Improving the production of podophyllotoxin in hairy roots of Hyptis suaveolens induced from regenerated plantlets

Improving the production of podophyllotoxin in hairy roots of Hyptis suaveolens induced from... Ten Hyptis suaveolens hairy root lines were established by infecting nodal explants with OPENACCESS K599+pGus-GFP+ and ATCC15834+pTDT strains from Agrobacterium rhizogenes. Genetic transformation was confirmed by epifluorescence and plagiotropic hairy root growth Citation: Bazaldua C, Cardoso-Taketa A, Trejo- Tapia G, Camacho-Diaz B, Arellano J, Ventura- in absence of growth regulators. Cytotoxicity was determined using the sulforhodamine B Zapata E, et al. (2019) Improving the production of method, and the production of podophyllotoxin (PTOX) was measured by high performance podophyllotoxin in hairy roots of Hyptis suaveolens thin layer chromatography scanning. Through these methodologies, HsTD10 was identified induced from regenerated plantlets. PLoS ONE 14 as the hairy root line with the highest cytotoxicity and PTOX production, which was corrobo- (9): e0222464. https://doi.org/10.1371/journal. pone.0222464 rated by liquid chromatography-mass spectrometry and micrOTOF-Q II. A suspension cul- ture of HsTD10 was established in which PTOX and carbohydrate consumption during Editor: Jen-Tsung Chen, National University of Kaohsiung, TAIWAN growth kinetics were quantified by high-performance liquid chromatography. Procedures to Received: June 19, 2019 increase the production and retrieval of PTOX in the HsTD10 line included selection of cul- ture medium, addition of thiamine, and modification of the PTOX extraction method. The Accepted: August 30, 2019 best combination of these variables was MS medium at 75% of its components with the Published: September 12, 2019 -1 addition of 2 mg L of thiamine, extraction with methanol-dichloromethane, and sonication Copyright:© 2019 Bazaldu ´a et al. This is an open at 40± 5˚C. During kinetics, growth-associated PTOX accumulation was observed. The access article distributed under the terms of the -1 specific growth rate (μ) was 0.11 d . The highest concentration of PTOX obtained with Creative Commons Attribution License, which -1 permits unrestricted use, distribution, and HsTD10 (5.6 mg g DW) was 100 times higher than that reported for roots of wild plants reproduction in any medium, provided the original and 56 times higher than that for in vitro nontransformed roots of H. suaveolens. author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This work was supported from a grant from CONACYT Ciencia Basica (222714) to MLV. Introduction The funder had no role in study design,data collecton and analysis,decision to publish, or Podophyllotoxin (PTOX) is a natural 2,7’-cyclolignan used to obtain the semisynthetic deriva- preparation of the manuscript. tives Etoposide1, Teniposide1 and Etopophos1, which are medicines widely used in cancer chemotherapy. The main source of PTOX is the speciesPodophyllumhexandrum [1], a wild Competing interests: The authors have declared that no competing interests exist. plant native to the Himalayas, which has been overexploited and is currently endangered [2]. PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 1 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin Annual PTOX production is 50–80 tons per year; however, demand exceeds 100 tons per year [3]. Chemical synthesis of PTOX is highly difficult and expensive due to the complexity of its molecular structure. The search for new natural sources that produce PTOX is mandatory, as is research for biotechnological alternatives, to achieve stable and controllable production of this compound [4]. Today, the most important PTOX producer species arePodophyllumhex- andrum,Juniperusbermudiana, andPodophyllumpeltatum, which produce 43, 22.6, and 4.7 -1 mg g dry weight (DW) of PTOX, respectively [1, 5, 6]. -1 The highest concentration of PTOX obtained frominvitro plant cultures (8.0 mg g DW) was reported in cell suspension cultures from the selected line Herm A ofLinumalbum [7], whereas the second and third were obtained from cell suspension cultures ofPodophyllumhex- -1 andrum andP.peltatum of 7.9 and 5.9 mg g DW, respectively [8, 9]. A high level of produc- tion of PTOX has been reported in hairy root cultures ofLinumalbum andL.flavum at -1 concentrations of 3.5 to 15 mg g DW [10–13]. Hyptissuaveolens is a wild shrub native to Mexico that was employed throughout history, mainly for the treatment of gastric and bile problems, cancer, fever, spasms, cough, malaria, yellow fever and other diseases [14]. As we previously reported, this plant produces PTOX [15], and its roots growinginvitro accumulate more PTOX than wild roots (0.57 vs 0.05 mg g DW, respectively) [16]. We also ascertained that the hairy roots of this plant, growing in sus- pension cultures in Gamborg’s B5 culture medium without hormones, produce PTOX or its analogs [17]. The aim of this research was to establish and select hairy root culture lines ofHyptissuaveo- lens, producing PTOX, as well as optimizing culture conditions to increase PTOX accumula- tion. This research demonstrates that the most effective factors for determining a good yield of -1 PTOX were the culture medium MS at 75% of its components, amended with 2 mg L of thia- mine. Similarly, sonication at 40 ± 5˚C using methanol-dichloromethane for extraction increased PTOX yield. The accumulation of PTOX in the hairy root suspension cultures ofH. suaveolens was growth-associated. Materials and methods Plant material Plants and seeds ofHyptissuaveolens were collected in Merida, Yucatan, Mexico, in 2006 and authenticated by German Carnevalli, Scientific Research Center of Yucatan Herbarium. A specimen of the plant was deposited (voucher number CICY 7086). The seeds were carefully washed under running tap water for 10 min and surface-sterilized by immersing in ethanol at 70% (v/v) for 1 min and in sodium hypochlorite at 1.5% (v/v) for 5 min. The sterilized seeds were immediately rinsed three times with sterile distilled water for 5 min. Subsequently, the seeds were placed in glass vessels containing Murashige and Skoog medium (MS, Sigma) [18], -1 amended with sucrose 3% (w/v), and 2.5 g L water-phytagel for germination. The plantlets were maintained until they were 5 cm in length in a growth chamber at 24 ± 2˚C with a photo- period of 16/8 h light/darkness. Light sources were cool white lamps of 40 watts. Obtaining and multiplying regenerated plantlets Theinvitro multiplication of plantlets was achieved by placing nodal segment explants mea- suring 3 cm in length under the same conditions described previously. During plantlet regen- eration, three concentrations were evaluated: three-quarters strength (TQS), half strength (HS), and full strength (FS) of MS medium. Plantlet height and root length were registered. PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 2 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin Induction and selection of hairy roots Root induction was performed by infection of nodal explants with two strains ofAgrobacter- iumrhizogenes, K599+pGus-GFP+ (facilitated by Dr. Federico Sanchez†, IBT-UNAM), and ATCC15834+pTDT, obtained in a previous work [17]. One hundred forty-four putative hairy roots were placed in Petri dishes with TQS MSB5 medium (M0404-Sigma), which contains the same amount of inorganic salts and myo-inositol as MS medium, but it is supplemented with -1 vitamins, as described by Gamborg (B5 medium): nicotinic acid (1 mg L ), piridoxine (1 mg -1 -1 L ), and thiamine (10 mg L ). This medium was supplemented with sucrose (3%, w/v) and -1 phytagel (2.5 g L ) without hormones. Putative hairy roots were maintained under these con- ditions for 45 days. From these cultures, ten hairy root lines were selected based on their fast autonomous growth, hairy high branched appearance and plagiotropic growth to corroborate their genetic transformation, cytotoxic activity, and PTOX production. These selected hairy root lines (HR- lines) were transferred to TQS MSB5 liquid culture medium and kept at constant agitation of 110 rpm under the same light, temperature and humidity conditions described for plantlet growth. Genetic transformation confirmation. To observe the fluorescence, 1 cm of the roots was cut and directly placed on microscope slides. An epifluorescence microscope with filters for blue and green light (460–490 nm, and 500–550 nm) (Nikon, Eclipse 80i, Tokyo, Japan) with an adapted camera (3CCD MTI, DC330, Michigan, USA) made it possible to corroborate hairy root transformation byAgrobacterium strains expressing the fluorescent green protein (GFP) or the tomato deaminase threonine protein (TDT- red fluorescence). PTOX extraction Biomasses of ten initially tested HR lines were filter-washed, oven dried at 60˚C for 72 h, finely ground, sieved through a 53-micron mesh, and extracted. Three methods of extraction were tested: 1) methanol-dichloromethane (MD-method) [19]: briefly, 2 mL of 80% (v/v) methanol was added to 100 mg of biomass dry weight (DW) and sonicated for 1 h, 4 mL of dichloro- methane and 4 mL of water were added and vortexed, and the organic phase was finally recov- ered and dried; 2) chloroformic method: 10 mL of 100% chloroform was added to 100 mg of biomass DW, stirred for 1 h, and dried; and 3) modified MD method, where the modification of the MD method [19] involved sonication at 40 ± 5˚C. All extracts were solubilized in HPLC grade MeOH and syringe-filtered (0.45 μm, Millex-Hv, Millipore) for PTOX identification through HPTLC, HPLC or micrOTOF QII. Identification of PTOX utilizing HPTLC-Scan Initially, PTOX was identified using the corresponding standard (Sigma) through high perfor- mance thin layer chromatography (HPTLC-Scanner TLC-3 CAMAG), as described in 2017 by Kamal et al. [20]. The retention factor from standard (PTOX Sigma) spot was compared with those spots from the extracts obtained through the MD and chloroformic methods. HPTLC scanning of these spots complemented the results. Cytotoxic evaluation To evaluate cytotoxicity, two types of extracts obtained through the MD and chloroformic methods from ten HR lines were tested. The cytotoxicity of crude extracts was determined using the sulforhodamine B method (SRB) [21] by quantifying the half-maximal inhibitory concentration (IC ) on four human carcinoma cell lines obtained from ATCC (HF6 = Colon, PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 3 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin MCF7 = Breast, PC3 = Prostate, and SiHa = Uterus). To continue exploring alternatives for increasing PTOX production, the most cytotoxic HsTD10 HR-line was selected, as established -1 by the US NCI, a plant-screening program for crude extracts (IC values less than 20 μg mL ) [22]. The positive controls were standards of etoposide and PTOX (Sigma). Identification of PTOX using HPLC-MS and MicrOTOF-Q II in the HsTD10 HR-line HPLC-MS. To quantify PTOX and to include information obtained from previous experi- ments, extracts were obtained through the chloroformic and modified MD methods. The presence of PTOX in the extracts was verified by HPLC-MS (negative mode). The retention time of the peak, UV absorption spectrum and m/z 413 molecular ion of the stan- dard PTOX (Sigma) were compared with those recorded for extracts from the HsTD10 selected HR-line. The HPLC-MS system (Shimadzu, Tokyo, Japan) was equipped with a sys- tem controller CBM-20 , 2 binary pumps LC-20AD, degasser DGU-20A3, autosampler SIL- 20AC, column oven CTO20A, UV-Vis diodes detector SPD-M20A, mass spectrometer (LCMS-2020), ionization source with electro spray (ESI), software version 5a. Dry gas (N2): 10 -1 -1 L min was employed. Also, scan m/z 330–440 nm, nebulizer gas flow 1.54 L min was employed. A Zorbax- Eclipse Plus 4.8 x 150 mm, 5 μm, RP C-18 column was connected to a -1 guard column. The injection volume was 10 μL, and a flow of 0.8 mL min at 25˚C was employed. An isocratic elution of the mobile phase consisting of methanol-acetonitrile-water- acetic acid (20:30:50:0.1) was used. MicrOTOF-Q II and competitive fragmentation modeling for metabolite identification platform (CFM-ID). The identification of PTOX in the HsTD10 HR-line extract was corrob- orated by micrOTOF- Q II 10392 by obtaining the ion m/z 415 (positive mode) of the standard PTOX (Sigma) and of the extract. The MS/MS partitioning profile of the m/z 415 and m/z 437 corresponded to PTOX-Na adduct (Na monoisotopic weight 23). The system was equipped with a micrOTOF-Q II 10392 (Bruker Daltonik, Germany), electrospray ionization source, set nebulizer: 0.4 bar, and drying oven at 180˚C. Analysis was performed at 50–3000 m/z, capillary -1 set 4500 V—500 V, and set of dry gas 4.0 L min . The predictive structures of the MS/MS par- titioning profile were established utilizing the CFM-ID platform from Wishart-lab (http:// cfmid3.wishartlab.com), which is referred to in the pubchem-ncbi site [23]. Modifying culture conditions for PTOX accumulation in HsTD10 HR-line Culture medium and method of extraction. The selected HsTD10 HR-line was cultured on three different media (MS, B5, and MSB5) at TQS for 30 days to evaluate root growth and PTOX production. Based on the results, the medium selected to increase PTOX production was MS at TQS, and the extraction was performed using the modified MD method (sonication at 40 ± 5˚C). Effect of thiamine addition. The effect of six different doses of thiamine (0.5, 1, 2, 4, 8, -1 and 10 mg L ) added to TQS MS medium was evaluated. Kinetic studies Growth and metabolite production was evaluated using Erlenmeyer flasks of 125 mL contain- -1 ing 30 mL of culture medium (MS amended with 2 mg L of thiamine and supplemented with -1 sucrose at 3%, w/v), where 1.3 g L (DW) of the HR-line HsTD10 were inoculated and then placed under constant agitation at 110 rpm. The following variables were measured every four days (three replicates) for 48 days: dry weight (fresh weight dried at 60˚C for 72 