molecules Article Production of Encecalin in Cell Cultures and Hairy Roots of Helianthella quinquenervis (Hook.) A. Gray J. Mabel Hernández-Altamirano 1,2 , Irene F. Ugidos 1 , Javier Palazón 3 , Mercedes Bonfill 3 , Penélope García-Angulo 1 , Jesús Álvarez 1 , José L. Acebes 1, *, Robert Bye 4 and Antonio Encina 1 1 Área de Fisiología Vegetal, Departamento de Ingeniería y Ciencias Agrarias, Universidad de León, 24071 León, Spain; mabel.hdez@gmail.com (J.M.H.-A.); iferu@unileon.es (I.F.U.); penelope.garcia@unileon.es (P.G.-A.); jmalvf@unileon.es (J.Á.); a.encina@unileon.es (A.E.) 2 Laboratorio de Cultivo de Tejidos Vegetales, Jardín Botánico, Instituto de Biología, Universidad Nacional Autónoma de México, Ciudad Universitaria, Mexico City 04510, Mexico 3 Unitat de Fisiologia Vegetal, Facultat de Farmàcia, Universitat de Barcelona, Avda Joan XXIII 27-31, 08028 Barcelona, Spain; javierpalazon@ub.edu (J.P.); mbonfill@ub.edu (M.B.) 4 Laboratorio de Etnobotánica, Jardín Botánico, Instituto de Biología, Universidad Nacional Autónoma de México, Ciudad Universitaria, Mexico City 04510, Mexico; bye.robert@gmail.com * Correspondence: jl.acebes@unileon.es Academic Editor: Natalizia Miceli   Received: 22 June 2020; Accepted: 14 July 2020; Published: 15 July 2020 Abstract: Plant cell and organ cultures of Helianthella quinquenervis, a medicinal plant whose roots are used by the Tarahumara Indians of Chihuahua, Mexico, to relieve several ailments, were established to identify and quantify some chromenes with biological activity, such as encecalin, and to evaluate their potential for biotechnological production. Gas chromatography–mass spectrometry (GC-MS) analysis corroborated the presence of quantifiable amounts of encecalin in H. quinquenervis cell cultures (callus and cell suspensions). In addition, hairy roots were obtained through three transformation protocols (prick, 45-s sonication and co-culture), using wild type Agrobacterium rhizogenes A4. After three months, cocultivation achieved the highest percentage of transformation (66%), and a comparable production (FW) of encecalin (110 µg/g) than the sonication assay (120 µg/g), both giving far higher yields than the prick assay (19 µg/g). Stable integration of rolC and aux1 genes in the transformed roots was confirmed by polymerase chain reaction (PCR). Hairy roots from cocultivation (six months-old) accumulated as much as 1086 µg/g (FW) of encecalin, over three times higher than the cell suspension cultures. The production of encecalin varied with growth kinetics, being higher at the stationary phase. This is the first report of encecalin production in hairy roots of H. quinquenervis, demonstrating the potential for a future biotechnological production of chromenes. Keywords: medicinal plant; encecalin; Helianthella quinquenervis; Agrobacterium rhizogenes A4; chromenes; sonication; secondary metabolites 1. Introduction Helianthella quinquenervis (Hook.) A. Gray (Asteraceae) is a perennial herb that grows in montane and subalpine coniferous forest of the Rocky Mountains of the western United States of America and Canada where it is known as the aspen sunflower, and in pine-oak forests of the Sierra Madre of Chihuahua and Nuevo Leon, Mexico [1] (Figure 1). The Tarahumara Indians of Chihuahua know it as rarésoa [2] or corsonero (probably derived from escorzonera) and employ it medicinally. Dry and powdered roots are used to alleviate coughs and muscle aches and to treat gastrointestinal diseases and ulcers [3]. Crude extracts of H. quinquenervis roots have shown antimicrobial activity against Trichophyton mentagrophytes, one of the fungi that cause ringworm infections in humans [4], and antiprotozoal activity [5]. Molecules 2020, 25, 3231; doi:10.3390/molecules25143231 www.mdpi.com/journal/molecules Molecules 2020, 25, x 2 of 21 Molecules 2020, 25, Trichophyton 3231 mentagrophytes, one of the fungi that cause ringworm infections in humans [4],2 and of 20 antiprotozoal activity [5]. Figure 1. Helianthella quinquenervis and chemical structures of encecalin and related compounds Figure 1. Helianthella quinquenervis and chemical structures of encecalin and related compounds demethylencecalin and euparin (photo of flowering plant: Bocoyna, Chihuahua; R. Bye). demethylencecalin and euparin (photo of flowering plant: Bocoyna, Chihuahua; R. Bye). Phytochemical Phytochemical analysis analysis of of the the roots roots and aerial parts and aerial parts ofof H. H. quinquenervis