 1 mL of 1 M KI. The absorbance of the mixture was measured at 390 nm using spectrophotometer (SmartSpec Plus spectrophotometer, Bio-Rad, Hercules, CA, USA). The content of H2O2 was calculated using a standard curve and expressed as µmol/g FW.
For the determination of lipid peroxidation in cell cultures, the thiobarbituric acid (TBA) was used for determines MDA as an end product of lipid peroxidation (Zaragoza-Martinez et al. 2016). Briefly, 1 mL 0.5% (w:v) TBA in 20% TCA were added to 0.5 mL of the supernatant. The mixture was boiled in a bath water for 30 min and incubated on ice in order to stop reaction. Then, the samples were centrifuged at 12000 rpm for 5 min, and the absorbance of supernatant was read at 532 and 600 nm (with triplicate extracts and reads). The amount of MDA-TBA complex (red pigment) was measured using the extinction coefficient (155/mM cm) and expressed as µmol/g FW.
2.5. Preparation of Protein extract and enzymes activity assay
100 mg FW of the cells were pulverized in liquid nitrogen by a mortar and pestle and homogenized in 100 mM phosphate buffer (pH = 7.8). The homogenate was centrifuged at 12000 rpm for 20 min at 4oC and the supernatant was used for antioxidant enzymes activity assay.
Protein concentration of the samples were determined spectrophotometrically using Bradford (1976) method, and quantified using bovine serum albumin (BCA) standard curve and expressed as mg/g FW.
The activity of catalase (CAT) enzyme was measured as decomposition of H2O2 and decrease in the absorbance at 240 nm according to Aebi (1984). The reaction mixture contained 50 µL of protein extract, 500 µL of 10 mM H2O2 and 2450 µL of 100 mM potassium phosphate buffer (pH = 7.0). Then, the absorbance of the reaction was measured for 2 min at 15 s intervals. CAT activity was defined as the unit/mg protein.
The guaiacol peroxidase (GPX) enzyme activity was measured using the H2O2-dependent oxidation of guaiacol with the increase in the absorbance at 470 nm, according to Putter (1974). The reaction mixture contained 100 mM potassium phosphate buffer (pH = 7.0), 20 mM guaiacol, and 10 mM H2O2. The reaction was initiated by addition 0.1 mL of protein extract. POX activity was defined as the unit/mg protein.
Polyphenol oxidase (PPO) activity was measured using Kar and Mishra (1976) method. Briefly, 100 µL protein extract were added to 1.5 mL of 0.2 M Tris base and 0.4 µL of 0.02 M pirogalol, and incubated at 25°C for 5 min. Then the absorbance of the reaction mixture at 420 nm was recorded. PPO activity expressed as the unit/mg protein.
2.6. Proline determination with colorimetric assay
Proline content of the cell suspensions was measured using a ninhydrin-based colorimetric assay according to Bates (1973). The proline concentration was quantified using a standard curve of L-proline; and was expressed as µg/g FW. Briefly, 5 mL of 3.3% sulfosalicylic acid (2-hydroxy-5-sulfobenzoic acid) was added to 100 mg of ground FW and was centrifuged at 4000 rpm, 4ºC for 10 min. Then, 1 mL extract was added to 1 mL regent of acidic ninhydrin (containing 1,2,3-indantrione monohydrate, glacial acetic acid and 6M ortho-phosphoric acid) and 1 mL glacial acetic acid. The mixture was incubated at 100ºC for 60 min. Then, was added 2 mL toluene to the reaction mixture, vortex the samples for 2 min, and was incubated at room temperature for 30 min. Finally, the absorbance was measured at 520 nm.
2.7. Extraction and determination of secondary metabolites
2.7.1. Spectrophotometric assay
100 mg FW of cell suspension was powdered in liquid nitrogen using a mortar and pestle, and homogenized in acidified methanol (99:1) (Wagner, 1979).
The total phenolic content (TPC) was measured using a modified colorimetric Folin-Ciocalteu method described by Al-Farsi et al. (2005), and quantified using standard curve of gallic acid. The total flavonoid content (TFC) of the cell suspensions was determined using a modified aluminum chloride (AlCl3) colorimetric method reported by Anjum et al. (2017). The standard curve of quercetin was used for TFC quantification, and TFC was expressed as mg of quercetin equivalents/g FW. For anthocyanin content measurement, the absorbance of the cells methanolic extract was recorded at 550 nm and quantified using A=ɛbc (Sopari, 2015). Where, A is the absorbance of the extracts, b is the width of cuvette (cm), and c is the concentration of anthocyanin (M), and ɛ is molar extinction of anthocyanin at 550 nm (ɛ=33000×106 /M cm). The anthocyanin content was reported as µM/g FW.
The measurement of the absorbance of the samples was done using Nanodrop (NanodropOne_C- Nanodrop scientific- AZY1706749, USA).
2.7.2. HPLC analysis
The cell suspension cultures were extracted for flavonoid compounds with 90% (v/v) ethanol based on Zu et al. (2006) method. Briefly, 0.5 g of FW was ground in liquid nitrogen and 1.5 ml ethanol were added and vortexed for 5–10 min. Then, the samples were sonicated in a bath sonicator (Bandelin electronic®, Germany) for 15 min at 35°C and incubated at room temperature for 3 h, the vortex and sonication were repeated three times. Finally, the samples were centrifuged (Sigma 1-14K, Germany) at 12000 rpm for 20 min. The supernatant was collected and filtered using a 0.22 µm filter. The filtrate was dried at 45°C, till final volume was up to 100 µL and stored at -20°C until analysis. HPLC analysis was performed on a Sykam HPLC system (SYKAM 1130, Sykam GmbH, Germany). Chromatographic analysis was carried out on a HIQ SIL C18V reversed-phase column (ø 4.6 mm×250 mm) packed with 5 µm diameter particles, the mobile phase was methanol–acetonitrile–water (40:15:45, v/v/v). RU, QE and KA were detected by DAD following HPLC (SYKAM 1130, Sykam GmbH, Germany) separation at 257 nm for RU, 368 nm for QE, KA. Flow rate and injection volume were 1.0 mL/min and 20 µL, respectively. All chromatographic operations were carried out at room temperature. The Calibration curves of flavonoids compounds standards including rutin (Sigma-Aldrich, USA) quercetin (Sigma-Aldrich, Q4951, CAS: 117-39-5) and kaempferol (Sigma-Aldrich, 60010, CAS: 520-18-3) were used for identification and quantification of these metabolites in the M. azedarach L. cell cultures extract. Calibration curves had great linearity to the concentration of corresponding compounds and were as y = 14.532x-0.785 for rutin (R2RU = 0.9999), y = 74.199x-118.98 for quercetin (R2QE = 1.0000), and y = 83.196x-74.023 (R2KA = 1.0000) for kaempferol.