h), pH, PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 4 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin conductivity (portable conductivity meter HM digital model AP2 precision ± 2%, resolution 1 μS), medium carbohydrates and PTOX production. Quantification of medium carbohydrates and PTOX was performed by HPLC. Duplication time was obtained by applying the formula TD = LN (2)/ μ, where “μ” is the slope grade from LN (final dry weight (DWf)–initial dry weight (DWi)/ final time (Tf)–initial time (Ti)). The specific rate growth (μ) is defined by μ = LN(2)/TD. Quantification of PTOX. This analysis was performed by the calibration of a Waters con- troller 600 HPLC-DAD analysis (Waters. Millipore Corp., Waters Chromatography Division, Milford, Ma, USA). The HPLC system was equipped with a Waters 600E multisolvent delivery system with a Waters W996 diode array detector, autosampler (Waters 717 Plus), and Software Millenium 32 (Waters). The analytical HPLC separations were carried out on a Zorbax eclipse RP C18 (150 mm x 46 mm x 5 μm) column. The mobile phase consisted of an isocratic elution -1 with methanol-acetonitrile-water (20:30:50) and a flow rate of 0.8 mL min . The standard PTOX was acquired from Sigma. Carbohydrate quantification. To evaluate the consumption of carbohydrates during kinetics, three carbohydrate standards (sucrose, fructose, and glucose from Sigma) were uti- lized. Two milliliters of the culture medium were collected every four days for 48 days and fro- zen until use. Samples of culture medium were diluted at 6% (v/v) with HPLC grade water and then syringe-filtered (0.45 μm, Millex-Hv, Millipore). Subsequently, 10 μL of the dilution was injected directly into the HPLC column. The calibration curve and retention time of the peak from each standard were established by HPLC (Waters, Millipore Corp., Waters Chromatog- raphy Division, Milford, Ma, USA). The HPLC system was equipped with a Waters 600E mul- tisolvent delivery system and a refractive index detector (Waters 2414). The analytical HPLC separations were performed using a CAPCELL PAK UG NH2 (150 mm x 46 mm x 5 μm) col- umn (Shiseido, Japan). The mobile phase consisted of an isocratic elution with acetonitrile- -1 water (75:25) at a flow rate of 0.7 mL min . Statistical analysis Results analyses were performed using ANOVA and posttest HSD of Tukey with a p < 0.01 by the VassarStats- online, Website for Statistical Computation. Results In vitro regeneration and effect of medium concentration on Hyptis suaveolens plantlets The concentration of MS [18] culture medium significantly influenced the budding and length of the regenerated plantlet roots, which grew faster on TQS MS (MS 5519 Sigma). Regarding the height of plantlets regenerated in full strength MS and those regenerated on TQS MS medium, significant differences (p<0.01) were observed after 20 days. Nevertheless, root sprouting and root length were significantly different (p<0.01) from day 10 onwards (Table 1). Induction of hairy roots and genetic transformation efficiency In response toAgrobacterium infection (strains K599+pGus-GFP+ and ATCC15834+pTDT), putative hairy roots sprouted after 8–12 days in culture. In total, 144 putative hairy roots were induced; 15 days after infection, the roots were excised and placed in Petri dishes (five to six per Petri dish) containing hormone-free MSB5 culture medium (M0404 Sigma). Ten induced roots that grew faster were individually transferred to Petri dishes. The roots showed the char- acteristic hairy root phenotype of theAgrobacteriumrhizogenes-transformed roots and high PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 5 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin Table 1. Effect of MS culture medium concentration on the growth of Hyptis suaveolens plantlets. Plantlets height (cm) Root length (cm) MS medium concentration 1 2 3 1 2 3 Days HS TQS FS HS TQS FS 10 0.18 ± 0.08 a 0.82 ± 0.15 a 0.75 ± 0.14 a 0 0.42 ± 0.1 a 0 15 0.48 ± 0.09 a 1.69 ± 0.25 a 1.59 ± 0.22 a 0 1.09 ± 0.19 a 0 20 0.87 ± 0.13 c 3.15 ± 0.24 a 2.66 ± 0.27 b 0 2.08 ± 0.23 a 0 25 1.22 ± 0.15 c 4.84 ± 0.18 a 4.07 ± 0.31 b 0 3.01 ± 0.24 a 0.56 ± 0.08 b 30 2.06 ± 0.19 c 6.63 ± 0.33 a 5.86 ± 0.37 b 0.37 ± 0.008 c 5.95 ± 0.4 a 1.23 ± 0.09 b Half strength Three quarters strength Full strength. ANOVA and HSD Tukey test post-hoc analysis. Different letter in the same line indicates statistically significant differences between treatments (mean ± SD, N = 10, Significance level was fixed at p<0.01. https://doi.org/10.1371/journal.pone.0222464.t001 branched, rapid and plagiotropic growth, which was observed in both semisolid (Fig 1 HR- lines 1a – 10a) and liquid culture medium (Fig 1 HR-lines 1b – 10b). The efficiency of transformation obtained with the ATCC15834+pTDT strain was 60% and with the K599+pGus-GFP+ strain was 46.7% (Table 2). One hundred forty-four putatively transformed roots were obtained, and ten HR lines with optimum growth were selected and further subjected to cytotoxic evaluation and PTOX identification by HPTLC. Green or red epifluorescence was utilized to corroborate the genetic transformation of hairy roots. Four HR-lines expressed the green fluorescent protein (HsGF1 –HsGF4), and six expressed the threonine deaminase of tomato protein (HsTD5 –HsTD10), which fluoresces in red (Fig 2). Extraction and identification of PTOX utilizing HPTLC-Scan The first extraction to identify PTOX included the use of both chloroformic and MD methods [19]. For the identification of PTOX by high performance thin layer chromatography (HPTLC) [20] of the ten HR lines, we considered the retention factor (Rf = 0.56) developed by the PTOX standard (Sigma). The displayed Rf = 0.53 of HsTD10 HR-line extracts was similar to the standard (Fig 3). The HPTLC profiling of HsTD10 HR-line extract visualized at 254 nm (Fig 3A and 3E) for the plate cerium sulfate-revealed and observed at 366 nm (Fig 3B and 3F), and we observed with white light (Fig 3C and 3G), indicated a very close RF with the PTOX standard (lane “s”). The identity of PTOX in the extracts was confirmed by the UV spectra pro- filing, generated by the Scanner–TLC 3-CAMAG) observed in Fig 3D (MeOH-CH Cl -extrac- 2 2 tion) and 3h (CHCl - extraction), displaying a clear result in the last one. Cytotoxic evaluation Both extracts from the ten HR lines were evaluated for cytotoxicity using the sulforhodamine (SRB) method. Cytotoxic evaluation showed that all of the extracts were cytotoxic for at least one carcinoma cell line. Depending on the extract, the HR-lines showed differential cyto- -1 toxic activity (Table 3). Some IC values were lower than 4 μg mL , a threshold value reported for active pure compounds [22]. The chloroformic and methanol-dichloromethane extracts from the HsTD10 HR-line showed the highest cytotoxic activity in three (SIHa, PC3, and MCF7) out of four carcinoma cell lines evaluated, with IC values ranging between 1.8 and -1 16.6 μg mL ; therefore, this line was selected for continued research exploring and improving PTOX accumulation. PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 6 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin Fig 1. Ten selected HR lines. Individualized in solid MSB5 culture medium (1a – 10a) and in liquid MSB5 liquid culture medium (1b – 10b) without hormones. https://doi.org/10.1371/journal.pone.0222464.g001 Identification of PTOX using HPLC-MS and MicrOTOF-Q II in the HsTD10 HR-line HPLC-MS. The identification of PTOX in the HsTD10 HR-line was also carried out by HPLC-MS. Fig 4 shows the chromatogram of the m/z 413 that corresponds to PTOX in a nega- tive mode. MicrOTOF-Q II. When the identification was performed by micrOTOF-Q II, there was overwhelming evidence that the selected HR-line produces PTOX, as the molecular ion m/z 415 corresponding to PTOX (positive mode), the molecular ion m/z 437 corresponding to the adduct PTOX-Na and the partitioning profile of m/z 415, were observed (Fig 5). Competitive fragmentation modeling for metabolite identification (CFM-ID) plat- form. To correlate the observed peaks in the partitioning profile obtained by MS/MS from Table 2. Number of induced and selected HR-lines. Strain Number of explants Explants with roots sprout Number of roots induced Number of selected lines K599+GFP-GUS+ 15 7 34 4 ATCC15834+pTDT 40 24 110 6 https://doi.org/10.1371/journal.pone.0222464.t002 PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 7 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin Fig 2. Confirmation of genetic transformation. Genetic transformation was corroborated by epifluorescence of induced hairy roots of Hyptissuaveolens with the strain K599+pGus-GFP+ (a, and b) and the strain ATCC15834+pTDT (c, and d). Observed in darkness and illuminated with blue (a), green (c), and white lights (b, d, and f). When nontransformed roots were illuminated with blue or green light, epifluorescence was not observed (e). All microphotographs are 20X. https://doi.org/10.1371/journal.pone.0222464.g002 m/z 415 with predictive structures, the CFM-ID (http://cfmid3.wishartlab.com) platform was utilized, showing resulting ionized structures (Fig 6). Modification of culture conditions and yield of PTOX in the HsTD10 HR line Effect of culture medium and of the method of extraction on yield of PTOX. The results showing PTOX accumulation in the HsTD10 HR line at 30 days of culture grown in PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 8 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin Fig 3. Identification of PTOX by HPTLC-Scan. Lane 10 indicates the HsTD10 HR-line extract, and the standard (POTX, Sigma) is referred to as letter “s”. The profiling of the different solvent extractions MeOH-CH Cl (a, b, and c) 2 2 and CHCl (e, f, and g) are presented. Developed-plate visualizations at 254 nm (a, and e), cerium sulfate revealed- plate at 366 nm (b, and f), and white light (c, and g) are shown. Absorbance spectra from the spots of PTOX in both extracts (d, and h) are shown. https://doi.org/10.1371/journal.pone.0222464.g003 MS, MSB5, and B5, all at TQS, are presented in Table 4. When the extraction of PTOX was performed using chloroform, a higher accumulation of this lignan was obtained from roots -1 Table 3. Cytotoxicity (IC μg mL ) of ten selected HR-lines of Hyptis suaveolens. HR-Lines Methanol–Dichloromethane extracts Chloroformic extracts � -1 Carcinoma cell lines (IC50 μg mL ) HF6 SIHa PC3 MCF7 HF6 SIHa PC3 MCF7 HsGF1 5.9 ± 0.3 >20 >20 4.2 ± 0.1 6.4 ± 0.5 17.4 ± 0.9 >20 5.0 ± 0.3 HsGF2 6.7 ± 0.1 >20 >20 4.1 ± 0.4 3.2 ± 0.2 >20 >20 5.2 ± 0.4 HsGF3 7.1 ± 0.3 >20 >20 4.6 ± 0.3 12.1 ± 0.3 6.6 ± 0.7 >20 6.2 ± 0.5 HsGF4 11.7 ± 0.2 >20 >20 3.8 ± 0.2 10.9 ± 0.8 6.1 ± 0.2 >20 4.0 ± 0.3 HsTD5 5.5 ± 0.5 >20 >20 8.9 ± 0.6 15.7 ± 0.7 6.3 ± 0.1 >20 7.4 ± 0.6 HsTD6 7.7 ± 0.6 12.2 ± 0.5 >20 6.3 ± 0.8 14.2 ± 0.9 16.4 ± 0.2 >20 7.0 ± 0.8 HsTD7 2.8 ± 0.1 13.5 ± 0.3 >20 9.6 ± 0.5 5.1 ± 0.2 9.0 ± 0.4 18.5 ± 0.8 3.5 ± 0.4 HsTD8 18.1 ± 0.5 >20 >20 >20 >20 9.69 ± 0.6 >20 5.5 ± 0.3 HsTD9 9.1 ± 0.2 16.8 ± 0.7 >20 4.9 ± 0.2 9.1 ± 0.7 4.1 ± 0.2 16.2 ± 0.7 2.9 ± 0.7 HsTD10 5.9 ± 0.2 7.1 ± 0.4 16.6 ± 0.7 3.3 ± 0.2 8.2 ± 0.4 1.8 ± 0.1 6.68 ± 0.3 2.5 ± 0.2 PTOX 0.011 1.66E-03 1.2 ± 0.01 4.23E-6 0.011 1.66E-03 1.2 ± 0.01 4.23E-6 Etoposide 1.8 ± 0.1 0.35 0.99 0.03 1.8 ± 0.1 0.35 0.99 0.03 Carcinoma cell lines: HF6 = Colon, SiHa = Uterine, PC3 = Prostate, MCF7 = Breast. The positive controls utilized were PTOX and Etoposide. https://doi.org/10.1371/journal.pone.0222464.t003 PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 9 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin Fig 4. Identification of PTOX by HPLC-MS. (a) PTOX standard (Sigma) showing 1) molecular ion m/z 413 in negative mode, 2) retention time of the peak (7.3 min), and 3) UV spectrum absorption; (b) HsTD10 HR-line 1) molecular ion m/z 413 corresponding to PTOX in negative mode, 2) retention time of the peak at 7.3 min, and 3) UV spectrum similar to PTOX. https://doi.org/10.1371/journal.pone.0222464.g004 PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 10 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 11 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin Fig 5. Identification of podophyllotoxin by micrOTOF-Q II in the HsTD10 HR-line. (a) spectrum of the extract showing the PTOX-Na and PTOX-K adducts, (b) partitioning profile (MS/MS) from molecular ion m/z 415 corresponding to PTOX (positive mode). https://doi.org/10.1371/journal.pone.0222464.g005 grown on MS medium followed by those grown on MSB5 and B5 media. However, when the extraction was undertaken using the modified MD method, the yield of PTOX increased con- siderably. Nevertheless, in this experiment, the best result was recorded in MSB5 followed by B5 and MS (Table 4). Effect of thiamine addition to TQS MS culture medium on modulating the yield of PTOX in the HsTD10 HR-line. The results regarding the effect on PTOX yield of extra addition of thiamine at various concentrations to the MS culture medium (0.5, 1, 2, 4, 8 and 10 -1 mg L ) are shown (Fig 7). When extraction was performed with sonication at 40 ± 5˚C (Koul- man modified), PTOX accumulation increased by 34% (Fig 7 black columns). Growth kinetics and PTOX accumulation Measurements of conductivity during growth kinetics showed that as anticipated, this parame- ter declined in inverse proportion to biomass increase. The highest dry weight biomass was -1 achieved at day 40 (7.2 g L DW), and the exponential phase of growth of the selected HR-line -1 occurred from day 8 to day 28 with a mean specific growth rate (μ) of 0.11 d (Fig 8A). The production of PTOX was growth associated, observing a maximum accumulation of 5.4 and -1 5.6 mg g between days 32 and 36 (Fig 8B). Concerning carbohydrate consumption, sucrose concentration on the medium decreased at a constant rate until day 20, when it was completely hydrolyzed; in contrast, fructose and glucose initially increased up to day 20, and the Fig 6. Structures predicted by CFM-ID related to each peak of partitioning profile from m/z 415 