quinquenervis isolated isolated and and identified identified different chromenes (benzopyrans) such as encecalin and demethylencecalin, different chromenes (benzopyrans) such as encecalin and demethylencecalin, and benzofurans such and benzofurans such as as euparin (Figure 1) [6–8]. Diverse bioassays have been used to verify the biological euparin (Figure 1) [6–8]. Diverse bioassays have been used to verify the biological activity of some of activity of some of thethe 200200 chromenes chromenes andand benzofurans benzofurans identified identified in H.inquinquenervis H. quinquenervis and other and other plantplant species, species, most most of of them them belonging to the family Asteraceae [9]. Many of these secondary metabolites belonging to the family Asteraceae [9]. Many of these secondary metabolites have insecticidal and have insecticidal and cytotoxic cytotoxic properties properties [10,11].[10,11]. Encecalin Encecalin and demethylencecalin and demethylencecalin inhibit theinhibit radicalthe radical growth growth of of seedlings of seedlings of several crop plants and weed, including Amaranthus hypochondriacus several crop plants and weed, including Amaranthus hypochondriacus and Echinochloa crusgalli [6,12–14]. and Echinochloa crusgalli In isolated[6,12–14]. spinachInchloroplasts, isolated spinach chloroplasts, they inhibit they inhibit ATP synthesis, ATP uptake proton synthesis, andproton electronuptake and flow and electron flow and disturb disturb photosystem II. Onphotosystem the other hand,II. On the other euparin hand,ATP inhibits euparin inhibits synthesis, ATP synthesis, proton uptake andproton basal uptake and basal and phosphorylating electron transport, suggesting and phosphorylating electron transport, suggesting that euparin suppresses phosphorylation in that euparin suppresses phosphorylation chloroplasts, acting in aschloroplasts, acting as an energy-transfer an energy‐transfer inhibitor [3]. Additionally,inhibitor [3]. Additionally, encecalin encecalin and demethylencecalin and demethylencecalin exhibit marginal cytotoxicity exhibit marginal against severalcytotoxicity human tumour against cell several lines [6].human tumour Encecalin cellbeen has also linesused [6]. Encecalin has also been used as a key intermediate for the synthesis of as a key intermediate for the synthesis of diverse ethers, amides and amines that were checked fordiverse ethers, amides and amines that were antiprotozoal checked activity [15]. for antiprotozoal activity [15]. The The source source of of these these compounds compounds continues continues to to be be wild wild medicinal medicinal plants plantswith withethnobotanical ethnobotanical value. value. However, the commercial production However, the commercial production of secondary metabolites from wild plants is of secondary metabolites from wild plants is hampered hampered by by fluctuating environmental conditions fluctuating environmental conditions andand lowlow yields. yields. InIn addition, addition, harvesting harvesting the the medicinal medicinal rootroot kills kills the plant, thus the plant, thus preventing preventing seed seed production, production, which which is critical for is critical for regeneration regeneration andand maintenance maintenance of of natural populations. Consequently, it is important to develop alternative natural populations. Consequently, it is important to develop alternative systems of production systems of production of of medicinal plants,especially medicinal plants, especially those those with with limited limited ecological ecological and geographical and geographical ranges,ranges, in case in case the demandthe demand for these plants suddenly overrides the local supply and drives the for these plants suddenly overrides the local supply and drives the wild populations into extinction. wild populations into Molecules 2020, 25, 3231 3 of 20 Such alternative methodology should be appropriate for transfer to institutions located in the region of the source plant material so that inhabitants can benefit from the biotechnological applications. An alternative approach to secondary metabolite production is organic synthesis, but this is frequently constrained by the complexity of the target structure [16]. More sustainable strategies to obtain plant secondary metabolites of interest for the agrochemical, pharmaceutical and food industries involve in vitro culture methodologies, together with the use of