2.8. Statistical analysis
All experiments conducted based on completely randomized design (CRD) in triplicate. Experimental data subjected to analysis of variance (ANOVA) (p < 0.05) and mean comparison using Duncan’s Multiple Range Test (DMRT) (at p < 0.05). Statistical analysis was performed using IBM SPSS Ver.21 statistical software (IBM Corporation and Others, Armonk, NY, USA). The results expressed as mean ± Standard error (SE) of triplicate experiments. The graphs produced using Microsoft Office Excel 2010.
3.1. Callus induction and biomass
In the present study the callus were successfully induced from the leaf, inflorescence and petiole explants on MS medium containing different concentration and combination of auxin and cytokinins (Table 1 and Fig. 1). However, as shown in the Table 1, the percent of callus induction, callus growth (biomass), color and structure of the callus were significantly different depending on the explant type and PGRs combination in the medium. The percentage of callus induction from the leaf, inflorescence and petiole explants were varied from 0.0 to 87.15, 63.89 to 100 and 46.43 to 100, respectively, depending on the PGRs combination in the medium. But the lowest percentage of callus induction from inflorescence explants was related to 1 mg/L NAA + 1 mg/L BAP (63.89%), which significantly lower than the other combinations of PGRs and had 1.55-fold lower as compared to the MS medium without PGRs (MS free) (Table 1). However there were no significant differences between the MS medium containing other concentration and combinations of PGRs and MS medium without PGRs in terms of the percentage of callus induction from inflorescence explants. The percentage of callus induction from petiole explants in all auxin and cytokinin combinations were statistically same, and significantly higher than that of the medium without PGRs (table 1). So, the callus induction from this explant in medium supplemented with 1 mg/L NAA + 1 mg/L Kin and 1 mg/L 2,4-D + 1 mg/L Kin (100%), was about 2.15-fold higher than that of the medium without PGRs (46.43%). On the other hands, the variation in the percentage of callus induction from leaf explant was higher as compared to the inflorescence and petiole explants. In this explant, no callus induction was occurred on the medium supplemented with 5 mg/L NAA + 5 mg/L Kin and 5 mg/L 2,4-D + 1 mg/L Kin. However, in the medium supplemented with 1, 3 or 5 mg/L NAA + 1 mg/L Kin, 5 mg/L NAA + 1 mg/L BAP and 3 mg/L 2,4-D + 1 mg/L Kin more than 77% of the leaf explants exhibited callus induction and production (Table 1).
The highest growth of callus (fresh weight) belonged to the leaf explants cultured on the MS medium supplemented with 5 mg/L NAA + 5 mg/L BAP (6.307 g/explant) and 5 mg/L NAA + 3 mg/L Kin (5.152 g/explant) and 5 mg/L 2,4-D + 5 mg/L Kin (3.977 g/explant), which was higher than those of other PGRs combinations and medium without PGRs. In this regards, in all PGR treatments, the growth of callus from the leaf explants was significantly higher than medium without PGRs (Table 1). Increasing the concentration of 2,4-D had no significant effect on callus growth in the leaf explants, but increasing the concentration of NAA from 1 mg/L to 5 mg/L significantly increased the callus growth from the M. azedarach L. leaf explants. Increasing the concentration of cytokinins was also significantly affected the callus growth, and as show in Fig. 2 increasing the concentration of Kin and BAP (from 1 to 3 mg/L) caused 1.5–2.5 fold 1.5–3.5 fold of increase in the callus growth, respectively. In the petiole and inflorescence explants was observed different significant between explants supplemented with 2,4-D and control based on the growth of callus, which the petiole explants supplemented with Kin 1.6-fold higher than BAP and the inflorescence explants supplemented with BAP 2.1-fold higher than Kin had the growth of callus (Table 1 and Fig. 2).
The texture and color of callus were varied depending on the explant type and PGRs combination of in the medium (Fig. 1 and Table 1). The leaf explants cultured on the MS medium containing 1 mg/L NAA, regardless of the cytokinin type, produced green and compact callus,, but on the medium supplemented with the high concentrations of NAA (3 and 5 mg/L) in combination with Kin produced the milky and friable callus. The leaf explants cultured on the medium containing different concentration of 2,4-D (1, 3 and 5 mg/L) in combination with Kin or BAP produced yellow-milky and friable callus (Fig. 1a, b, c and d). The inflorescence explants cultured on the MS medium supplemented with NAA had a milky compact callus; but, the calli produced on the MS medium along with 2,4-D (1 and 3 mg/L) were milky and yellow friable callus.