ion. (a-d) structures for m/z 385, (e) structure for m/z 357, (f) structure for m/z 301, (g) structure for m/z 297, (h) structure for m/z 217. https://doi.org/10.1371/journal.pone.0222464.g006 PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 12 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin Table 4. Influence of culture medium and extraction method in PTOX yield by the HsTD10 HR-line. -1 Culture medium at TQS Concentration of PTOX (mg g DW) Extraction method Chloroform Koulman Koulman Modified MS 1.52 ± 0.011 a 1.99 ± 0.021 b 2.89 ± 0.035 b MSB5 0.63 ± 0.017 b 2.97 ± 0.027 a 4.50 ± 0.032 a B5 0.58 ± 0.006 b 2.20 ± 0.014 b 3.24 ± 0.032 b ANOVA and HSD Tukey test post-hoc analysis. Different letter in the same column indicates statistically significant differences between treatments (mean ± SD, N = 3, Significance level was fixed at p<0.01). https://doi.org/10.1371/journal.pone.0222464.t004 concentration of glucose began to diminish thereafter, while fructose concentration remained unchanged (Fig 8C). Discussion Effect of medium components on plantlet regeneration The best result for regenerated plantlets evaluated by height and root length was obtained with TQS MS medium. Regenerated plantlet height was similar for 15 days, but subsequently, sig- nificant differences (p<0.01) between all treatments emerged. In contrast, roots were sprout- ing on TQS MS at day 10, although when half-strength (HS) or full-strength (FS) MS were used, root sprouting occurred at 25 and 30 days, respectively. Likewise, sprouted roots in TQS MS at 30 days grew up to five to fifteen times the length compared to those grown at FS MS and HS MS, respectively. This result is similar to that reported in 2011 by Borges et al. [24] for the root length and plantlet height ofDiscoreaalata but different from that reported in 2002 by Espinosa et al. [25] and in 2016 by Jimenez-Mariña et al. [26], who obtained better results Fig 7. Effect of thiamine extra addition to TQS MS and of the extraction method in the production of PTOX by the HsTD10 HR- line at 30 days in culture. Koulman method (Gray) and modified Koulman method (Black) ANOVA, and HSD Tukey test post hoc analysis. Different letters indicate statistically significant differences between treatments (mean ± SD, N = 3, Significance level was fixed at p<0.01). https://doi.org/10.1371/journal.pone.0222464.g007 PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 13 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin Fig 8. Kinetic parameters from the HsTD10 HR line during 48 days of growth in TQS MS medium supplemented with thiamine (2 mg -1 L ). (a) Correlation between dry weight (DW) and conductivity, (b) relation between biomass and PTOX accumulation, and (c) relation between carbohydrate consumption and biomass. (■ conductivity, —▲— DW, —●— PTOX, ^ fructose, D glucose,□ sucrose). https://doi.org/10.1371/journal.pone.0222464.g008 with FS MS in terms of plantlet height forIpomeabatatas andDianthuscaryophyllus, respec- tively. There is a direct relationship between plantlet height and root length. Genetic transformation efficiency and its corroboration Transformation efficiency (defined as the number of responsive explants regarding the total number of infected explants) obtained in the present work using ATCC15834+pTDT (60%) and K599+pGus-GFP+ (46.7%) strains was greater than that obtained in 2013 by Ooi et al. [27] with the A4Agrobacterium strain infectingSolanummammosum (21.4%) but lower than that reported in 2015 by Thilip et al. [28], with the R1000Agrobacterium strain infectingWith- aniasomnifera (93.3%). The corroboration of transformation has traditionally been reported by Southern blotting or using PCR. However, in this work, the use of K599+pGus-GFP+ and ATCC15834+pTDT strains enabled confirmation of transformation by visual observation of fluorescence through an epifluorescence microscope. The most important advantage of this method is that it reduces the carbon footprint and does not damage the obtained transformed tissues. Moreover, this approach has been successfully utilized for the structural elucidation of biological molecules and their interactions, as well as forinvitro assays orinvivo monitoring cellular research [29]. Extraction and identification of PTOX utilizing HPTLC-Scan The identification of PTOX in wild roots ofH.suaveolens has been reported using high-perfor- mance liquid chromatography coelution [16] and liquid chromatography-masses (m/z 415, positive mode) [15]. Similarly, the adduct PTOX-Na (m/z 437) has also been reported in leaves ofJuniperusbermudiana [5]. In the present work, we used the methodology described by Kamal et al. [20], who reported the identification and quantification of PTOX using an HPTLC system. It was possible to confirm the presence of PTOX in the extracts by the devel- opment of spots with similar Rfs: 0.56 for the PTOX standard and 0.53 for the extracts. The PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 14 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin slight difference in Rfs can be explained because the extract is a complex matrix composed of many metabolites which, in turn, influence chromatographic development. Nevertheless, the unequivocal identity of PTOX in the extracts was also confirmed through spectroscopic (maxi- mum UV absorption) and spectrometric (LC-MS/MS) evidence. Cytotoxic evaluation All HR-lines were cytotoxic for at least one of the carcinoma cell lines utilized. Interestingly, -1 IC values were lower than 4 μg mL in five out of ten evaluated HR lines, which is propi- tious, as this value is a predicted value for a pure compound [22]. The HsTD10 HR-line was the most cytotoxic in three (SIHa, PC3, and MCF7) out of four carcinoma cell lines, and the one that showed activity against all the evaluated carcinoma cell lines. Identification of PTOX using HPLC-MS and MicrOTOF-Q II in the HsTD10 HR-line To corroborate the identification and quantification of PTOX in the extracts of the HsTD10 HR-line, we used HPLC-MS equipment in negative mode (m/z 413) because under our ioniza- tion conditions, the m/z 415 (positive mode) appeared to be notably low. The m/z 415 molecu- lar ion was identified with a micrOTOF-Q II mass spectrometer. This equipment permitted the identification of the adduct PTOX-Na (m/z 437), as reported in 2011 by Renouard et al. [5], and the partitioning profile during the MS/MS experiment of the m/z 415. This result is similar to several spectra available at the https://pubchem.ncbi.nlm. nih.gov/compound/podophyllotoxin#section=MS-MS site [23]. Seven of the eight predictive structures proposed as resulting from ionization of the PTOX molecule in this work can also be observed, following links (4.3.2. MS/MS spectrum 108322, 108323, 175510, and 175511). In both cases, prediction was carried out utilizing the Competitive Fragmentation Modeling for Metabolite Identification platform (CFM-ID) from Wishart-lab (http://cfmid3.wishartlab. com). Selection of culture conditions and yield of PTOX in the HsTD10 HR-line Under our conditions, the culture medium affected the concentration of PTOX, which was determined on the HsTD10 HR-line growing on three different media. It has been reported that the best culture medium for improving PTOX production depends on the plant species and the type ofinvitro cultivation employed. The influence of MS and B5 (Gamborg’s) culture media for biomass growth and PTOX accumulation on transformed cell suspension and hairy root cultures ofLinumalbum [11], as well as in hairy root cultures ofL.flavum, higher biomass accumulation and PTOX production were accomplished with B5 medium [13]. In contrast, MS medium has been utilized for the production of PTOX and MPTOX in adventitious root cultures ofPodophyllumpeltatum [9], hairy root cultures ofLinummucronatum ssp.mucrona- tum [30], and hairy root cultures ofLinumstrictum ssp.strictum [31]. Effect of culture medium and the extra addition of thiamine on PTOX accumulation. Considering the results described in this study, it was necessary to ascertain how much the cul- ture medium or the method of extraction affected the yield of PTOX in the HsTD10 HR-line. When the HsTD10 HR-line was established in three culture media and extraction was per- formed using chloroform, the best result was obtained in roots grown in MS. However, when the extraction was performed using the modified MD method, the best result was obtained from roots grown in MSB5 medium (7 times more PTOX than that obtained in chloroform extracts from the same line, growing in the same culture medium). The differences between MS and MSB5 culture medium pertain to the concentration of vitamins: 100 times more PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 15 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin thiamine and twice the amount of pyridoxine and nicotinic acid in the MSB5 medium. Then, the question was whether thiamine could modulate PTOX production. The addition of thia- mine to TQS MS medium increased the yield of PTOX in the HR-line. Our results showed that there was no significant difference in biomass production, but there was a clear difference in PTOX accumulation. The increase in PTOX production was -1 dose dependent on the thiamine concentration up to a maximum of 2 mg L ; at this point, production began to decrease with each succeeding increased dose of thiamine. To date, the relationship between the addition of thiamine to the culture medium and the increase in PTOX production has not been reported. Lignans are phenylpropanoid com- pounds that have been identified as plant-defenders [32–34]. Regarding the phenylpropanoid pathway, Boubakri et al. [35] reported that thiamine increased the expression of genes along this route in grape vines. This vitamin increased plant defense activity [36], induced systemic acquired resistance (SAR) inArabidopsis, rice, tobacco, and cucumber [37], and increased the concentration of callose and lignin inArabidopsis [38]. -1 As the accumulation of PTOX in the HsTD10 HR-line was dose-dependent up to 2 mg L of thiamine, and higher concentrations notably reduced the accumulation of PTOX, in future experiments, it would be interesting to evaluate enzyme activity in the cultures of this HR-line. The increase of thiamine in the HsTD10 HR-line ofHyptissuaveolens may activate a defense mechanism, possibly increasing the concentration of some phenylpropanoids but definitely that of PTOX. Effect of sonication at 40 ± 5˚C in PTOX extraction. Several reports have analyzed the effect of temperature on PTOX extraction. In 2002, Bedir et al. [6] reported greater PTOX extraction from leaves ofPodophyllumpeltatum and modified leaves and twigs ofJuniperus virginiana using hot methanol. However, in 2011, Renouard et al. [5] analyzed the effect of temperature but did not report any significant differences inJuniperusbermudiana or in 12 other species from the same genus; nevertheless, in these reports, sonication was not employed. It is important to note that the modified MD method was performed as described in 2003 by Koulman et al. [19] with one change, as instead of performing sonication for one hour at room temperature, we sonicated for one hour at 40 ± 5˚C. This change significantly increased PTOX extraction. In our work, it is clear that the culture medium and the extraction method were factors that determined the level of PTOX production. Growth kinetics The highest dry weight biomass obtained under our culture conditions at day 40 was 9.4 times higher than the inoculum. The specific growth rate of the HsTD10 HR-line suspension cultures -1 -1 (0.11 d ) is higher than that reported in 2006 by Nader et al. (0.08 d ) [39] but lower than that -1 reported in 2005 by Caspeta et al. (0.12 d ) [40] for hairy root cultures ofGalphimiaglauca and Solanumchrysotrichum, respectively. Moreover, the production of PTOX associated with growth observed in this work is similar to that observed in hairy roots and in cell suspension cultures ofL.album [11, 41], as well as in cell suspension cultures ofP.hexandrum [42]. In this work, the consumption of carbohydrates was selective for sucrose and glucose because fructose was not consumed. This result is similar to that reported in 1998 by Ham- mouri et al. [43], who found that the hairy roots ofBetavulgaris were unable to use fructose as a source of energy when using the same initial concentration of sucrose (3%). Nonetheless, this result differs from that reported in 2000 by Shimon-Kerner et al. [44] because they found similar levels of glucose and fructose in hairy root cultures ofSymphytumofficinale after 28 days in culture, a difference which is probably due to the time period of their kinetic experiment. PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 16 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin It is also important to note that the concentration of PTOX between days 32 and 36 at the -1 beginning of the stationary growth phase was the highest obtained (5.4 and 5.6 mg g , respectively). PTOX accumulation in wild plants and plant material obtained byinvitro cultures (cell, callus, tissues, plantlets, transformed tissues) show a different pattern. To date, the greatest -1 supply of PTOX has been obtained from the wild plantPodophyllumhexandrum (43 mg g DW) [1]; however, this yield diminishes when PTOX is obtained from cell suspension cultures -1 of this species (7.9 mg g DW) [8]. Similarly, the concentration of PTOX in wildPodophyllum -1 peltatum plants is similar to that obtained in cell suspension cultures (4.7 and 5.9 mg g DW, respectively) [6, 9]. The accumulation of PTOX inLinumalbum has been reported to be higher -1 -1 in cell suspension cultures (8 mg g DW) [7] than in wild roots of the same species (0.3 mg g DW) [45]. However, the highest accumulation of PTOX in this species was obtained from -1 hairy root extracts (15 mg g DW) [10]. The highest concentration of PTOX reported usinginvitro cultures was obtained in hairy -1 -1 root cultures ofLinumalbum (15 and 