modified tissues, organs and whole plants [17–19]. Transformed hairy roots cultures, obtained by the infection of plant cells with Agrobacterium rhizogenes, are an option for the biotechnological exploitation of metabolites due to their high growth rate, genetic stability and capacity for growing in hormone-free media. In addition, this biotechnological strategy allows the production and/or biotransformation of highly diverse molecules [20,21]. A. rhizogenes is a gram-negative soil bacterium that can cause the hairy root syndrome in many plants. Infections in wound sites are followed by the transfer, integration and expression of T-DNA from the root-inducing (Ri) plasmid and the subsequent development of the hairy root phenotype [22]. The hairy roots, once separated from the tissue of origin, can continue to grow in hormone-free media. The Agrobacterium genes implicated in the rhizogenesis, known as rol and aux genes, are located in two regions which are independently transferred to the plant cell [23]: the left transferred (TL -DNA) and right transferred (TR -DNA) regions, respectively. The aux genes are involved in auxin biosynthesis and/or sensitivity. In the last decade, the use of hairy root culture technologies for pharmaceutical production has progressed from laboratory-scale to an industrial level [24]. The secondary metabolite yields of transformed root cultures can be comparable with those of intact plants, and in some species may be increased by elicitation [25,26]. Although encecalin and related compounds are relatively simple molecules, little is known about their production in in vitro culture systems. Furthermore, the biological importance of these chromenes and benzofurans, calls for new production strategies with potential to be optimized for their large-scale and cost-effective production. In this work, callus, cell suspension and hairy-root cultures of H. quinquenervis were obtained in order to evaluate the production of encecalin and other related secondary metabolites with potential value in the agricultural and pharmaceutical area. 2. Results and Discussion 2.1. Germination Treatments and Callus Induction Mechanical scarification (Figure 2a) was a precondition for the germination of H. quinquenervis seeds, which present an extra-embryonic dormancy due to the hardness and poor permeability of the pericarp. Scarification under two different conditions (in vitro and soil) clearly affected the germination rate, increasing it from 13% and 38%, respectively, for unscarified seeds to 90% for scarified seeds after thirteen days (data not shown). A similar result was reported in seeds of Anthemis chrysantha (Asteraceae); after breaking seed dormancy by mechanical scarification or excising the embryo, 71% germination was achieved [27]. These results indicated that the presence of the pericarp and testa inhibits seed germination. H. quinquenervis scarified seeds were germinated in vitro and cultured in axenic conditions. Two months after germination, plants were 5 to 8 cm in height showing balanced stem and leaf development (Figure 2b). These stems and leaf were selected as explants (Figure 2c) for callus induction. After testing both types of explants with different combinations and ratios of auxin/cytokinin, the selected medium was 6-benzylaminopurine (BAP, 1 mg/L) + 2,4-dichlorophenoxyacetic acid (2,4-d) (0.1 mg/L). Leaf explants were chosen for their high percentage of callogenesis (90%) and low oxidation rate (data not shown). The biomass produced by callus from leaf explants was 0.273 mg FW/explant, showing a friable consistency and pale-yellow color. The treatment with BAP (1 mg/L) + naphthaleneacetic acid (NAA, 2 mg/L) on leaf explants yielded a higher quantity of callus biomass (0.318 mg FW/explant), but it was discarded because of an oxidation process that appeared during subculturing, and the compact consistency of the calli. Molecules 2020, 25, 3231 4 of 20 Molecules 2020, 25, x 4 of 21 Figure Figure 2. 