3.2. Cell suspension culture
The calli derived from leaf, inflorescence and petiole explants were transferred into the liquid MS medium supplemented with the same concentration and combination of PGRs used in the callus induction. Data analysis of suspension culture showed that there were considerable differences between different PGR combinations in terms of the cell growth and fresh and dry weights of the cells (Table 1). The highest fresh weight of the cells from suspension cultures were obtained from the leaf and inflorescence explant- derived callus transferred into the liquid MS medium supplemented with 1 mg/L 2,4-D + 1 mg/L Kin (8.489 g/50 mL suspension) and 1 mg/L 2,4-D + 1 mg/L BAP (6.852 g/50 mL suspension), respectively (Table 1). These suspension cultures were yellow and milky in color (Fig. 1 e, f, g and h). Furthermore, the highest dry weight of the cells were obtained with inflorescence explant-derived callus in the liquid medium containing 1 mg/L 2,4-D + 1 mg/L BAP and leaf explant derived- callus in the liquid medium supplemented with 1 mg/L 2,4-D + 1 mg/L Kin and 3 mg/L Kin + 1 mg/L Kin (Table 1). The cell suspension cultured on the 1 mg/L NAA + 1 mg/L BAP, 3 and 5 NAA + 1 and 3 mg/L mg/L Kin medium were include the green, yellow or brown granular small cells, which were embryogenesis cells.
Increasing the 2,4-D and NAA level from 1 to 3 mg/L significantly reduced the growth of the suspension cultures of M. azedarach L. in terms of the fresh weight and dry weight of the cell. Generally, orthogonal analysis showed that low concentration of 2,4-D and BAP was showed the highest FW and DW of cell suspensions (Fig. 3). So, The MS medium supplemented with 1 mg/L 2,4-D and 1 mg/L Kin or BAP was chosen to evaluate the cell growth curve of suspension cultures stablished form the leaf and inflorescence callus.
3.3. Cell growth and growth efficiency in suspension cultures
The cell growth curve was monitored using the cell count, SCV and PCV in two different medium and two types of explants of M. azedarach L. As showed in Fig. 4, both cell cultures showed four distinct growth phases including lag, exponential (log), linear and stationary phases. The cell culture in medium containing 1 mg/L 2,4-D + 1 mg/L Kin had a remarkable increase in growth on the log phase from the day 6–21 and after the growth declined. The highest cell count was obtained on day 21 (892×104 cell/mL suspension). The growth curve derived from cell suspension of leaf explants compared to inflorescence explants had a better and more process from cell count, SCV and PCV (Fig. 4). Applying 1 mg/L 2,4-D + 1 mg/L BAP on cell derived from inflorescence explants were showed different results, so that, had an increase in growth from the day 4–12 on the cell growth by cell count (×104) and day 0–12 on the cell growth based on SCV and PCV (%) and after the growth decreased and then started stationary phase (Fig. 4). Based on the present study, 2,4-D (1 mg/L) combination with kin resulted in better cell suspension growth responses compared to its combination with BAP. The growth efficiency of the cell cultures were evaluated using growth index (GI), specific growth rate (GSR) and doubling time (Dt) parameters. The leaf cell cultures in liquid medium containing 1 mg/L 2,4-D + 1 mg/L Kin exhibited growth efficiency higher than that of the inflorescence cell cultures in the medium containing 1 mg/L 2,4-D + 1 mg/L BAP (Table 2).
3.4. Antioxidant enzymes activity
There were significant differences between PGRs treatments in terms of CAT, GPX and PPO activity, protein, proline, H2O2 and MDA content in M. azedarach L. cell cultures. The highest CAT (3721.04 U/mg pro.), GPX (1720710.23 U/mg pro.) and PPO (184314.93 U/mg pro.) activity was related to the leaf cell suspensions cultures in 3 mg/L NAA + 3 mg/L BAP treatment. The protein content of inflorescence cell suspension cultures in 3 mg/L NAA + 3 mg/L Kin and 3 mg/L 2,4-D + 3 mg/L BAP and leaf cell suspension cultures in 3 mg/L 2,4-D + 1 mg/L BAP was higher than that of other PGRs treatments. Also, the inflorescence cell suspension cultures in the medium supplemented with 3 mg/L NAA + 1 mg/L Kin had the highest proline content, which was not significantly different from inflorescence and leaf cell suspension cultures in 3 mg/L NAA + 3 mg/L Kin and 1 mg/L NAA + 1 mg/L BAP, respectively. The highest H2O2 content were observed in the leaf and inflorescence cell suspension cultures in the medium containing 3 mg/L NAA and 3 mg/L 2,4-D, respectively. However, the highest MDA content was belonged to the inflorescence cell suspension cultures in the medium supplemented with 3 mg/LNAA regardless the types and concentration of cytokinin (Table 3).
3.5. Secondary metabolites
Quantitative analysis showed that the highest amount of TFC and TPC was observed in the inflorescence (2.93 mg QE/g FW and 1.455 mg GAE/g FW) and leaf (2.90 mg QE/g FW and 1.570 mg GAE/g FW) cell suspension cultures in 3 mg/L NAA + 1 mg/L BAP and 3 mg/L NAA + 1 mg/L Kin treatments, respectively, followed by the inflorescence cell suspension culture in 3 mg/L NAA + 1 mg/L Kin and 1 mg/L 2,4-D + 1 mg/L Kin (TFC) and the leaf cell suspension cultures in 1 mg/L NAA + 1 mg/L BAP treatments (TFC and TPC) (Table 3). Generally, the amount of TFC and TPC in treatments NAA were slightly higher than those in treatments containing 2,4-D. The highest amount of AC belonged to the inflorescence cell suspension culture in 3 mg/L 2,4-D + 3 mg/L BAP. The highest amount of AC in inflorescence cell suspension of cultures was significantly higher than that of leaf cell suspension of cultures. Furthermore, there was no difference between the types of auxins in the inflorescence cell suspension cultures in term of AC content, where in leaf cell suspension cultures, treatments containing NAA produced the higher AC in comparison with treatments containing 2,4-D (Table 3).