5.1 mg g DW) [10, 11] andL.flavum (4.5 mg g DW) -1 [13]. Under our conditions, the optimum yield obtained for the HsTD10 HR-line (5.6 mg g DW) is similar to that reported in 2008 by Baldi et al. [11] inL.album, which is 100 times higher than that obtained from roots of wild plants and 56 times higher than that obtained from nontransformed roots ofHyptissuaveolens growninvitro [16]. Conclusions Nodal explants ofHyptissuaveolens infected with K599+pGus-GFP+ and ATCC15834+pTDT strains fromAgrobacteriumrhizogenes were suitable explants for obtaining ten hairy root line cultures. The HsTD10 HR-line was the greatest cytotoxic and podophyllotoxin producer. The culture medium did not significantly affect biomass growth but was a determining factor for the accumulation of PTOX in the hairy roots ofH.suaveolens, an influence that was greatest when using MS at three-quarters strength. Thiamine modulated the production of PTOX, -1 obtaining the highest values when it was added at 2 L to MS medium. Sonication at 40 ± 5˚C using MD extraction increased PTOX extraction. PTOX accumulation in kinetic suspension culture ofH.suaveolens-hairy roots was growth-associated. The accumulation of PTOX in the HsTD10 HR-line was greater than that obtained from the roots of wild plants orinvitro non- transformed cultured roots. Acknowledgments C. Bazaldu ´ a acknowledge Instituto Polite ´cnico Nacional (IPN) for work partial licenses during his PhD research. We recognize Centro de Nanociencias y Micro y Nanotecnologı ´as (CNMN- IPN) for the experimental service with the micrOTOF-Q II spectrometer. We thank Dr. Feder- ico Sa ´nchez from Instituto de Biotecnologı ´a, (IBT-UNAM) for facilitating theAgrobacterium strain K599+pGus-GFP+, thanks also go to Ariadna Zenil for her help with the HPLC Waters. Thanks to Juan Manuel Solares for his help with the Scanner-TLC-3-CAMAG. Author Contributions Conceptualization: Crescencio Bazaldu ´ a. Formal analysis: Crescencio Bazaldua, Alexandre Cardoso-Taketa. Funding acquisition: Marı ´a Luisa Villarreal. ´ ´ Investigation: Crescencio Bazaldua, Alexandre Cardoso-Taketa, Marıa Luisa Villarreal. PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 17 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin Methodology: Crescencio Bazaldu ´ a, Alexandre Cardoso-Taketa, Gabriela Trejo-Tapia, Brenda Camacho-Diaz, Jesu ´ s Arellano, Elsa Ventura-Zapata. Resources: Gabriela Trejo-Tapia. Supervision: Marı ´a Luisa Villarreal. Validation: Alexandre Cardoso-Taketa. Writing – original draft: Crescencio Bazaldu ´ a, Marı ´a Luisa Villarreal. Writing – review & editing: Crescencio Bazaldu ´ a, Jesu ´ s Arellano, Marı ´a Luisa Villarreal. References 1. Giri A, Narasu ML. Production of podophyllotoxin from Podophyllum hexandrum: a potential natural product for clinically useful anticancer drugs. Cytotechnol. 2000; 34:17–26. https://doi.org/10.1023/ A:1008138230896 PMID: 19003377 2. Arroo RRJ, Alfermann AW, Medarde M, Petersen M, Pras N, Woolley JG. Plant cell factories as a source for anti-cancer lignans. Phytochem Rev. 2002; 1(1):27–35. https://doi.org/10.1023/ A:1015824000904 3. Alam A, Naik PK, Mishra GP. Congruence of RAPD and ISSR markers for evaluation of genomic rela- tionship among 28 populations of Podophyllum hexandrum Royle from Himachal Pradesh, India. Turk J Bot. 2009; 33:1–12. https://doi.org/10.3906/bot-0711-10 4. Moraes RM, Burandt Ch, Ganzera M, Li X, Khan I, Canel C. The American mayapple revisited–Podo- phyllum peltatum- still a potential cash crop? Econ Bot. 2000; 54(4):471–76. 5. Renouard S, Lopez T, Hendrawati O, Dupre P, Doussot J, Falguieres A, et al. Podophyllotoxin and deoxypodophyllotoxin in Juniperus bermudiana and 12 other Juniperus species: Optimization of extrac- tion, method validation, and quantification. J Agric Food Chem. 2011; 59:8101–07. https://doi.org/10. 1021/jf201410p PMID: 21702435 6. Bedir E, Khan I, Moraes RM. Bioprospecting for podophyllotoxin In: Trends in New Crops and New Uses; Janick J. and Whipkey A., ASHS Press; Alexandria, VA, USA. 2002 pp 545–49. ISBN 0- 970756-5-5. 7. Wink M, Alfermann AW, Franke R, Wetterauer B, Distl M, Windho ¨ vel J, et al. Sustainable bioproduction of phytochemicals by plant in vitro cultures: Anticancer agents. Plant Gen Res.2005; 3(2):90–100. https://doi.org/10.1079/PGR200575 8. Ahmad R, Sharma VK, Rai AK, Shivananda RD, Shivananda BG. Production of lignans in callus culture of Podophyllum hexandrum. Trop J Pharm Res. 2007; 6(4):803–08. www.bioline.org.br/pdf?pr07022. 9. Anbazhagan VR, Ahn CH, Harada E, Kim YS, Choi YE. Podophyllotoxin production via cell and adventi- tious root cultures of Podophyllum peltatum. In Vitro Cell Dev Biol-Plant. 2008; 44:494–501. https://doi. org/10.1007/s11627-008-9134-1 10. Farkya S, Bisaria VS. Exogenous hormones affecting morphology and biosynthetic potential of hairy root line (LYR2i) of Linum album. J Biosci Bioeng. 2008; 105(2):140–6. https://doi.org/10.1263/jbb.105. 140 PMID: 18343341 11. Baldi A, Srivastava AK, Bisaria VS. Improved podophyllotoxin production by transformed cultures of Linum album. Biotechnol J. 2008; 3:1256–63. https://doi.org/10.1002/biot.200800173 PMID: 18932162 12. Cong LH, Dauwe R, Lequart M, Vinchon S, Renouard S, Fliniaux O, et al. Kinetics of glucosylated and non-glucosylated aryltetralin lignans in Linum hairy root cultures. Phytochemistry. 2015; 115(1):70–8. https://doi.org/10.1016/J.PHYTOCHEM.2015.01.001 13. Renouard S, Corbin C, Drouet S, Medvedec B, Doussot J, Colas C, et al. Investigation of Linum flavum (L.) hairy root cultures for the production of anticancer aryltetralin lignans. Int J Mol Sci. 2018; 19 (4):990. https://doi.org/10.3390/ijms19040990 14. Prince SP, Ram RK, Anurag C, Dinesh G, Sharma VK. Hyptis suaveolens (L.) Poit.: A phyto pharmaco- logical review. Int J Chem Pharm Sci. 2013; 4:1–11. www.researchgate.net/publication/255982083. 15. Lautie ´ E, Quintero R, Fliniaux MA, Villarreal ML. Selection methodology with scoring system: Applica- tion to Mexican plants producing podophyllotoxin related lignans. J Ethnopharmacol. 2008; 120:402– 12. https://doi.org/10.1016/j.jep.2008.09.014 PMID: 18930796 16. Veloz RA, Cardoso-Taketa A, Villarreal ML. Production of podophyllotoxin from roots and plantlets of Hyptis suaveolens cultivated in vitro. Pharmacogn Res. 2013; 5(2):93–102. https://doi.org/10.4103/ 0974-8490.110538 PMID: 23798883 PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 18 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin 17. Bazaldu ´ a C, Cardoso-Taketa A, Arellano J, Camacho-Diaz B, Ventura-Zapata E, Villarreal ML. Podo- phyllotoxin-like lignans production through hairy roots of Hyptis suaveolens. J Chem Biol Phy Sci. 2014; 4(5):37–47. www.researchgate.net/publication/271827761. 18. Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 1962; 15:473–97. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x 19. Koulman A, Kubbinga ME, Batterman S, Woerdenbag HJ, Pras N, Woolley JG, et al. A phytochemical study of lignans in whole plants and cell suspension cultures of Anthriscus sylvestris. Planta Med. 2003; 69(8):733–8. https://doi.org/10.1055/s-2003-42776 PMID: 14531024 20. Kamal A, Singh M, Ahmad FJ, Saleem K, Ahmad S. A validated HPTLC method for the quantification of podophyllotoxin in Podophyllum hexandrum and etoposide in marketed formulation. Arab J Chem. 2017; 10:2539–46. https://doi.org/org/10.1016/j.arabjc.2013.09.027 21. Vanicha V, Kanyawim K. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat Protoc. 2006; 1(3):1112–16. https://doi.org/10.1038/nprot.2006.179 PMID: 17406391 22. John Boik. Natural compounds in cancer therapy. Oregon Medical Press, Princeton, MN, USA. 2001. 23. Pubchem.ncbi—Podophyllotoxin. Chemical and physical properties: Spectral properties, MS-MS. Avail- able online: https://pubchem.ncbi.nlm.nih.gov/compound/podophyllotoxin#section=MS-MS (accessed on 18 October 2018). 24. Borges GM, Destrade BR, Meneses RS, 5. Go ´ mez KR, Malaurie B, Hamon P, et al. Optimization of a culture medium for micropropagated plants of Dioscorea alata L. Rev Colomb Biotecnol. 2011; 13 (2):221–8. Spanish. 25. Espinosa RA, Salas HL, Gonza ´ lez PO, Silva PJJ. Empleo de a ´ cido abscı ´sico, manitol y la disminucio ´ n de la concentracio ´ n de las sales del medio de cultivo en la conservacio ´ n in vitro de Ipomoea batatas. Biotecnol Veg. 2002; 2(1):39–42. https://revista.ibp.co.cu/index.php/BV/rt/printerFriendly/128/html Spanish. 26. Jime ´ nez-Mariña L, Silva-Pupo JJ, Borges-Garcı ´a M, Fonseca-Arias M. Conservacio ´ n in vitro del cultivo de clavel español (Dianthus caryophyllus L.) a partir de sales minerales. Agron Mesoam. 2016; 27 (1):177–81. https://doi.org/org/10.15517/am.v27i1.2189 Spanish. 27. Ooi CT, Syahida A, Stanslas J, Maziah M. Efficiency of different Agrobacterium rhizogenes strains on hairy roots induction in Solanum mammosum. World J Microbiol Biotechnol. 2013; 29(3):421–30. https://doi.org/10.1007/s11274-012-1194-z PMID: 23090845 28. Thilip C, Raju SC, Varutharaju K, Aslam A, Shajahan A. Improved Agrobacterium rhizogenes-mediated hairy root culture system of Withania somnifera (L.) Dunal using sonication and heat treatment. 3 Bio- tech. 2015; 5(6):949–56. https://doi.org/10.1007/s13205-015-0297-2 PMID: 28324399 29. Chen G, Song F, Xiong X, Peng X. Fluorescent nanosensors based on fluorescence resonance energy transfer (FRET). Ind Eng Chem Res. 2013; 52:11228−45. https://doi.org/10.1021/ie303485n 30. Samadi A, Jafari M, Nejhad NM, Hossenian F. Podophyllotoxin and 6-methoxy podophyllotoxin produc- tion in hairy root cultures of Linum mucronatum ssp. Mucronatum. Pharmacogn Mag. 2014; 10 (38):154–60. https://doi.org/10.4103/0973-1296.131027 PMID: 24914281 31. Vasilev NP, Ionkova I. Cytotoxic activity of extracts from Linum cell cultures. Fitoterapia 2005; 76:50–3. https://doi.org/10.1016/j.fitote.2004.10.008 PMID: 15664462 32. Gang RD, Kasahara H, Zhi-Qiang X, Mijnsbrugge VK, Bauw G, Boerjan W, et al. Evolution of plant defense mechanisms relationships of phenylcoumaran benzylic ether reductases to pinoresinol-laricire- sinol and isoflavone reductases. J Biol Chem. 1999; 274(11):7516–27. https://doi.org/10.1074/jbc.274. 11.7516 PMID: 10066819 33. Dixon RA, Achnine L, Kota P, Chang-Jun L, Srinivasa RMS, Liangjang W. The phenylpropanoid path- way and plant defense—a genomics perspective. Molec Plant Pathol. 2002; 3(5):371–90. https://doi. org/10.1046/j.1364-3703.2002.00131.x PMID: 20569344 34. Hano C, Addi M, Bensaddek L, Cro ˆ nier D, Baltora-Rosset S, Doussot J, et al. Differential accumulation of monolignol-derived compounds in elicited flax (Linum usitatissimum) cell suspension cultures. Planta. 2006; 223(5):975–89. http://www.jstor.org/stable/23389388. https://doi.org/10.1007/s00425- 005-0156-1 PMID: 16292660 35. Boubakri H, Poutaraud A, Ali WM, Clayeux C, Baltenweck-Guyot R, Steyer D, et al. Thiamine modu- lates metabolism of the phenylpropanoid pathway leading to enhanced resistance to Plasmopara viti- cola in grapevine. BMC Plant Biol 2013; 13:31. https://doi.org/10.1186/1471-2229-13-31 PMID: 36. Abdel-Monaim MF. Role of riboflavin and thiamine in induced resistance against charcoal rot disease of soybean. Afr J Biotechnol. 2011; 10(53):10842–55. https://doi.org/10.5897/AJB11.253 37. Ahn IP, Kim S, Lee YH. Vitamin B1 functions as an activator of plant disease resistance. Plant Physiol. 2005; 138:1505–15. https://doi.org/10.1104/pp.104.058693 PMID: 15980201 PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 19 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin 38. Zhou J, Sun A, Xing D. Modulation of cellular redox status by thiamine-activated NADPH oxidase con- fers Arabidopsis resistance to Sclerotinia sclerotiorum. J Exp Bot. 2013; 64(11):3261–72. https://doi. org/10.1093/jxb/ert166 PMID: 23814275 39. Nader BL, Cardoso-Taketa AT, Pereda-Miranda R, Villarreal ML. Production of triterpenoids in liquid- cultivated hairy roots of Galphimia glauca. Planta Med. 2006; 72:842–4. https://doi.org/10.1055/s-2006- 931601 PMID: 16732534 40. Caspeta L, Nieto I, Zamilpa A, Alvarez L, Quintero R, Villarreal ML. Solanum chrysotrichum hairy root cultures: characterization, scale-up and production of five antifungal saponins for human use. Planta Med. 2005; 71:1084–7. https://doi.org/10.1055/s-2005-873170 PMID: 16320218 41. Seidel V, Windho ¨ vel J, Eaton G, Alfermann AW, Arroo RRJ, Medarde M, et al. Biosynthesis of podo- phyllotoxin in Linum album cell cultures. Planta. 2002; 215(6):1031–9. https://doi.org/10.1007/s00425- 002-0834-1 PMID: 12355164 42. Chattopadhyay S, Srivastava A, Bhojwani S, Bisaria VS. Development of suspension culture of Podo- phyllum hexandrum for production of podophyllotoxin. Biotechnol Lett. 2001; 23(24):2063–6. https://doi. org/10.1023/A:1013704116860 43. Hammouri MK, Ereifej KI, Shibli RA, Al-Karaki GN. Quantitative analysis of fructose fate in a plant fer- mentation system. J Agric Food Chem. 1998; 46(4):1428−32. https://doi.org/10.1021/jf970437z 44. Shimon-Kerner N, Mills D, Merchuk JC. Sugar utilization and invertase activity in hairy-root and cell-sus- pension cultures of Symphytum officinale. Plant Cell Tissue Organ Cult. 2000; 62(2):89–94. 45. Mohagheghzadeh A, Hemmati S, Alfermann AW. Quantification of aryltetralin lignans in Linum album organs and in vitro cultures. Iran J Pharm Sci. 2006: 2(1):47–56. www.ijps.ir/article_1909_451.html. PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 20 / 20 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png PLoS ONE Public Library of Science (PLoS) Journal

Improving the production of podophyllotoxin in hairy roots of Hyptis suaveolens induced from regenerated plantlets

PLoS ONE, Volume 14 (9) – Sep 12, 2019

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Copyright: © 2019 Bazaldúa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper. Funding: This work was supported from a grant from CONACYT Ciencia Basica (222714) to MLV. The funder had no role in study design,data collecton and analysis,decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.