2. Seeds Seeds and and cell cell cultures cultures of of Helianthella Helianthella quinquenervis: quinquenervis: (a) (a) seeds seeds with withand andwithout withoutscarification; scarification; (b) two months in vitro plants; (c) leaf explant with forming callus; (d) callus in darkness; (e) (b) two months in vitro plants; (c) leaf explant with forming callus; (d) callus in darkness; (e) callus callus in in photoperiod; (f) cell suspension culture. photoperiod; (f) cell suspension culture. A similar When stemresult waswere explants reported in seeds exposed of Anthemisofchrysantha to combinations (Asteraceae); auxin/cytokinin, after breaking they generated 58% ofseed calli dormancy by mechanical in the presence of BAP (1 mg/L) + NAA (2 scarification ormg/L) excising andthe 60%embryo, with BAP 71% + 2,4-d was germination (1 mg/L) achieved (0.1 mg/L). [27]. Because These results indicated both combinations that theless generated presence biomass of than the pericarp in the caseandoftesta leafinhibits explantseed germination. and resulted in oxidation and inH.the quinquenervis productionscarified seeds calli, of less friable were the germinated in vitro stem explants anddiscarded were cultured in foraxenic futureconditions. experiments.Two monthsOnce the optimum conditions for callus induction were established, one set of calli was kept inleaf after germination, plants were 5 to 8 cm in height showing balanced stem and the development dark (Figure 2d) (Figure 2b). These and another stems set under and leaf were a photoperiod selected of 16:8 (Figureas2e). explants The calli(Figure cultured2c)under for callus light induction. were greenish and friable, with some compact and hard areas. On the other hand, those maintained in After the dark were testing both typescolor a pale-yellow of explants and more with different friable. For thiscombinations reason, darkand ratioscalli cultured of auxin/cytokinin, were chosen for the selected obtaining cellmedium was 6‐benzylaminopurine suspension cultures. (BAP, 1 mg/L) + 2,4‐dichlorophenoxyacetic acid (2,4‐ D) (0.1 mg/L). Leaf explants were chosen for their high percentage of callogenesis (90%) and low 2.2. Characterization oxidation rate (dataofnotCellshown). Suspension TheCultures biomass produced by callus from leaf explants was 0.273 mg FW/explant, showingcultures Cell suspension a friable consistency (Figure 2f) wereand pale‐yellow derived from leaf color. calliThe treatment growing in MSwith BAPwith medium (1 mg/L) BAP + naphthaleneacetic acid (NAA, 2 mg/L) on leaf explants yielded a higher quantity (1 mg/L) + 2,4-d (0.1 mg/L) in the dark. To achieve these, calli were transferred to a liquid medium of callus biomass of (0.318 mg FW/explant), but it was discarded because of an oxidation process the same composition and allowed to grow on a rotary shaker in the dark. Figure 3 shows the growth that appeared during subculturing, kinetics of the and the compact consistency suspension-cultured of the cells. After calli. a lag phase of two days, the biomass dry weight (DW) When stem explants were exposed to combinations progressively increased, reaching the stationary phase at day of 8. auxin/cytokinin, they generated Once the cell suspension cultures58% of grew calli in the presence stable—after more thanof BAP (1 mg/L) + NAA 8 subcultures—a 70%(2viability mg/L) and was60% with BAPduring determined (1 mg/L) + 2,4‐ active D (0.1 mg/L). growth. Because both combinations generated less biomass than in the case of leaf explant and resulted in oxidation and in the production of less friable calli, the stem explants were discarded for future experiments. Once the optimum conditions for callus induction were established, one set of calli was kept in the dark (Figure 2d) and another set under a photoperiod of 16:8 (Figure 2e). The calli cultured under light were greenish and friable, with some compact and hard areas. On the other hand, those maintained in the dark were a pale‐yellow color and more friable. For this reason, dark cultured calli were chosen for obtaining cell suspension cultures. 2.2. Characterization of Cell Suspension Cultures Cell suspension cultures (Figure 2f) were derived from leaf calli growing in MS medium with BAP (1 mg/L) + 2,4‐D (0.1 mg/L) in the dark. To achieve these, calli were transferred to a liquid medium of the same composition and allowed to grow on a rotary shaker in the dark. Figure 3 shows Molecules 2020, 25, x 5 of 21 the growth kinetics of the suspension‐cultured cells. After a lag phase of two days, the biomass dry weight (DW) progressively increased, reaching the stationary phase at day 8. Once the cell Molecules 2020,cultures suspension 25, 3231 grew stable—after more than 8 subcultures—a 70% viability was determined 5 of 20 during active growth. Figure 3. Kinetic growth of cell suspension culture of Helianthella quinquenervis expressed in dry weight Figure 3. Kinetic (DW) g/150 growth mL. Values areofmeans cell