By increasing the concentration of 2,4-D from 1 to 3 mg/L was not observed TFC significantly reduced in leaf explants, but in the inflorescence explants likely leaf explants treated by NAA, TFC significantly decreased (1.3-fold). In the total of leaf, inflorescence and petiole explants, decreasing of TFC from concentration of 3 to 5 mg/L NAA is the highest. Also, leaf and inflorescence explants treated by BAP (1.09 and 1.46-fold) and Kin (1.22 and 1.30-fold) induced low by increasing concentration from 1 to 3 mg/L, that this reduction was lowest in leaf explants, respectively. But, in the total of leaf, inflorescence and petiole explants, decreasing of TFC was no different in treated by Kin or BAP. Like that TFC, there was a decline in TPC of cell suspension of leaf explants in M. azedarach L. with an enhancement of NAA concentration from 1 to 3 with a it had lower slope than 3 to 5 mg/L (3 and 5-fold, respectively). This decreasing in overall explants, leaf, inflorescence and petiole explants was obtained up to 5.76-fold by increasing 3 to 5 mg/L of NAA. The cell suspension of inflorescence explants supplemented with 2,4-D (increasing concentration from 1 to 3 mg/L) 1.65-fold significant decreasing were indicated. By applying 2,4-D in the cell suspension of leaf explants showed increasing of AC about 2 fold simultaneously by increasing concentration of 2,4-D (from 1 to 3 mg/L). This content by increasing concentration of NAA from 1 to 3 mg/L and 3 to 5 mg/L had about 1.5 and 2-fold inhibited, respectively. The variation of AC in the increasing trend of cytokinin concentrations for cell suspension of leaf explants of M. azedarach L. is higher than inflorescence explants. So that, in the leaf explants had an increasing trend for Kin (2.52-fold) and a declining trend for BAP (2.31-fold). There is no significant difference the enhancing trend between type of cytokinins in the inflorescence explants, and these changes in AC of the inflorescence explants treated with Kin and BAP ranged from 1.13-fold and 1.40-fold, respectively.
Production and accumulation of rutin, quercetin and kaempferol in the cell suspension of M. azedarach L. were measured by HPLC. The chromatogram of rutin, quercetin and kaempferol standards and samples prepared from leaf, inflorescence and petiole cell suspension cultures were presented in Fig. 5. The results indicated that the amount of these metabolites were significantly different depending of the PGRs combination and explant type (Fig. 6). The inflorescence cell suspension cultures in the medium containing 3 mg/L NAA + 1 mg/L BAP produced the highest amount of rutin (47.536 mg/g FW) which was 1.84-fold of the high-rutin production in the leaf cell suspension cultures (25.845 mg/g FW) (3 mg/L NAA + 3 mg/L Kin) and 3.93-fold of the high-rutin production in the petiole cell suspension culture (12.092 mg/g FW) (1 mg/L 2,4-D + 1 mg/L Kin). The highest amount of quercetin (8.570 mg/g FW) and kaempferol (5.420 mg/g FW) were produced in the petiole cell suspension cultures in the medium supplemented with 1 mg/L 2,4-D + 1 mg/L Kin (Fig. 6).
On average of different explants an cytokinins, increasing the concentration of 2,4-D from 1 to 3 mg/L significantly reduced the quercetin and kaempferol content in cell cultures (Fig. 7). In contrast, increasing the concentration of NAA from 1 to 3 mg/L significantly enhanced the production and accumulation of rutin, quercetin and kaempferol in the leaf cell suspension cultures (Fig. 7). Increasing the concentration of NAA from 3 to 5 mg/L significantly repressed the production and accumulation of rutin and quercetin in the leaf cell suspension cultures, but had no significant effect on kaempferol production and accumulation.
3.7. Correlation analysis
Correlation analysis showed significant coefficients between biochemical and phytochemical characteristics of the cell cultures. The amount of protein had a positive significant correlation with H2O2, MDA, proline and AC content in the cell cultures, where significantly reduced GPX and PPO activity. Also, increasing CAT activity was affected significantly negative on the GPX and PPO activity. There was a significant a positive correlation between GPX with PPO and H2O2, TFC and TPC, H2O2 with MDA, and MDA with the proline content. on the other hand, the proline content had a direct relationship with TFC and TPC, and these characteristic significantly correlated with amount of rutin in the M. azedarach L. cell cultures. A positive and significant correlation was detected between the amount of quercetin with kaempferol (95.3%) and TFC with TPC (92.7%). But, the correlation of rutin with quercetin and kaempferol was not statistically significant (Table 4). There was significant and positive correlation between FW with DW (93.6%), but they had no correlation with enzyme activity and secondary metabolites (data not shown).