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Abstract

Ten Hyptis suaveolens hairy root lines were established by infecting nodal explants with OPENACCESS K599+pGus-GFP+ and ATCC15834+pTDT strains from Agrobacterium rhizogenes. Genetic transformation was confirmed by epifluorescence and plagiotropic hairy root growth Citation: Bazaldua C, Cardoso-Taketa A, Trejo- Tapia G, Camacho-Diaz B, Arellano J, Ventura- in absence of growth regulators. Cytotoxicity was determined using the sulforhodamine B Zapata E, et al. (2019) Improving the production of method, and the production of podophyllotoxin (PTOX) was measured by high performance podophyllotoxin in hairy roots of Hyptis suaveolens thin layer chromatography scanning. Through these methodologies, HsTD10 was identified induced from regenerated plantlets. PLoS ONE 14 as the hairy root line with the highest cytotoxicity and PTOX production, which was corrobo- (9): e0222464. https://doi.org/10.1371/journal. pone.0222464 rated by liquid chromatography-mass spectrometry and micrOTOF-Q II. A suspension cul- ture of HsTD10 was established in which PTOX and carbohydrate consumption during Editor: Jen-Tsung Chen, National University of Kaohsiung, TAIWAN growth kinetics were quantified by high-performance liquid chromatography. Procedures to Received: June 19, 2019 increase the production and retrieval of PTOX in the HsTD10 line included selection of cul- ture medium, addition of thiamine, and modification of the PTOX extraction method. The Accepted: August 30, 2019 best combination of these variables was MS medium at 75% of its components with the Published: September 12, 2019 -1 addition of 2 mg L of thiamine, extraction with methanol-dichloromethane, and sonication Copyright:© 2019 Bazaldu ´a et al. This is an open at 40± 5˚C. During kinetics, growth-associated PTOX accumulation was observed. The access article distributed under the terms of the -1 specific growth rate (μ) was 0.11 d . The highest concentration of PTOX obtained with Creative Commons Attribution License, which -1 permits unrestricted use, distribution, and HsTD10 (5.6 mg g DW) was 100 times higher than that reported for roots of wild plants reproduction in any medium, provided the original and 56 times higher than that for in vitro nontransformed roots of H. suaveolens. author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This work was supported from a grant from CONACYT Ciencia Basica (222714) to MLV. Introduction The funder had no role in study design,data collecton and analysis,decision to publish, or Podophyllotoxin (PTOX) is a natural 2,7’-cyclolignan used to obtain the semisynthetic deriva- preparation of the manuscript. tives Etoposide1, Teniposide1 and Etopophos1, which are medicines widely used in cancer chemotherapy. The main source of PTOX is the speciesPodophyllumhexandrum [1], a wild Competing interests: The authors have declared that no competing interests exist. plant native to the Himalayas, which has been overexploited and is currently endangered [2]. PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 1 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin Annual PTOX production is 50–80 tons per year; however, demand exceeds 100 tons per year [3]. Chemical synthesis of PTOX is highly difficult and expensive due to the complexity of its molecular structure. The search for new natural sources that produce PTOX is mandatory, as is research for biotechnological alternatives, to achieve stable and controllable production of this compound [4]. Today, the most important PTOX producer species arePodophyllumhex- andrum,Juniperusbermudiana, andPodophyllumpeltatum, which produce 43, 22.6, and 4.7 -1 mg g dry weight (DW) of PTOX, respectively [1, 5, 6]. -1 The highest concentration of PTOX obtained frominvitro plant cultures (8.0 mg g DW) was reported in cell suspension cultures from the selected line Herm A ofLinumalbum [7], whereas the second and third were obtained from cell suspension cultures ofPodophyllumhex- -1 andrum andP.peltatum of 7.9 and 5.9 mg g DW, respectively [8, 9]. A high level of produc- tion of PTOX has been reported in hairy root cultures ofLinumalbum andL.flavum at -1 concentrations of 3.5 to 15 mg g DW [10–13]. Hyptissuaveolens is a wild shrub native to Mexico that was employed throughout history, mainly for the treatment of gastric and bile problems, cancer, fever, spasms, cough, malaria, yellow fever and other diseases [14]. As we previously reported, this plant produces PTOX [15], and its roots growinginvitro accumulate more PTOX than wild roots (0.57 vs 0.05 mg g DW, respectively) [16]. We also ascertained that the hairy roots of this plant, growing in sus- pension cultures in Gamborg’s B5 culture medium without hormones, produce PTOX or its analogs [17]. The aim of this research was to establish and select hairy root culture lines ofHyptissuaveo- lens, producing PTOX, as well as optimizing culture conditions to increase PTOX accumula- tion. This research demonstrates that the most effective factors for determining a good yield of -1 PTOX were the culture medium MS at 75% of its components, amended with 2 mg L of thia- mine. Similarly, sonication at 40 ± 5˚C using methanol-dichloromethane for extraction increased PTOX yield. The accumulation of PTOX in the hairy root suspension cultures ofH. suaveolens was growth-associated. Materials and methods Plant material Plants and seeds ofHyptissuaveolens were collected in Merida, Yucatan, Mexico, in 2006 and authenticated by German Carnevalli, Scientific Research Center of Yucatan Herbarium. A specimen of the plant was deposited (voucher number CICY 7086). The seeds were carefully washed under running tap water for 10 min and surface-sterilized by immersing in ethanol at 70% (v/v) for 1 min and in sodium hypochlorite at 1.5% (v/v) for 5 min. The sterilized seeds were immediately rinsed three times with sterile distilled water for 5 min. Subsequently, the seeds were placed in glass vessels containing Murashige and Skoog medium (MS, Sigma) [18], -1 amended with sucrose 3% (w/v), and 2.5 g L water-phytagel for germination. The plantlets were maintained until they were 5 cm in length in a growth chamber at 24 ± 2˚C with a photo- period of 16/8 h light/darkness. Light sources were cool white lamps of 40 watts. Obtaining and multiplying regenerated plantlets Theinvitro multiplication of plantlets was achieved by placing nodal segment explants mea- suring 3 cm in length under the same conditions described previously. During plantlet regen- eration, three concentrations were evaluated: three-quarters strength (TQS), half strength (HS), and full strength (FS) of MS medium. Plantlet height and root length were registered. PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 2 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin Induction and selection of hairy roots Root induction was performed by infection of nodal explants with two strains ofAgrobacter- iumrhizogenes, K599+pGus-GFP+ (facilitated by Dr. Federico Sanchez†, IBT-UNAM), and ATCC15834+pTDT, obtained in a previous work [17]. One hundred forty-four putative hairy roots were placed in Petri dishes with TQS MSB5 medium (M0404-Sigma), which contains the same amount of inorganic salts and myo-inositol as MS medium, but it is supplemented with -1 vitamins, as described by Gamborg (B5 medium): nicotinic acid (1 mg L ), piridoxine (1 mg -1 -1 L ), and thiamine (10 mg L ). This medium was supplemented with sucrose (3%, w/v) and -1 phytagel (2.5 g L ) without hormones. Putative hairy roots were maintained under these con- ditions for 45 days. From these cultures, ten hairy root lines were selected based on their fast autonomous growth, hairy high branched appearance and plagiotropic growth to corroborate their genetic transformation, cytotoxic activity, and PTOX production. These selected hairy root lines (HR- lines) were transferred to TQS MSB5 liquid culture medium and kept at constant agitation of 110 rpm under the same light, temperature and humidity conditions described for plantlet growth. Genetic transformation confirmation. To observe the fluorescence, 1 cm of the roots was cut and directly placed on microscope slides. An epifluorescence microscope with filters for blue and green light (460–490 nm, and 500–550 nm) (Nikon, Eclipse 80i, Tokyo, Japan) with an adapted camera (3CCD MTI, DC330, Michigan, USA) made it possible to corroborate hairy root transformation byAgrobacterium strains expressing the fluorescent green protein (GFP) or the tomato deaminase threonine protein (TDT- red fluorescence). PTOX extraction Biomasses of ten initially tested HR lines were filter-washed, oven dried at 60˚C for 72 h, finely ground, sieved through a 53-micron mesh, and extracted. Three methods of extraction were tested: 1) methanol-dichloromethane (MD-method) [19]: briefly, 2 mL of 80% (v/v) methanol was added to 100 mg of biomass dry weight (DW) and sonicated for 1 h, 4 mL of dichloro- methane and 4 mL of water were added and vortexed, and the organic phase was finally recov- ered and dried; 2) chloroformic method: 10 mL of 100% chloroform was added to 100 mg of biomass DW, stirred for 1 h, and dried; and 3) modified MD method, where the modification of the MD method [19] involved sonication at 40 ± 5˚C. All extracts were solubilized in HPLC grade MeOH and syringe-filtered (0.45 μm, Millex-Hv, Millipore) for PTOX identification through HPTLC, HPLC or micrOTOF QII. Identification of PTOX utilizing HPTLC-Scan Initially, PTOX was identified using the corresponding standard (Sigma) through high perfor- mance thin layer chromatography (HPTLC-Scanner TLC-3 CAMAG), as described in 2017 by Kamal et al. [20]. The retention factor from standard (PTOX Sigma) spot was compared with those spots from the extracts obtained through the MD and chloroformic methods. HPTLC scanning of these spots complemented the results. Cytotoxic evaluation To evaluate cytotoxicity, two types of extracts obtained through the MD and chloroformic methods from ten HR lines were tested. The cytotoxicity of crude extracts was determined using the sulforhodamine B method (SRB) [21] by quantifying the half-maximal inhibitory concentration (IC ) on four human carcinoma cell lines obtained from ATCC (HF6 = Colon, PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 3 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin MCF7 = Breast, PC3 = Prostate, and SiHa = Uterus). To continue exploring alternatives for increasing PTOX production, the most cytotoxic HsTD10 HR-line was selected, as established -1 by the US NCI, a plant-screening program for crude extracts (IC values less than 20 μg mL ) [22]. The positive controls were standards of etoposide and PTOX (Sigma). Identification of PTOX using HPLC-MS and MicrOTOF-Q II in the HsTD10 HR-line HPLC-MS. To quantify PTOX and to include information obtained from previous experi- ments, extracts were obtained through the chloroformic and modified MD methods. The presence of PTOX in the extracts was verified by HPLC-MS (negative mode). The retention time of the peak, UV absorption spectrum and m/z 413 molecular ion of the stan- dard PTOX (Sigma) were compared with those recorded for extracts from the HsTD10 selected HR-line. The HPLC-MS system (Shimadzu, Tokyo, Japan) was equipped with a sys- tem controller CBM-20 , 2 binary pumps LC-20AD, degasser DGU-20A3, autosampler SIL- 20AC, column oven CTO20A, UV-Vis diodes detector SPD-M20A, mass spectrometer (LCMS-2020), ionization source with electro spray (ESI), software version 5a. Dry gas (N2): 10 -1 -1 L min was employed. Also, scan m/z 330–440 nm, nebulizer gas flow 1.54 L min was employed. A Zorbax- Eclipse Plus 4.8 x 150 mm, 5 μm, RP C-18 column was connected to a -1 guard column. The injection volume was 10 μL, and a flow of 0.8 mL min at 25˚C was employed. An isocratic elution of the mobile phase consisting of methanol-acetonitrile-water- acetic acid (20:30:50:0.1) was used. MicrOTOF-Q II and competitive fragmentation modeling for metabolite identification platform (CFM-ID). The identification of PTOX in the HsTD10 HR-line extract was corrob- orated by micrOTOF- Q II 10392 by obtaining the ion m/z 415 (positive mode) of the standard PTOX (Sigma) and of the extract. The MS/MS partitioning profile of the m/z 415 and m/z 437 corresponded to PTOX-Na adduct (Na monoisotopic weight 23). The system was equipped with a micrOTOF-Q II 10392 (Bruker Daltonik, Germany), electrospray ionization source, set nebulizer: 0.4 bar, and drying oven at 180˚C. Analysis was performed at 50–3000 m/z, capillary -1 set 4500 V—500 V, and set of dry gas 4.0 L min . The predictive structures of the MS/MS par- titioning profile were established utilizing the CFM-ID platform from Wishart-lab (http:// cfmid3.wishartlab.com), which is referred to in the pubchem-ncbi site [23]. Modifying culture conditions for PTOX accumulation in HsTD10 HR-line Culture medium and method of extraction. The selected HsTD10 HR-line was cultured on three different media (MS, B5, and MSB5) at TQS for 30 days to evaluate root growth and PTOX production. Based on the results, the medium selected to increase PTOX production was MS at TQS, and the extraction was performed using the modified MD method (sonication at 40 ± 5˚C). Effect of thiamine addition. The effect of six different doses of thiamine (0.5, 1, 2, 4, 8, -1 and 10 mg L ) added to TQS MS medium was evaluated. Kinetic studies Growth and metabolite production was evaluated using Erlenmeyer flasks of 125 mL contain- -1 ing 30 mL of culture medium (MS amended with 2 mg L of thiamine and supplemented with -1 sucrose at 3%, w/v), where 1.3 g L (DW) of the HR-line HsTD10 were inoculated and then placed under constant agitation at 110 rpm. The following variables were measured every four days (three replicates) for 48 days: dry weight (fresh weight dried at 60˚C for 72 