suspension culture ± S.D. of the of Helianthella measurement quinquenervis from three flasks. expressed in dry weight (DW) g/150 mL. Values are means ± S.D. of the measurement from three flasks. H. quinquenervis suspension cultured cells grew forming aggregates, which seemed to function H. quinquenervis as generators of newsuspension cultured cells. Filtration cellsdifferent through grew forming aggregates, sized meshes which resulted inseemed to sized diversely function cell as generators of new cells. Filtration through different sized meshes resulted in diversely aggregates in the stationary phase (Figure 4a), ranging from large (greater than 2 mm and accounting sized cell aggregates for over 40%in the stationary of the DW) to phase (Figure very small 4a), ranging (between from 120 and 400large (greater µm and than 2 mm accounting and accounting for 30% of the DW), for over 40% of the DW) as well as individual cells.to very small (between 120 and 400 μm and accounting for 30% of the DW), as well as individual cells. Although cell suspension cultures of H. quinquenervis presented great variation in the size of cellular aggregates, this did not affect the culture viability. In samples obtained by differential filtration, large cell aggregates with high viability were observed (Figure 4d). These appeared to be the generators of cells that would be part of a suspension with cells of more homogeneous size. The establishment and characterization of suspension cultures to produce compounds of interest has been reported in cultures of Stevia rebaudiana in shake flasks, as a strategy to obtain an in vitro cell line producing stevioside [28]. In the case of medicinal plants, many strategies have been developed to induce high-yield cultures [29], including elicitation to stimulate production [30] and the transformation of metabolic pathways [31]. With this taken into account, it is believed that the establishment of suspension cultures of H. quinquenervis would allow the study of chromenes. Molecules 2020, 25, 3231 6 of 20 Molecules 2020, 25, x 6 of 21 (a) Figure 4. (a) Size of clusters of cell suspension culture of Helianthella quinquenervis analyzed at the Figure 4. (a) Size of clusters of cell suspension culture of Helianthella quinquenervis analyzed at the stationary phase (eighth day). Results are expressed as the percentage of DW retained in each filter. stationary phase (eighth day). Results are expressed as the percentage of DW retained in each filter. Values are means ± S.D. of five measurements; (b–e) viability of two suspension culture fractions Values are means ± S.D. of five measurements; (b–e) viability of two suspension culture fractions obtained by differential filtration with 2 mm mesh (b,d), and homogenous cultures obtained with 22 obtained by differential filtration with 2 mm mesh (b,d), and homogenous cultures obtained with 22 µm μm mesh (c,e) during the end of the exponential phase and the start of the stationary phase; b and c, mesh (c,e) during the end of the exponential phase and the start of the stationary phase; (b,c) with light with light field; d and e, with fluorescence microscopy. field; (d,e) with fluorescence microscopy. Although cell suspension cultures of H. quinquenervis presented great variation in the size of cellular aggregates, this did not affect the culture viability. In samples obtained by differential The establishment and characterization of suspension cultures to produce compounds of interest has been reported in cultures of Stevia rebaudiana in shake flasks, as a strategy to obtain an in vitro cell line producing stevioside [28]. In the case of medicinal plants, many strategies have been developed to induce high‐yield cultures [29], including elicitation to stimulate production [30] and Molecules 2020, 25, 3231 of metabolic pathways [31]. With this taken into account, it is believed that the transformation 7 ofthe 20 establishment of suspension cultures of H. quinquenervis would allow the study of chromenes. 