Callus induction and production provides an important approach for study of developmental processes and metabolic pathways in the plant cells, and is a practicable way to plant regeneration, cell suspension culture and production of secondary metabolites (Ahmadpour et al. 2016; Firoozi et al. 2019). Different factors including plant growth regulators especially auxins and cytokinins, plant genotype and explant type influence the callus induction, cell growth and development, and secondary metabolites production in the plant cells (Zare et al., 2009; Firoozi et al, 2019; Farjaminezhad and Garoosi, 2019) In the present study, we investigated the effect of different PGRs combinations including auxins (NAA and 2,4-D) and cytokinins (Kin and BAP) on callus induction and growth, and cell suspension culture establishment, growth and secondary metabolites production from leaf, inflorescence and petiole explants of M. azedarach L. The percentage of callus induction, callus growth (biomass), color and texture of the calli were different depending on the M. azedarach L. explant type and applied PGRs combinations. Furthermore, in a given plant species different types of explants show diverse in vitro response, especially depending on PGRs combination. So, selection of the most responsive explant play a vital role in callus induction and growth, somatic embryogenesis and plant regeneration from in vitro cultures (Zare et al., 2009; Mostafa et al, 2020). Here, in leaf explants of M. azedarach L. the percentage of callus induction and callus fresh weight were varied from 0.0 to 87.15% and 0.0 to 6.307 g/explant, respectively; where the callus induction and fresh weight in inflorescence explants were 63.89–100% and 0.115–1.01 g/explant, respectively. In the leaf explant, the callus induction in 1 mg/L NAA + 1 mg/L Kin (86.11%), 3 mg/L NAA + 1 mg/L Kin (87.15%), 3 mg/L 2,4-D + 1 mg/L Kin (80.95%), 5 mg/L NAA + 1 mg/L Kin (77.09%) and 5 mg/L NAA + 1 mg/L BAP (83.33%) treatments were higher than that of the other PGRs treatments. These results are accordance with those reported by Bernabe-Antonio et al. (2017), in which the highest percentage of callus induction (100 %) from leaf explants of Eysenhardtia polystachya (Ortega) was reported on MS medium supplemented with 8.28 µM NAA + 0.41 µM kinetin. Farjaminezhad and Garoosi (2019) reported that the highest percentage of callus induction from leaf explants of Azadirachta indica was obtained in the MS medium supplemented with 2 mg/L 2,4-D + 1 mg/L kinetin, 2 mg/L 2,4-D + 3 mg/L kinetin, 2 mg/L 2,4-D + 3 mg/L BAP, 0.5 mg/L NAA + 2 mg/L BAP, 1 mg/L NAA with 3 mg/L BAP, 1 mg/L picloram with 2 mg/L kinetin, and 1 mg/L picloram with 3 mg/L kinetin. On the other hands, Kin was more than effective in callus induction from the leaf explants of M. azedarach L. However, in inflorescence explant, the callus induction in all assayed PGR combinations, except 1 mg/L NAA + 1 mg/L BAP (63.89%), was higher than 87.83%; but callus growth (fresh weight) in this explant was lower than that of the leaf explant. It has been proven that the optimal PGRs combination for callus induction and growth is varied depending on the plant species, genotype and explant type, and should be experimentally optimized (Zare et al., 2009; Ahmadpour et al. 2016; Mostafa et al. 2020). In our study, the calli produced from M. azedarach L. leaf explants exhibited a great growth and biomass, and produced higher than 5 g fresh weight per explant on MS medium supplemented with 5 mg/L NAA + 3 mg/L Kin and 5 mg/L NAA + 5 mg/L BAP. While, in Azadirachta indica the highest fresh weight of calli produced from the leaf explants was 0.26 g per explants, which was reported in MS medium containing 1.5 mg/L NAA + 3 mg/L Kin (Farjaminezhad and Garoosi, 2019).
Kaviani (2014) reported that the embryonic axes and cotyledon explants of M. azedarach L. did not showed any callus induction and growth in the medium without 2,4-D. In contrast, in our study, 59.04%, 98.81% and 46.43% of leaf, inflorescence and petiole explants cultured on the MS medium without any PGRs exhibited callus induction, indicating the important role of explant type in the callus induction and growth in M. azedarach L. in vitro cultures. It has been shown that the concentration of endogenous hormones in the various parts of the plants is different. This endogenous factor in interaction with exogenous applied PGRs can be influence the plant cells division, growth and development as well as callus induction (Mostafa et al. 2020). The higher percentage of callus induction from the inflorescence explants may be contributed to the higher level of the endogenous phyto-hormones in this explant as compared to the leaf explant. Because, 98.81% of inflorescence explant produced callus on the MS medium without PGRs, while only 59.04% of leaf explants showed callus induction on this medium. Mostafa et al. (2020) showed that the level of the endogenous auxins including indole-3-acetic acid and methyl indole-3-acetic acetate, and cytokinins including trans-zeatin and dihydrozeatin in the high callus-producing explants (tip explants) of garlic were significantly greater than those in the low-callus producing explants (upper leaf parts). In addition, Centeno et al. (1996) reported that callus induction and proliferation from different sections of petiole explants of Actinidia deliciosa is correlated with their levels of endogenous IAA and zeatin.
In the present study, callus and cell suspension cultures of M. azedarach L. were achieved for the first time from different explants and PGRs combinations that could be used in biotechnological and molecular studies, and secondary metabolites investigations and production. Oure findings confirm the previous studies showing that PGRs combination of the medium and explant type and source play a crucial role in plant cell suspension culture establishment and growth (Farjaminezhad et al. 2013; Bernabe-Antonio et al. 2017; Farjaminezhad and Garoosi, 2019), so the growth of the M. azedarach L. cell cultures were varied depending on the explant type and PGRs. Although the callus induction from petiole explants was high compared to leaf explants, growth rate of the callus and cell suspension cultures derived from the petiole explants were very low as compared to the cell cultures derived from leaf callus. From our findings, the friable callus from leaf explant in liquid MS medium containing 1 mg/L 2,4-D + 1 mg/L Kin establish an efficient cell suspension culture with the highest growth rate. In a study in Azadirachta indica cell culture, Farjaminezhad and Garoosi (2019) reported the highest cell proliferation and density in the MS medium supplemented with 1.5 mg/L 2,4-D and 3 mg/L zeatin riboside. Furthermore, in the present study, increasing 2,4-D level from 1 to 3 mg/L in cell suspension cultures derived from leaf M. azedarach L. caused a significant reduction in the cell growth (FW and DW) of the suspension cultures, where increasing of NAA level from 1 to 3 mg/L slightly increased the suspension cells growth. Contrary to the present results, in Azadirachta indica cell cultures, increased concentrations of NAA (from 1 to 3 mg/L) significantly decrease the growth (FW) of the suspension cultures (Farjaminezhad and Garoosi, 2019). However, in accordance with the results reported in Azadirachta indica (Farjaminezhad and Garoosi 2019), increasing the Kin level increases the growth (FW) of cell suspension, but increasing the BAP decrease the growth (FW) of M. azedarach L. cell suspension cultures.