h), pH, PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 4 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin conductivity (portable conductivity meter HM digital model AP2 precision ± 2%, resolution 1 μS), medium carbohydrates and PTOX production. Quantification of medium carbohydrates and PTOX was performed by HPLC. Duplication time was obtained by applying the formula TD = LN (2)/ μ, where “μ” is the slope grade from LN (final dry weight (DWf)–initial dry weight (DWi)/ final time (Tf)–initial time (Ti)). The specific rate growth (μ) is defined by μ = LN(2)/TD. Quantification of PTOX. This analysis was performed by the calibration of a Waters con- troller 600 HPLC-DAD analysis (Waters. Millipore Corp., Waters Chromatography Division, Milford, Ma, USA). The HPLC system was equipped with a Waters 600E multisolvent delivery system with a Waters W996 diode array detector, autosampler (Waters 717 Plus), and Software Millenium 32 (Waters). The analytical HPLC separations were carried out on a Zorbax eclipse RP C18 (150 mm x 46 mm x 5 μm) column. The mobile phase consisted of an isocratic elution -1 with methanol-acetonitrile-water (20:30:50) and a flow rate of 0.8 mL min . The standard PTOX was acquired from Sigma. Carbohydrate quantification. To evaluate the consumption of carbohydrates during kinetics, three carbohydrate standards (sucrose, fructose, and glucose from Sigma) were uti- lized. Two milliliters of the culture medium were collected every four days for 48 days and fro- zen until use. Samples of culture medium were diluted at 6% (v/v) with HPLC grade water and then syringe-filtered (0.45 μm, Millex-Hv, Millipore). Subsequently, 10 μL of the dilution was injected directly into the HPLC column. The calibration curve and retention time of the peak from each standard were established by HPLC (Waters, Millipore Corp., Waters Chromatog- raphy Division, Milford, Ma, USA). The HPLC system was equipped with a Waters 600E mul- tisolvent delivery system and a refractive index detector (Waters 2414). The analytical HPLC separations were performed using a CAPCELL PAK UG NH2 (150 mm x 46 mm x 5 μm) col- umn (Shiseido, Japan). The mobile phase consisted of an isocratic elution with acetonitrile- -1 water (75:25) at a flow rate of 0.7 mL min . Statistical analysis Results analyses were performed using ANOVA and posttest HSD of Tukey with a p < 0.01 by the VassarStats- online, Website for Statistical Computation. Results In vitro regeneration and effect of medium concentration on Hyptis suaveolens plantlets The concentration of MS [18] culture medium significantly influenced the budding and length of the regenerated plantlet roots, which grew faster on TQS MS (MS 5519 Sigma). Regarding the height of plantlets regenerated in full strength MS and those regenerated on TQS MS medium, significant differences (p<0.01) were observed after 20 days. Nevertheless, root sprouting and root length were significantly different (p<0.01) from day 10 onwards (Table 1). Induction of hairy roots and genetic transformation efficiency In response toAgrobacterium infection (strains K599+pGus-GFP+ and ATCC15834+pTDT), putative hairy roots sprouted after 8–12 days in culture. In total, 144 putative hairy roots were induced; 15 days after infection, the roots were excised and placed in Petri dishes (five to six per Petri dish) containing hormone-free MSB5 culture medium (M0404 Sigma). Ten induced roots that grew faster were individually transferred to Petri dishes. The roots showed the char- acteristic hairy root phenotype of theAgrobacteriumrhizogenes-transformed roots and high PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 5 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin Table 1. Effect of MS culture medium concentration on the growth of Hyptis suaveolens plantlets. Plantlets height (cm) Root length (cm) MS medium concentration 1 2 3 1 2 3 Days HS TQS FS HS TQS FS 10 0.18 ± 0.08 a 0.82 ± 0.15 a 0.75 ± 0.14 a 0 0.42 ± 0.1 a 0 15 0.48 ± 0.09 a 1.69 ± 0.25 a 1.59 ± 0.22 a 0 1.09 ± 0.19 a 0 20 0.87 ± 0.13 c 3.15 ± 0.24 a 2.66 ± 0.27 b 0 2.08 ± 0.23 a 0 25 1.22 ± 0.15 c 4.84 ± 0.18 a 4.07 ± 0.31 b 0 3.01 ± 0.24 a 0.56 ± 0.08 b 30 2.06 ± 0.19 c 6.63 ± 0.33 a 5.86 ± 0.37 b 0.37 ± 0.008 c 5.95 ± 0.4 a 1.23 ± 0.09 b Half strength Three quarters strength Full strength. ANOVA and HSD Tukey test post-hoc analysis. Different letter in the same line indicates statistically significant differences between treatments (mean ± SD, N = 10, Significance level was fixed at p<0.01. https://doi.org/10.1371/journal.pone.0222464.t001 branched, rapid and plagiotropic growth, which was observed in both semisolid (Fig 1 HR- lines 1a – 10a) and liquid culture medium (Fig 1 HR-lines 1b – 10b). The efficiency of transformation obtained with the ATCC15834+pTDT strain was 60% and with the K599+pGus-GFP+ strain was 46.7% (Table 2). One hundred forty-four putatively transformed roots were obtained, and ten HR lines with optimum growth were selected and further subjected to cytotoxic evaluation and PTOX identification by HPTLC. Green or red epifluorescence was utilized to corroborate the genetic transformation of hairy roots. Four HR-lines expressed the green fluorescent protein (HsGF1 –HsGF4), and six expressed the threonine deaminase of tomato protein (HsTD5 –HsTD10), which fluoresces in red (Fig 2). Extraction and identification of PTOX utilizing HPTLC-Scan The first extraction to identify PTOX included the use of both chloroformic and MD methods [19]. For the identification of PTOX by high performance thin layer chromatography (HPTLC) [20] of the ten HR lines, we considered the retention factor (Rf = 0.56) developed by the PTOX standard (Sigma). The displayed Rf = 0.53 of HsTD10 HR-line extracts was similar to the standard (Fig 3). The HPTLC profiling of HsTD10 HR-line extract visualized at 254 nm (Fig 3A and 3E) for the plate cerium sulfate-revealed and observed at 366 nm (Fig 3B and 3F), and we observed with white light (Fig 3C and 3G), indicated a very close RF with the PTOX standard (lane “s”). The identity of PTOX in the extracts was confirmed by the UV spectra pro- filing, generated by the Scanner–TLC 3-CAMAG) observed in Fig 3D (MeOH-CH Cl -extrac- 2 2 tion) and 3h (CHCl - extraction), displaying a clear result in the last one. Cytotoxic evaluation Both extracts from the ten HR lines were evaluated for cytotoxicity using the sulforhodamine (SRB) method. Cytotoxic evaluation showed that all of the extracts were cytotoxic for at least one carcinoma cell line. Depending on the extract, the HR-lines showed differential cyto- -1 toxic activity (Table 3). Some IC values were lower than 4 μg mL , a threshold value reported for active pure compounds [22]. The chloroformic and methanol-dichloromethane extracts from the HsTD10 HR-line showed the highest cytotoxic activity in three (SIHa, PC3, and MCF7) out of four carcinoma cell lines evaluated, with IC values ranging between 1.8 and -1 16.6 μg mL ; therefore, this line was selected for continued research exploring and improving PTOX accumulation. PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 6 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin Fig 1. Ten selected HR lines. Individualized in solid MSB5 culture medium (1a – 10a) and in liquid MSB5 liquid culture medium (1b – 10b) without hormones. https://doi.org/10.1371/journal.pone.0222464.g001 Identification of PTOX using HPLC-MS and MicrOTOF-Q II in the HsTD10 HR-line HPLC-MS. The identification of PTOX in the HsTD10 HR-line was also carried out by HPLC-MS. Fig 4 shows the chromatogram of the m/z 413 that corresponds to PTOX in a nega- tive mode. MicrOTOF-Q II. When the identification was performed by micrOTOF-Q II, there was overwhelming evidence that the selected HR-line produces PTOX, as the molecular ion m/z 415 corresponding to PTOX (positive mode), the molecular ion m/z 437 corresponding to the adduct PTOX-Na and the partitioning profile of m/z 415, were observed (Fig 5). Competitive fragmentation modeling for metabolite identification (CFM-ID) plat- form. To correlate the observed peaks in the partitioning profile obtained by MS/MS from Table 2. Number of induced and selected HR-lines. Strain Number of explants Explants with roots sprout Number of roots induced Number of selected lines K599+GFP-GUS+ 15 7 34 4 ATCC15834+pTDT 40 24 110 6 https://doi.org/10.1371/journal.pone.0222464.t002 PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 7 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin Fig 2. Confirmation of genetic transformation. Genetic transformation was corroborated by epifluorescence of induced hairy roots of Hyptissuaveolens with the strain K599+pGus-GFP+ (a, and b) and the strain ATCC15834+pTDT (c, and d). Observed in darkness and illuminated with blue (a), green (c), and white lights (b, d, and f). When nontransformed roots were illuminated with blue or green light, epifluorescence was not observed (e). All microphotographs are 20X. https://doi.org/10.1371/journal.pone.0222464.g002 m/z 415 with predictive structures, the CFM-ID (http://cfmid3.wishartlab.com) platform was utilized, showing resulting ionized structures (Fig 6). Modification of culture conditions and yield of PTOX in the HsTD10 HR line Effect of culture medium and of the method of extraction on yield of PTOX. The results showing PTOX accumulation in the HsTD10 HR line at 30 days of culture grown in PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 8 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin Fig 3. Identification of PTOX by HPTLC-Scan. Lane 10 indicates the HsTD10 HR-line extract, and the standard (POTX, Sigma) is referred to as letter “s”. The profiling of the different solvent extractions MeOH-CH Cl (a, b, and c) 2 2 and CHCl (e, f, and g) are presented. Developed-plate visualizations at 254 nm (a, and e), cerium sulfate revealed- plate at 366 nm (b, and f), and white light (c, and g) are shown. Absorbance spectra from the spots of PTOX in both extracts (d, and h) are shown. https://doi.org/10.1371/journal.pone.0222464.g003 MS, MSB5, and B5, all at TQS, are presented in Table 4. When the extraction of PTOX was performed using chloroform, a higher accumulation of this lignan was obtained from roots -1 Table 3. Cytotoxicity (IC μg mL ) of ten selected HR-lines of Hyptis suaveolens. HR-Lines Methanol–Dichloromethane extracts Chloroformic extracts � -1 Carcinoma cell lines (IC50 μg mL ) HF6 SIHa PC3 MCF7 HF6 SIHa PC3 MCF7 HsGF1 5.9 ± 0.3 >20 >20 4.2 ± 0.1 6.4 ± 0.5 17.4 ± 0.9 >20 5.0 ± 0.3 HsGF2 6.7 ± 0.1 >20 >20 4.1 ± 0.4 3.2 ± 0.2 >20 >20 5.2 ± 0.4 HsGF3 7.1 ± 0.3 >20 >20 4.6 ± 0.3 12.1 ± 0.3 6.6 ± 0.7 >20 6.2 ± 0.5 HsGF4 11.7 ± 0.2 >20 >20 3.8 ± 0.2 10.9 ± 0.8 6.1 ± 0.2 >20 4.0 ± 0.3 HsTD5 5.5 ± 0.5 >20 >20 8.9 ± 0.6 15.7 ± 0.7 6.3 ± 0.1 >20 7.4 ± 0.6 HsTD6 7.7 ± 0.6 12.2 ± 0.5 >20 6.3 ± 0.8 14.2 ± 0.9 16.4 ± 0.2 >20 7.0 ± 0.8 HsTD7 2.8 ± 0.1 13.5 ± 0.3 >20 9.6 ± 0.5 5.1 ± 0.2 9.0 ± 0.4 18.5 ± 0.8 3.5 ± 0.4 HsTD8 18.1 ± 0.5 >20 >20 >20 >20 9.69 ± 0.6 >20 5.5 ± 0.3 HsTD9 9.1 ± 0.2 16.8 ± 0.7 >20 4.9 ± 0.2 9.1 ± 0.7 4.1 ± 0.2 16.2 ± 0.7 2.9 ± 0.7 HsTD10 5.9 ± 0.2 7.1 ± 0.4 16.6 ± 0.7 3.3 ± 0.2 8.2 ± 0.4 1.8 ± 0.1 6.68 ± 0.3 2.5 ± 0.2 PTOX 0.011 1.66E-03 1.2 ± 0.01 4.23E-6 0.011 1.66E-03 1.2 ± 0.01 4.23E-6 Etoposide 1.8 ± 0.1 0.35 0.99 0.03 1.8 ± 0.1 0.35 0.99 0.03 Carcinoma cell lines: HF6 = Colon, SiHa = Uterine, PC3 = Prostate, MCF7 = Breast. The positive controls utilized were PTOX and Etoposide. https://doi.org/10.1371/journal.pone.0222464.t003 PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 9 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin Fig 4. Identification of PTOX by HPLC-MS. (a) PTOX standard (Sigma) showing 1) molecular ion m/z 413 in negative mode, 2) retention time of the peak (7.3 min), and 3) UV spectrum absorption; (b) HsTD10 HR-line 1) molecular ion m/z 413 corresponding to PTOX in negative mode, 2) retention time of the peak at 7.3 min, and 3) UV spectrum similar to PTOX. https://doi.org/10.1371/journal.pone.0222464.g004 PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 10 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 11 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin Fig 5. Identification of podophyllotoxin by micrOTOF-Q II in the HsTD10 HR-line. (a) spectrum of the extract showing the PTOX-Na and PTOX-K adducts, (b) partitioning profile (MS/MS) from molecular ion m/z 415 corresponding to PTOX (positive mode). https://doi.org/10.1371/journal.pone.0222464.g005 grown on MS medium followed by those grown on MSB5 and B5 media. However, when the extraction was undertaken using the modified MD method, the yield of PTOX increased con- siderably. Nevertheless, in this experiment, the best result was recorded in MSB5 followed by B5 and MS (Table 4). Effect of thiamine addition to TQS MS culture medium on modulating the yield of PTOX in the HsTD10 HR-line. The results regarding the effect on PTOX yield of extra addition of thiamine at various concentrations to the MS culture medium (0.5, 1, 2, 4, 8 and 10 -1 mg L ) are shown (Fig 7). When extraction was performed with sonication at 40 ± 5˚C (Koul- man modified), PTOX accumulation increased by 34% (Fig 7 black columns). Growth kinetics and PTOX accumulation Measurements of conductivity during growth kinetics showed that as anticipated, this parame- ter declined in inverse proportion to biomass increase. The highest dry weight biomass was -1 achieved at day 40 (7.2 g L DW), and the exponential phase of growth of the selected HR-line -1 occurred from day 8 to day 28 with a mean specific growth rate (μ) of 0.11 d (Fig 8A). The production of PTOX was growth associated, observing a maximum accumulation of 5.4 and -1 5.6 mg g between days 32 and 36 (Fig 8B). Concerning carbohydrate consumption, sucrose concentration on the medium decreased at a constant rate until day 20, when it was completely