The PGR treatments of leaf explants with the high concentration of 2,4-D (3 mg/L) caused to significantly increase in the proline and decrease in the protein (1.24-fold), H2O2 (1.39-fold) and MDA (2.22-fold) content; and Kin decreased proline and protein content, but decreasing H2O2 and MDA content had no significant for type of cytokinin. While the protein concentration of inflorescence explants was significantly increased with the high concentration of 2,4-D (1.17-fold), and significantly decreased with the high concentration of Kin. Also, an increasing 2,4-D concentration from 1 to 3 mg/L results to significantly enhance of endogenous production proline and H2O2 (1.37-fold) and MDA (1.78-fold) in cell suspension of inflorescence explants of M. azedarach L. While in cell suspension of leaf explants, proline and protein content were significantly decreased by increase NAA concentration (1 to 3 mg/L, 2.44-fold and 3 to 5 mg/L, 1.46-fold) and this decreasing for proline was effective by BAP compared to Kin; but for leaf explants protein had no different significant between Kin and BAP. H2O2 content was not more difference, when NAA increased from 1 to 3 mg/L, but there was a significant increment by BAP. On the other hand, while 5 mg/L NAA treatment caused a significant reduction H2O2 content of inflorescence explants compared to 3 mg/L NAA (1.22-fold), Kin caused a significant decrease H2O2 content leaf and inflorescence explants. The moderate concentration of NAA decreased leaf explants MDA, while the high concentration of NAA (5 mg/L) showed significantly enhance; Kin and BAP concentrations had no different significant in production of MDA content in cell suspension of M. azedarach L.
Pearson’s correlation analysis showed that an increase in production of H2O2 has been associated with a significantly enhancement in accumulation of protein and MDA content in M. azedarach L. This is in agreement with the results reported by Bao et al. (2017), who observed a markedly increase in the MDA level in cells treated with H2O2 compared with the control. In addition to protective effects of antioxidant enzymes against oxidative stress, previous studies have also showed that the oxidative stress plays an important role for the production of secondary metabolites in plant culture systems (Shohael et al. 2006). Moyer et al. (2002) and Sgherri et al. (2004) proved that the production and accumulation of secondary metabolites including phenolic compounds is associated with oxidative stress. For manipulation of production and accumulation of secondary metabolites in vitro culture, PGRs were known as one of the main factors (Firoozi et al. 2019) that can activate the synthesis of phytochemicals components such as phenolic compounds and flavonoids (Sharma et al. 2012).
Here we optimized for a first time an efficient protocol for callus induction and cells suspension culture in M. azedarach L. both callus induction and growth, and cell culture establishment and growth were influenced by explant types and PGRs combinations. Although callus induction from inflorescence explants was high, this explant showed a low potential for callus growth rate and cell suspension culture establishment compared to leaf and petiole explants. Furthermore, the biochemical properties including antioxidant enzymes activity and proline, MDA and H2O2 content and metabolome characteristics of the M. azedarach L. cell cultures were different depending on the explant type, and type and concentration of PGRs. Therefore, it could elucidate that the production and accumulation of high-value secondary metabolites such as rutin, quercetin and kaempferol in the cell suspension of M. azedarach L. can be enhanced by handling the medium combination.
Funding
This work was supported by the University of Mohaghegh Ardabili Grant numbers [51-1796, 1395.08.03].
Conflict of interest
The authors declare that they have no conflict of interest.
Authors' contributions
Conceived and designed the experiments: FA and NZ. Performed the experiments: FA. Analyzed the data: FA and NZ. Contributed reagents/materials/analysis tools: NZ, RAZ and PSM. Wrote the paper: FA.
Acknowledgments
This work was supported by the University of Mohaghegh Ardabili Grant numbers [51-1796, 1395.08.03].
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Table 1. Some of qualitative and quantitative characteristics of calli and cell suspensions on different type and concentration of PGRs and explants
|
Explant |
PGRs (mg/L) |
|
Callus culture |
|
Cell suspension culture |
|||||
|
Auxin |
Cytokinin |
% of callus induction |
Texture of callus |
FW of callus (g/explant) |
Growth of suspension |
FW of cell (g/ 50 mL suspension) |
DW of cell (g/ 50 mL suspension) |
|||
|
Leaf |
MS free |
|
59.04±5.27 |
Friable |
0.225±0.031 |
|
NA |
NA |
NA |
|
|
1 NAA |
1 Kin |
|
86.11±13.89 |
Compact |
0.465±0.050 |
|
- |
0.069±0.002 |
0.008±0.001 |
|
|
1 NAA |
1 BAP |
|
68.22±13.57 |
Compact |
0.744±0.039 |
|
+ |
0.754±0.294 |
0.117±0.044 |
|
|
1 2,4-D |
1 Kin |
|
55.47±9.46 |
Friable |
1.373±0.141 |
|
+++ |
8.489±1.043 |
0.463±0.063 |
|
|
3 NAA |
1 Kin |
|
87.15±14.61 |
Friable |
1.842±0.073 |
|
++ |
2.042±0.506 |
0.177±0.026 |
|
|
3 NAA |
1 BAP |
|
60.85±12.64 |
Compact |
1.630±0.156 |
|
+ |
0.290±0.086 |
0.027±0.008 |
|
|
3 2,4-D |
1 Kin |
|
80.95±12.30 |
Friable |
0.368±0.012 |
|
+ |
0.202±0.003 |
0.019±0.001 |
|
|
3 2,4-D |
1 BAP |
|
69.50±10.58 |
Friable |
0.466±0.016 |
|
+ |
0.853±0.029 |
0.08±0.001 |
|
|
3 NAA |
3 Kin |
|
41.32±1.43 |
Friable |
2.894±0.139 |
|
++ |
5.885±2.257 |
0.427±0.137 |
|
|
3 NAA |
3 BAP |
|
53.49±13.15 |
Compact |
2.147±0.298 |
|
+ |
0.28±0.005 |
0.018±0.001 |
|
|
5 NAA |
1 Kin |
|
77.09±10.41 |
Friable |
0.901±0.063 |
|
NA |
NA |
NA |
|
|
5 NAA |
1 BAP |
|
83.33±0.02 |
Compact |
0.369±0.003 |
|
NA |
NA |
NA |
|
|
5 NAA |
3 Kin |
|
58.84±14.47 |
Friable |
5.152±0.276 |
|
++ |
2.170±0.153 |
0.176±0.012 |
|
|
5 NAA |
3 BAP |
|
31.04±2.29 |
Compact |
0.333±0.205 |
|
NA |
NA |
NA |
|
|
5 NAA |
5 Kin |
|
0.00±0.00 |
- |
- |
|
- |
- |
- |
|
|
5 NAA |
5 BAP |
|
50.00±8.00 |
Compact |
6.307±1.343 |
|
NA |
NA |
NA |
|
|
5 2,4-D |
1 Kin |
|
0.00±0.00 |
- |
- |
|
- |
- |
- |
|
|
5 2,4-D |
1 BAP |
|
50.42±9.47 |
Friable |
0.768±0.278 |
|
NA |
NA |
NA |
|
|
5 2,4-D |
3 Kin |
|
45.00±5.00 |
Friable |
0.296±0.108 |
|
NA |
NA |
NA |
|
|
5 2,4-D |
3 BAP |
|
18.26±3.97 |
Friable |
0.186±0.053 |
|
NA |
NA |
NA |
|
|
5 2,4-D |
5 Kin |
|
51.43±5.40 |
Friable |
3.977±1.211 |
|
NA |
NA |
NA |
|
|
5 2,4-D |
5 BAP |
|
14.29±1.00 |
Friable |
0.177±0.002 |
|
NA |
NA |
NA |
|
|
Inflorescence |
MS free |
|
98.81±1.19 |
Friable |
0.263±0.043 |
|
NA |
NA |
NA |
|
|
1 NAA |
1 Kin |
|
100.00±0.00 |
Compact |
0.358±0.001 |
|
NA |
NA |
NA |
|
|
1 NAA |
1 BAP |
|
63.89±20.97 |
Compact |
0.183±0.003 |
|
NA |
NA |
NA |
|
|
1 2,4-D |
1 Kin |
|
100.00±0.00 |
Friable |
0.664±0.030 |
|
++ |
4.977±0.956 |
0.248±0.022 |
|
|
1 2,4-D |
1 BAP |
|
99.55±1.64 |
Friable |
0.853±0.059 |
|
++ |
6.852±0.972 |
0.725±0.157 |
|
|
3 NAA |
1 Kin |
|
96.30±3.70 |
Compact |
0.461±0.076 |
|
+ |
1.098±0.012 |
0.012±0.002 |
|
|
3 NAA |
1 BAP |
|
100.00±0.00 |
Compact |
0.387±0.015 |
|
+ |
1.141±0.009 |
0.019±0.001 |
|
|
3 NAA |
3 Kin |
|
87.83±5.49 |
Compact |
0.167±0.069 |
|
+ |
1.027±0.016 |
0.112±0.006 |
|
|
3 NAA |
3 BAP |
|
98.34±1.47 |
Compact |
0.312±0.001 |
|
NA |
NA |
NA |
|
|
5 2,4-D |
1 Kin |
|
100.00±0.00 |
Friable |
0.115±0.005 |
|
NA |
NA |
NA |
|
|
5 2,4-D |
1 BAP |
|
100.00±0.00 |
Friable |
0.165±0.001 |
|
NA |
NA |
NA |
|
|
3 2,4-D |
3 Kin |
|
99.52±15.29 |
Friable |
0.510±0.021 |
|
+ |
0.166±0.001 |
0.003±0.001 |
|
|
3 2,4-D |
3 BAP |
|
94.19±3.68 |
Friable |
1.010±0.052 |
|
++ |
0.661±0.011 |
0.071±0.013 |
|
|
Petiole |
MS free |
|
46.43±2.92 |
Friable |
0.208±0.015 |
|
NA |
NA |
NA |
|
|
1 NAA |
1 Kin |
|
100.00±0.00 |
Compact |
0.209±0.046 |
|
+ |
0.292±0.008 |
0.036±0.001 |
|
|
1 NAA |
1 BAP |
|
92.50±7.50 |
Compact |
0.234±0.135 |
|
NA |
NA |
NA |
|
|
1 2,4-D |
1 Kin |
|
100.00±0.00 |
Friable |
1.058±0.058 |
|
+ |
0.492±0.019 |
0.048±0.021 |
|
|
1 2,4-D |
1 BAP |
|
97.50±2.50 |
Friable |
0.657±0.379 |
|
NA |
NA |
NA |
|
FW of callus (g/explant) = Fresh weight of callus 3 month after culture
FW and DW of cell (gr) = Fresh and Dry weight of cell suspension 3 month after establishment on suspension culture, respectively.
-: Weak, +: Moderate, ++: Goog and +++: Excellent callus growth.