hydrolyzed; in contrast, fructose and glucose initially increased up to day 20, and the Fig 6. Structures predicted by CFM-ID related to each peak of partitioning profile from m/z 415 ion. (a-d) structures for m/z 385, (e) structure for m/z 357, (f) structure for m/z 301, (g) structure for m/z 297, (h) structure for m/z 217. https://doi.org/10.1371/journal.pone.0222464.g006 PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 12 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin Table 4. Influence of culture medium and extraction method in PTOX yield by the HsTD10 HR-line. -1 Culture medium at TQS Concentration of PTOX (mg g DW) Extraction method Chloroform Koulman Koulman Modified MS 1.52 ± 0.011 a 1.99 ± 0.021 b 2.89 ± 0.035 b MSB5 0.63 ± 0.017 b 2.97 ± 0.027 a 4.50 ± 0.032 a B5 0.58 ± 0.006 b 2.20 ± 0.014 b 3.24 ± 0.032 b ANOVA and HSD Tukey test post-hoc analysis. Different letter in the same column indicates statistically significant differences between treatments (mean ± SD, N = 3, Significance level was fixed at p<0.01). https://doi.org/10.1371/journal.pone.0222464.t004 concentration of glucose began to diminish thereafter, while fructose concentration remained unchanged (Fig 8C). Discussion Effect of medium components on plantlet regeneration The best result for regenerated plantlets evaluated by height and root length was obtained with TQS MS medium. Regenerated plantlet height was similar for 15 days, but subsequently, sig- nificant differences (p<0.01) between all treatments emerged. In contrast, roots were sprout- ing on TQS MS at day 10, although when half-strength (HS) or full-strength (FS) MS were used, root sprouting occurred at 25 and 30 days, respectively. Likewise, sprouted roots in TQS MS at 30 days grew up to five to fifteen times the length compared to those grown at FS MS and HS MS, respectively. This result is similar to that reported in 2011 by Borges et al. [24] for the root length and plantlet height ofDiscoreaalata but different from that reported in 2002 by Espinosa et al. [25] and in 2016 by Jimenez-Mariña et al. [26], who obtained better results Fig 7. Effect of thiamine extra addition to TQS MS and of the extraction method in the production of PTOX by the HsTD10 HR- line at 30 days in culture. Koulman method (Gray) and modified Koulman method (Black) ANOVA, and HSD Tukey test post hoc analysis. Different letters indicate statistically significant differences between treatments (mean ± SD, N = 3, Significance level was fixed at p<0.01). https://doi.org/10.1371/journal.pone.0222464.g007 PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 13 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin Fig 8. Kinetic parameters from the HsTD10 HR line during 48 days of growth in TQS MS medium supplemented with thiamine (2 mg -1 L ). (a) Correlation between dry weight (DW) and conductivity, (b) relation between biomass and PTOX accumulation, and (c) relation between carbohydrate consumption and biomass. (■ conductivity, —▲— DW, —●— PTOX, ^ fructose, D glucose,□ sucrose). https://doi.org/10.1371/journal.pone.0222464.g008 with FS MS in terms of plantlet height forIpomeabatatas andDianthuscaryophyllus, respec- tively. There is a direct relationship between plantlet height and root length. Genetic transformation efficiency and its corroboration Transformation efficiency (defined as the number of responsive explants regarding the total number of infected explants) obtained in the present work using ATCC15834+pTDT (60%) and K599+pGus-GFP+ (46.7%) strains was greater than that obtained in 2013 by Ooi et al. [27] with the A4Agrobacterium strain infectingSolanummammosum (21.4%) but lower than that reported in 2015 by Thilip et al. [28], with the R1000Agrobacterium strain infectingWith- aniasomnifera (93.3%). The corroboration of transformation has traditionally been reported by Southern blotting or using PCR. However, in this work, the use of K599+pGus-GFP+ and ATCC15834+pTDT strains enabled confirmation of transformation by visual observation of fluorescence through an epifluorescence microscope. The most important advantage of this method is that it reduces the carbon footprint and does not damage the obtained transformed tissues. Moreover, this approach has been successfully utilized for the structural elucidation of biological molecules and their interactions, as well as forinvitro assays orinvivo monitoring cellular research [29]. Extraction and identification of PTOX utilizing HPTLC-Scan The identification of PTOX in wild roots ofH.suaveolens has been reported using high-perfor- mance liquid chromatography coelution [16] and liquid chromatography-masses (m/z 415, positive mode) [15]. Similarly, the adduct PTOX-Na (m/z 437) has also been reported in leaves ofJuniperusbermudiana [5]. In the present work, we used the methodology described by Kamal et al. [20], who reported the identification and quantification of PTOX using an HPTLC system. It was possible to confirm the presence of PTOX in the extracts by the devel- opment of spots with similar Rfs: 0.56 for the PTOX standard and 0.53 for the extracts. The PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 14 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin slight difference in Rfs can be explained because the extract is a complex matrix composed of many metabolites which, in turn, influence chromatographic development. Nevertheless, the unequivocal identity of PTOX in the extracts was also confirmed through spectroscopic (maxi- mum UV absorption) and spectrometric (LC-MS/MS) evidence. Cytotoxic evaluation All HR-lines were cytotoxic for at least one of the carcinoma cell lines utilized. Interestingly, -1 IC values were lower than 4 μg mL in five out of ten evaluated HR lines, which is propi- tious, as this value is a predicted value for a pure compound [22]. The HsTD10 HR-line was the most cytotoxic in three (SIHa, PC3, and MCF7) out of four carcinoma cell lines, and the one that showed activity against all the evaluated carcinoma cell lines. Identification of PTOX using HPLC-MS and MicrOTOF-Q II in the HsTD10 HR-line To corroborate the identification and quantification of PTOX in the extracts of the HsTD10 HR-line, we used HPLC-MS equipment in negative mode (m/z 413) because under our ioniza- tion conditions, the m/z 415 (positive mode) appeared to be notably low. The m/z 415 molecu- lar ion was identified with a micrOTOF-Q II mass spectrometer. This equipment permitted the identification of the adduct PTOX-Na (m/z 437), as reported in 2011 by Renouard et al. [5], and the partitioning profile during the MS/MS experiment of the m/z 415. This result is similar to several spectra available at the https://pubchem.ncbi.nlm. nih.gov/compound/podophyllotoxin#section=MS-MS site [23]. Seven of the eight predictive structures proposed as resulting from ionization of the PTOX molecule in this work can also be observed, following links (4.3.2. MS/MS spectrum 108322, 108323, 175510, and 175511). In both cases, prediction was carried out utilizing the Competitive Fragmentation Modeling for Metabolite Identification platform (CFM-ID) from Wishart-lab (http://cfmid3.wishartlab. com). Selection of culture conditions and yield of PTOX in the HsTD10 HR-line Under our conditions, the culture medium affected the concentration of PTOX, which was determined on the HsTD10 HR-line growing on three different media. It has been reported that the best culture medium for improving PTOX production depends on the plant species and the type ofinvitro cultivation employed. The influence of MS and B5 (Gamborg’s) culture media for biomass growth and PTOX accumulation on transformed cell suspension and hairy root cultures ofLinumalbum [11], as well as in hairy root cultures ofL.flavum, higher biomass accumulation and PTOX production were accomplished with B5 medium [13]. In contrast, MS medium has been utilized for the production of PTOX and MPTOX in adventitious root cultures ofPodophyllumpeltatum [9], hairy root cultures ofLinummucronatum ssp.mucrona- tum [30], and hairy root cultures ofLinumstrictum ssp.strictum [31]. Effect of culture medium and the extra addition of thiamine on PTOX accumulation. Considering the results described in this study, it was necessary to ascertain how much the cul- ture medium or the method of extraction affected the yield of PTOX in the HsTD10 HR-line. When the HsTD10 HR-line was established in three culture media and extraction was per- formed using chloroform, the best result was obtained in roots grown in MS. However, when the extraction was performed using the modified MD method, the best result was obtained from roots grown in MSB5 medium (7 times more PTOX than that obtained in chloroform extracts from the same line, growing in the same culture medium). The differences between MS and MSB5 culture medium pertain to the concentration of vitamins: 100 times more PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 15 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin thiamine and twice the amount of pyridoxine and nicotinic acid in the MSB5 medium. Then, the question was whether thiamine could modulate PTOX production. The addition of thia- mine to TQS MS medium increased the yield of PTOX in the HR-line. Our results showed that there was no significant difference in biomass production, but there was a clear difference in PTOX accumulation. The increase in PTOX production was -1 dose dependent on the thiamine concentration up to a maximum of 2 mg L ; at this point, production began to decrease with each succeeding increased dose of thiamine. To date, the relationship between the addition of thiamine to the culture medium and the increase in PTOX production has not been reported. Lignans are phenylpropanoid com- pounds that have been identified as plant-defenders [32–34]. Regarding the phenylpropanoid pathway, Boubakri et al. [35] reported that thiamine increased the expression of genes along this route in grape vines. This vitamin increased plant defense activity [36], induced systemic acquired resistance (SAR) inArabidopsis, rice, tobacco, and cucumber [37], and increased the concentration of callose and lignin inArabidopsis [38]. -1 As the accumulation of PTOX in the HsTD10 HR-line was dose-dependent up to 2 mg L of thiamine, and higher concentrations notably reduced the accumulation of PTOX, in future experiments, it would be interesting to evaluate enzyme activity in the cultures of this HR-line. The increase of thiamine in the HsTD10 HR-line ofHyptissuaveolens may activate a defense mechanism, possibly increasing the concentration of some phenylpropanoids but definitely that of PTOX. Effect of sonication at 40 ± 5˚C in PTOX extraction. Several reports have analyzed the effect of temperature on PTOX extraction. In 2002, Bedir et al. [6] reported greater PTOX extraction from leaves ofPodophyllumpeltatum and modified leaves and twigs ofJuniperus virginiana using hot methanol. However, in 2011, Renouard et al. [5] analyzed the effect of temperature but did not report any significant differences inJuniperusbermudiana or in 12 other species from the same genus; nevertheless, in these reports, sonication was not employed. It is important to note that the modified MD method was performed as described in 2003 by Koulman et al. [19] with one change, as instead of performing sonication for one hour at room temperature, we sonicated for one hour at 40 ± 5˚C. This change significantly increased PTOX extraction. In our work, it is clear that the culture medium and the extraction method were factors that determined the level of PTOX production. Growth kinetics The highest dry weight biomass obtained under our culture conditions at day 40 was 9.4 times higher than the inoculum. The specific growth rate of the HsTD10 HR-line suspension cultures -1 -1 (0.11 d ) is higher than that reported in 2006 by Nader et al. (0.08 d ) [39] but lower than that -1 reported in 2005 by Caspeta et al. (0.12 d ) [40] for hairy root cultures ofGalphimiaglauca and Solanumchrysotrichum, respectively. Moreover, the production of PTOX associated with growth observed in this work is similar to that observed in hairy roots and in cell suspension cultures ofL.album [11, 41], as well as in cell suspension cultures ofP.hexandrum [42]. In this work, the consumption of carbohydrates was selective for sucrose and glucose because fructose was not consumed. This result is similar to that reported in 1998 by Ham- mouri et al. [43], who found that the hairy roots ofBetavulgaris were unable to use fructose as a source of energy when using the same initial concentration of sucrose (3%). Nonetheless, this result differs from that reported in 2000 by Shimon-Kerner et al. [44] because they found similar levels of glucose and fructose in hairy root cultures ofSymphytumofficinale after 28 days in culture, a difference which is probably due to the time period of their kinetic experiment. PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 16 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin It is also important to note that the concentration of PTOX between days 32 and 36 at the -1 beginning of the stationary growth phase was the highest obtained (5.4 and 5.6 mg g , respectively). PTOX accumulation in wild plants and plant material obtained byinvitro cultures (cell, callus, tissues, plantlets, transformed tissues) show a different pattern. To date, the greatest -1 supply of PTOX has been obtained from the wild plantPodophyllumhexandrum (43 mg g DW) [1]; however, this yield diminishes when PTOX is obtained from cell suspension cultures -1 of this species (7.9 mg g DW) [8]. Similarly, the concentration of PTOX in wildPodophyllum -1 peltatum plants is similar to that obtained in cell suspension cultures (4.7 and 5.9 mg g DW, respectively) [6, 9]. The accumulation of PTOX inLinumalbum has been reported to be higher -1 -1 in cell suspension cultures (8 mg g DW) [7] than in wild roots of the same species (0.3 mg g DW) [45]. However, the highest accumulation of PTOX in this species was obtained from -1 hairy root extracts (15 mg g DW) [10]. The highest concentration of PTOX reported usinginvitro cultures was obtained in hairy -1 -1 root cultures ofLinumalbum (15 and 5.1 mg g DW) [10, 11] andL.flavum (4.5 mg g DW) -1 [13]. Under our conditions, the optimum yield obtained for the HsTD10 HR-line (5.6 mg g DW) is similar to that reported in 2008 by Baldi et al. [11] inL.album, which is 100 times higher than that obtained from roots of wild plants and 56 times higher than that obtained from nontransformed roots ofHyptissuaveolens growninvitro [16]. Conclusions Nodal explants ofHyptissuaveolens infected with K599+pGus-GFP+ and ATCC15834+pTDT strains fromAgrobacteriumrhizogenes were suitable explants for obtaining ten hairy root line cultures. The HsTD10 HR-line was the greatest cytotoxic and podophyllotoxin producer. The culture medium did not significantly affect biomass growth but was a determining factor for the accumulation of PTOX in the hairy roots ofH.suaveolens, an influence that was greatest when using MS at three-quarters strength. Thiamine modulated the production of PTOX, -1 obtaining the highest values when it was added at 2 L to MS medium. Sonication at 40 ± 5˚C using MD extraction increased PTOX extraction. PTOX accumulation in kinetic suspension culture ofH.suaveolens-hairy roots was growth-associated. The accumulation of PTOX in the HsTD10 HR-line was greater than that obtained from the roots of wild plants orinvitro non- transformed cultured roots. Acknowledgments C. Bazaldu ´ a acknowledge Instituto Polite ´cnico Nacional (IPN) for work partial licenses during his PhD research. We recognize Centro de Nanociencias y Micro y Nanotecnologı ´as (CNMN- IPN) for the experimental service with the micrOTOF-Q II spectrometer. We thank Dr. Feder- ico Sa ´nchez from Instituto de Biotecnologı ´a, (IBT-UNAM) for facilitating theAgrobacterium strain K599+pGus-GFP+, thanks also go to Ariadna Zenil for her help with the HPLC Waters. Thanks to Juan Manuel Solares for his help with the Scanner-TLC-3-CAMAG. Author Contributions Conceptualization: Crescencio Bazaldu ´ a. Formal analysis: Crescencio Bazaldua, Alexandre Cardoso-Taketa. Funding acquisition: Marı ´a Luisa Villarreal. ´ ´ Investigation: Crescencio Bazaldua, Alexandre Cardoso-Taketa, Marıa Luisa Villarreal. PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 17 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin Methodology: Crescencio Bazaldu ´ a, Alexandre Cardoso-Taketa, Gabriela Trejo-Tapia, Brenda Camacho-Diaz, Jesu ´ s Arellano, Elsa Ventura-Zapata. Resources: Gabriela Trejo-Tapia. Supervision: Marı ´a Luisa Villarreal. Validation: Alexandre Cardoso-Taketa. Writing – original draft: Crescencio Bazaldu ´ a, Marı ´a Luisa Villarreal. Writing – review & editing: Crescencio Bazaldu ´ a, Jesu ´ s Arellano, Marı ´a Luisa Villarreal. References 1. Giri A, Narasu ML. Production of podophyllotoxin from Podophyllum hexandrum: a potential natural product for clinically useful anticancer drugs. Cytotechnol. 2000; 34:17–26. https://doi.org/10.1023/ A:1008138230896 PMID: 19003377 2. Arroo RRJ, Alfermann AW, Medarde M, Petersen M, Pras N, Woolley JG. Plant cell factories as a source for anti-cancer lignans. Phytochem Rev. 2002; 1(1):27–35. https://doi.org/10.1023/ A:1015824000904 3. Alam A, Naik PK, Mishra GP. Congruence of RAPD and ISSR markers for evaluation of genomic rela- tionship among 28 populations of Podophyllum hexandrum Royle from Himachal Pradesh, India. Turk J Bot. 2009; 33:1–12. https://doi.org/10.3906/bot-0711-10 4. Moraes RM, Burandt Ch, Ganzera M, Li X, Khan I, Canel C. The American mayapple revisited–Podo- phyllum peltatum- still a potential cash crop? Econ Bot. 2000; 54(4):471–76. 5. Renouard S, Lopez T, Hendrawati O, Dupre P, Doussot J, Falguieres A, et al. Podophyllotoxin and deoxypodophyllotoxin in Juniperus bermudiana and 12 other Juniperus species: Optimization of extrac- tion, method validation, and quantification. J Agric Food Chem. 2011; 59:8101–07. https://doi.org/10. 1021/jf201410p PMID: 21702435 6. Bedir E, Khan I, Moraes RM. Bioprospecting for podophyllotoxin In: Trends in New Crops and New Uses; Janick J. and Whipkey A., ASHS Press; Alexandria, VA, USA. 2002 pp 545–49. ISBN 0- 970756-5-5. 7. Wink M, Alfermann AW, Franke R, Wetterauer B, Distl M, Windho ¨ vel J, et al. Sustainable bioproduction of phytochemicals by plant in vitro cultures: Anticancer agents. Plant Gen Res.2005; 3(2):90–100. https://doi.org/10.1079/PGR200575 8. Ahmad R, Sharma VK, Rai AK, Shivananda RD, Shivananda BG. Production of lignans in callus culture of Podophyllum hexandrum. Trop J Pharm Res. 2007; 6(4):803–08. www.bioline.org.br/pdf?pr07022. 9. Anbazhagan VR, Ahn CH, Harada E, Kim YS, Choi YE. Podophyllotoxin production via cell and adventi- tious root cultures of Podophyllum peltatum. In Vitro Cell Dev Biol-Plant. 2008; 44:494–501. https://doi. org/10.1007/s11627-008-9134-1 10. Farkya S, Bisaria VS. Exogenous hormones affecting morphology and biosynthetic potential of hairy root line (LYR2i) of Linum album. J Biosci Bioeng. 2008; 105(2):140–6. https://doi.org/10.1263/jbb.105. 140 PMID: 18343341 11. Baldi A, Srivastava AK, Bisaria VS. Improved podophyllotoxin production by transformed cultures of Linum album. Biotechnol J. 2008; 3:1256–63. https://doi.org/10.1002/biot.200800173 PMID: 18932162 12. Cong LH, Dauwe R, Lequart M, Vinchon S, Renouard S, Fliniaux O, et al. Kinetics of glucosylated and non-glucosylated aryltetralin lignans in Linum hairy root cultures. Phytochemistry. 2015; 115(1):70–8. https://doi.org/10.1016/J.PHYTOCHEM.2015.01.001 13. Renouard S, Corbin C, Drouet S, Medvedec B, Doussot J, Colas C, et al. Investigation of Linum flavum (L.) hairy root cultures for the production of anticancer aryltetralin lignans. Int J Mol Sci. 2018; 19 (4):990. https://doi.org/10.3390/ijms19040990 14. Prince SP, Ram RK, Anurag C, Dinesh G, Sharma VK. Hyptis suaveolens (L.) Poit.: A phyto pharmaco- logical review. Int J Chem Pharm Sci. 2013; 4:1–11. www.researchgate.net/publication/255982083. 15. Lautie ´ E, Quintero R, Fliniaux MA, Villarreal ML. Selection methodology with scoring system: Applica- tion to Mexican plants producing podophyllotoxin related lignans. J Ethnopharmacol. 2008; 120:402– 12. https://doi.org/10.1016/j.jep.2008.09.014 PMID: 18930796 16. Veloz RA, Cardoso-Taketa A, Villarreal ML. Production of podophyllotoxin from roots and plantlets of Hyptis suaveolens cultivated in vitro. Pharmacogn Res. 2013; 5(2):93–102. https://doi.org/10.4103/ 0974-8490.110538 PMID: 23798883 PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 18 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin 17. Bazaldu ´ a C, Cardoso-Taketa A, Arellano J, Camacho-Diaz B, Ventura-Zapata E, Villarreal ML. Podo- phyllotoxin-like lignans production through hairy roots of Hyptis suaveolens. J Chem Biol Phy Sci. 2014; 4(5):37–47. www.researchgate.net/publication/271827761. 18. Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 1962; 15:473–97. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x 19. Koulman A, Kubbinga ME, Batterman S, Woerdenbag HJ, Pras N, Woolley JG, et al. A phytochemical study of lignans in whole plants and cell suspension cultures of Anthriscus sylvestris. Planta Med. 2003; 69(8):733–8. https://doi.org/10.1055/s-2003-42776 PMID: 14531024 20. Kamal A, Singh M, Ahmad FJ, Saleem K, Ahmad S. A validated HPTLC method for the quantification of podophyllotoxin in Podophyllum hexandrum and etoposide in marketed formulation. Arab J Chem. 2017; 10:2539–46. https://doi.org/org/10.1016/j.arabjc.2013.09.027 21. Vanicha V, Kanyawim K. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat Protoc. 2006; 1(3):1112–16. https://doi.org/10.1038/nprot.2006.179 PMID: 17406391 22. John Boik. Natural compounds in cancer therapy. Oregon Medical Press, Princeton, MN, USA. 2001. 23. Pubchem.ncbi—Podophyllotoxin. Chemical and physical properties: Spectral properties, MS-MS. Avail- able online: https://pubchem.ncbi.nlm.nih.gov/compound/podophyllotoxin#section=MS-MS (accessed on 18 October 2018). 24. Borges GM, Destrade BR, Meneses RS, 5. Go ´ mez KR, Malaurie B, Hamon P, et al. Optimization of a culture medium for micropropagated plants of Dioscorea alata L. Rev Colomb Biotecnol. 2011; 13 (2):221–8. Spanish. 25. Espinosa RA, Salas HL, Gonza ´ lez PO, Silva PJJ. Empleo de a ´ cido abscı ´sico, manitol y la disminucio ´ n de la concentracio ´ n de las sales del medio de cultivo en la conservacio ´ n in vitro de Ipomoea batatas. Biotecnol Veg. 2002; 2(1):39–42. https://revista.ibp.co.cu/index.php/BV/rt/printerFriendly/128/html Spanish. 26. Jime ´ nez-Mariña L, Silva-Pupo JJ, Borges-Garcı ´a M, Fonseca-Arias M. Conservacio ´ n in vitro del cultivo de clavel español (Dianthus caryophyllus L.) a partir de sales minerales. Agron Mesoam. 2016; 27 (1):177–81. https://doi.org/org/10.15517/am.v27i1.2189 Spanish. 27. Ooi CT, Syahida A, Stanslas J, Maziah M. Efficiency of different Agrobacterium rhizogenes strains on hairy roots induction in Solanum mammosum. World J Microbiol Biotechnol. 2013; 29(3):421–30. https://doi.org/10.1007/s11274-012-1194-z PMID: 23090845 28. Thilip C, Raju SC, Varutharaju K, Aslam A, Shajahan A. Improved Agrobacterium rhizogenes-mediated hairy root culture system of Withania somnifera (L.) Dunal using sonication and heat treatment. 3 Bio- tech. 2015; 5(6):949–56. https://doi.org/10.1007/s13205-015-0297-2 PMID: 28324399 29. Chen G, Song F, Xiong X, Peng X. Fluorescent nanosensors based on fluorescence resonance energy transfer (FRET). Ind Eng Chem Res. 2013; 52:11228−45. https://doi.org/10.1021/ie303485n 30. Samadi A, Jafari M, Nejhad NM, Hossenian F. Podophyllotoxin and 6-methoxy podophyllotoxin produc- tion in hairy root cultures of Linum mucronatum ssp. Mucronatum. Pharmacogn Mag. 2014; 10 (38):154–60. https://doi.org/10.4103/0973-1296.131027 PMID: 24914281 31. Vasilev NP, Ionkova I. Cytotoxic activity of extracts from Linum cell cultures. Fitoterapia 2005; 76:50–3. https://doi.org/10.1016/j.fitote.2004.10.008 PMID: 15664462 32. Gang RD, Kasahara H, Zhi-Qiang X, Mijnsbrugge VK, Bauw G, Boerjan W, et al. Evolution of plant defense mechanisms relationships of phenylcoumaran benzylic ether reductases to pinoresinol-laricire- sinol and isoflavone reductases. J Biol Chem. 1999; 274(11):7516–27. https://doi.org/10.1074/jbc.274. 11.7516 PMID: 10066819 33. Dixon RA, Achnine L, Kota P, Chang-Jun L, Srinivasa RMS, Liangjang W. The phenylpropanoid path- way and plant defense—a genomics perspective. Molec Plant Pathol. 2002; 3(5):371–90. https://doi. org/10.1046/j.1364-3703.2002.00131.x PMID: 20569344 34. Hano C, Addi M, Bensaddek L, Cro ˆ nier D, Baltora-Rosset S, Doussot J, et al. Differential accumulation of monolignol-derived compounds in elicited flax (Linum usitatissimum) cell suspension cultures. Planta. 2006; 223(5):975–89. http://www.jstor.org/stable/23389388. https://doi.org/10.1007/s00425- 005-0156-1 PMID: 16292660 35. Boubakri H, Poutaraud A, Ali WM, Clayeux C, Baltenweck-Guyot R, Steyer D, et al. Thiamine modu- lates metabolism of the phenylpropanoid pathway leading to enhanced resistance to Plasmopara viti- cola in grapevine. BMC Plant Biol 2013; 13:31. https://doi.org/10.1186/1471-2229-13-31 PMID: 36. Abdel-Monaim MF. Role of riboflavin and thiamine in induced resistance against charcoal rot disease of soybean. Afr J Biotechnol. 2011; 10(53):10842–55. https://doi.org/10.5897/AJB11.253 37. Ahn IP, Kim S, Lee YH. Vitamin B1 functions as an activator of plant disease resistance. Plant Physiol. 2005; 138:1505–15. https://doi.org/10.1104/pp.104.058693 PMID: 15980201 PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 19 / 20 Hairy roots of Hyptis suaveolens improving production of podophyllotoxin 38. Zhou J, Sun A, Xing D. Modulation of cellular redox status by thiamine-activated NADPH oxidase con- fers Arabidopsis resistance to Sclerotinia sclerotiorum. J Exp Bot. 2013; 64(11):3261–72. https://doi. org/10.1093/jxb/ert166 PMID: 23814275 39. Nader BL, Cardoso-Taketa AT, Pereda-Miranda R, Villarreal ML. Production of triterpenoids in liquid- cultivated hairy roots of Galphimia glauca. Planta Med. 2006; 72:842–4. https://doi.org/10.1055/s-2006- 931601 PMID: 16732534 40. Caspeta L, Nieto I, Zamilpa A, Alvarez L, Quintero R, Villarreal ML. Solanum chrysotrichum hairy root cultures: characterization, scale-up and production of five antifungal saponins for human use. Planta Med. 2005; 71:1084–7. https://doi.org/10.1055/s-2005-873170 PMID: 16320218 41. Seidel V, Windho ¨ vel J, Eaton G, Alfermann AW, Arroo RRJ, Medarde M, et al. Biosynthesis of podo- phyllotoxin in Linum album cell cultures. Planta. 2002; 215(6):1031–9. https://doi.org/10.1007/s00425- 002-0834-1 PMID: 12355164 42. Chattopadhyay S, Srivastava A, Bhojwani S, Bisaria VS. Development of suspension culture of Podo- phyllum hexandrum for production of podophyllotoxin. Biotechnol Lett. 2001; 23(24):2063–6. https://doi. org/10.1023/A:1013704116860 43. Hammouri MK, Ereifej KI, Shibli RA, Al-Karaki GN. Quantitative analysis of fructose fate in a plant fer- mentation system. J Agric Food Chem. 1998; 46(4):1428−32. https://doi.org/10.1021/jf970437z 44. Shimon-Kerner N, Mills D, Merchuk JC. Sugar utilization and invertase activity in hairy-root and cell-sus- pension cultures of Symphytum officinale. Plant Cell Tissue Organ Cult. 2000; 62(2):89–94. 45. Mohagheghzadeh A, Hemmati S, Alfermann AW. Quantification of aryltetralin lignans in Linum album organs and in vitro cultures. Iran J Pharm Sci. 2006: 2(1):47–56. www.ijps.ir/article_1909_451.html. PLOS ONE | https://doi.org/10.1371/journal.pone.0222464 September 12, 2019 20 / 20

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