NA=Not Assessed
Table 2. Growth efficiency based on cell count in cell suspensions of leaf and inflorescence in M. azedarach L.
|
Explants |
PGRs (mg/L) |
GI |
SGR |
Dt |
|
|
Auxin |
Cytokinin |
||||
|
Leaf |
1 2,4-D |
1 Kin |
0.897 |
0.108 |
6.397 |
|
Inflorescence |
1 2,4-D |
1 BAP |
0.622 |
0.081 |
19.732 |
Table 3. Mean comparisons of PGRs treatment on antioxidant enzymes activity and secondary metabolites in M. azedarach L. cell suspensions.
|
Explants |
Concentration of PGRs (mg/L) |
CAT (U/mg pro.) |
GPX |
PPO (U/mg pro.) |
Protein (mg/g FW) |
Proline (μg/g FW) |
H2O2 |
MDA (μmol/g FW) |
TFC |
TPC |
AC |
|
Leaf |
1 NAA+1 BAP |
389.044d |
165976.08d-h |
34835.44g |
0.242bc |
444.11ab |
20.40ef |
8.37d-g |
2.69ab |
1.049ab |
48.317abc |
|
1 2,4-D+1 Kin |
1222.672cd |
255418.80c-g |
33218.09g |
0.247bc |
277.19de |
21.95de |
9.72cd |
1.68e |
0.243c |
3.535f |
|
|
3 NAA+1 Kin |
2180.026a-d |
411944.18bc |
104496.45bc |
0.078e |
290.99cd |
17.72fg |
6.42f-i |
2.90a |
1.570a |
36.549b-e |
|
|
3 NAA+1 BAP |
3415.344ab |
554422.47b |
90678.15bc |
0.101de |
124.76f |
20.61ef |
6.12hij |
1.52e |
0.108c |
27.205c-f |
|
|
3 NAA+3 Kin |
2710.646abc |
296479.32c-f |
73170.94cde |
0.129de |
126.69f |
20.77ef |
6.28g-j |
1.69e |
0.311c |
59.950ab |
|
|
3 NAA+3 BAP |
3721.039a |
1720710.24a |
184314.93a |
0.041e |
184.49def |
30.12a |
12.57b |
1.79e |
0.264c |
11.902ef |
|
|
3 2,4-D+1 Kin |
742.987cd |
87609.34fgh |
82463.78bcd |
0.068e |
204.35def |
15.42g |
4.59ij |
1.60e |
0.186c |
6.616f |
|
|
3 2,4-D+1 BAP |
359.694d |
24600.35h |
26675.09g |
0.329ab |
401.66bc |
16.24g |
4.16j |
1.63e |
0.276c |
6.835f |
|
|
5 NAA+3 Kin |
2159.295a-d |
41931.30gh |
1099706.37b |
0.0612e |
124.26f |
18.25efg |
8.52def |
1.48e |
0.107c |
18.636def |
|
|
Inflorescence |
1 2,4-D+1 Kin |
1640.048bcd |
342950.41b-e |
51213.63efg |
0.191cd |
133.68f |
15.08g |
4.70ij |
2.40a-d |
0.538c |
58.165ab |
|
1 2,4-D+1 BAP |
2579.295abc |
130990.26e-h |
32682.64g |
0.255bc |
139.30f |
21.54ef |
6.61f-i |
2.01cde |
0.235c |
64.192ab |
|
|
3 NAA+1 Kin |
1084.996cd |
363763.88bcd |
46993.85fg |
0.172cd |
539.18a |
20.23ef |
17.43a |
2.45abc |
0.613c |
39.647bcd |
|
|
3 NAA+1 BAP |
544.327d |
302576.24c-f |
42489.38fg |
0.240bc |
270.45de |
25.30cd |
17.16a |
2.93a |
1.455a |
40.101bcd |
|
|
3 NAA+3 Kin |
613.965d |
119156.67e-h |
23117.22g |
0.350a |
458.40ab |
29.33ab |
15.89a |
2.32bcd |
0.503c |
54.916ab |
|
|
3 2,4-D+3 Kin |
1343.527cd |
165907.95d-h |
34319.02g |
0.255bc |
159.90ef |
18.43efg |
9.00de |
1.65e |
0.250c |
55.387ab |
|
|
3 2,4-D+3 BAP |
2090.755a-d |
148574.96d-h |
27564.84g |
0.266abc |
214.21def |
31.58a |
11.17bc |
1.69e |
0.220c |
72.525a |
|
|
Petiole |
1 2,4-D+1 Kin |
2205.867a-d |
343715.63b-e |
45565.40fg |
0.235bc |
115.89f |
26.32bc |
7.18e-h |
1.90de |
0.382c |
36.869b-e |
Means for groups in homogeneous subsets are displayed. Based on observed means.
Table 4. Pearson correlations coefficients between antioxidant enzymes activity and secondary metabolites in M. azedarach L. cell suspensions.
|
Correlations |
protein |
CAT |
GPX |
PPO |
H2O2 |
MDA |
Proline |
AC |
TFC |
TPC |
Rutin |
Quercetin |
|
CAT |
0.019 |
|||||||||||
|
GPX |
-0.481* |
0.674** |
||||||||||
|
PPO |
-0.817** |
0.660** |
0.714** |
|||||||||
|
H2O2 |
0.683** |
0.254 |
0.474* |
0.037 |
||||||||
|
MDA |
0.754** |
-0.206 |
0.201 |
-0.057 |
0.551* |
|||||||
|
Proline |
0.409* |
-0.345 |
0.101 |
-0.303 |
0.320 |
0.702** |
||||||
|
AC |
0.480* |
0.338 |
0.169 |
-0.194 |
0.362 |
0.098 |
-0.044 |
|||||
|
TFC |
0.133 |
-0.367 |
0.552* |
0.064 |
-0.007 |
0.369 |
0.554* |
0.197 |
||||
|
TPC |
0.133 |
-0.334 |
0.521* |
0.237 |
-0.056 |
0.303 |
0.504* |
0.017 |
0.927** |
|||
|
Rutin |
0.197 |
-0.153 |
0.364 |
-0.049 |
0.111 |
0.242 |
-0.117 |
0.221 |
0.542* |
0.537* |
||
|
Quercetin |
0.171 |
0.244 |
0.306 |
-0.058 |
0.396 |
-0.070 |
-0.342 |
0.034 |
-0.061 |
-0.012 |
0.153 |
|
|
Kaempferol |
-0.03 |
0.346 |
0.359 |
0.160 |
0.208 |
-0.209 |
-0.355 |
-0.045 |
-0.066 |
0.013 |
-0.014 |
0.953** |
* and** significant at the 0.05 and 0.01 probability level, respectively.