2.3. Hairy Root Induction 2.3. Hairy Root Induction The leaf explants, taken from in vitro-germinated plants, transformed by A. rhizogenes strain A4 The leaf explants, taken from in vitro‐germinated plants, transformed by A. rhizogenes strain A4 and maintained in hormone-free solid medium with cefotaxime, began to develop small roots after and maintained in hormone‐free solid medium with cefotaxime, began to develop small roots after twelve days of transformation, sprouting from the wound sites in all three of the transformation assays twelve days of transformation, sprouting from the wound sites in all three of the transformation (Figure 5a). After 20 days (Figure 5b), the length and number of the roots had increased. The roots assays (Figure 5a). After 20 days (Figure 5b), the length and number of the roots had increased. The were pale yellow, and callus formation at the base of the roots was observed after 30 days (Figure 5c), roots were pale yellow, and callus formation at the base of the roots was observed after 30 days but root growth was not affected. After several subcultures in 50% liquid MS medium with antibiotics, (Figure 5c), but root growth was not affected. After several subcultures in 50% liquid MS medium the roots were subcultured in liquid MS medium without antibiotics, in which they continued to with antibiotics, the roots were subcultured in liquid MS medium without antibiotics, in which they grow. The roots showed typical transformed root morphology: plagiotropic growth, thickness and the continued to grow. The roots showed typical transformed root morphology: plagiotropic growth, presence of root hairs (Figure 5d). thickness and the presence of root hairs (Figure 5d). Figure 5. Figure 5. Hairy Hairy roots roots from from Helianthella Helianthella quinquenervis quinquenervis in in hormone‐free hormone-free MS MS medium medium at at (a) (a) 12 12 d; d; (b) (b) 20 20 d; d; (c) 30 d; (d) 90 d after transformation. (c) 30 d; (d) 90 d after transformation. The susceptibility The susceptibility ofof H. quinquenervis plants plants to to transformation transformation by by A. rhizogenes was was tested tested using using three techniques. Besides the commonly used methods of pricking and co‐cultivation, three techniques. Besides the commonly used methods of pricking and co-cultivation, sonication sonication was also also was triedtried in which the wounded in which the wounded explants werewere explants exposed to ultrasound exposed [32]. The to ultrasound [32].results of theof The results three the different three assays different (prick, assays sonication (prick, and and sonication co‐culture) showed co-culture) thatthat showed H. quinquenervis waswas H. quinquenervis susceptible susceptible to transformation by A. rhizogenes strain A4 (Table 1). After forty days, the lowest to transformation by A. rhizogenes strain A4 (Table 1). After forty days, the lowest response to the response to the transformation was transformation was observed observedin inthe theprick prickassay assay(17%), (17%),ininthe thenumber number ofof infected explants infected explantsas as well as well root‐generating explants. Moreover, the infected explants showed oxidation as root-generating explants. Moreover, the infected explants showed oxidation over time. In the over time. In the sonication assay, sonication the transformation assay, the transformation percentage percentage waswas higher higher (40%), (40%), and and numerous numerous hairy hairy roots roots (56) (56) were obtained. were obtained. In In the the same same way, the sonication way, the sonication technique technique was was efficiently efficiently used used to to obtain obtain hairy hairy roots roots from explants from explants of of Verbascum Verbascum xanthophoeniceum, xanthophoeniceum, with with a a 75% 75% transformation transformation rate rate [32]. [32]. However, However, in in the the H. quinquenervis H. quinquenervis explants, explants, thethe co-culture co‐culture gave gave the the highest highest percentage percentage of of transformation transformation (66%) (66%) ofof the the three assays. Although the number of roots per explant varied among the assays, no differences were observed in the morphology of roots, which were generally thin and pale yellow. In all three experiments, the remainder of the explants were necrosed or reinfected (i.e., subsequently infected by A. rhizogenes), which prevented their subsequent use. Those transformed roots in which the infection by A. rhizogenes could not be eliminated were discarded. Molecules 2020, 25, 3231 8 of 20 Table 1. Hairy root formation on Helianthella quinquenervis leaf explants 40 days after transformation with A. rhizogenes. Number of Explants Method of Transformation Range of Roots/Explant Transformed/Total Explant Prick 6/35 1–3 Sonication 18/45 1–5 Co-culture 20/30 1–9 After forty days, several hairy roots from each assay were selected and maintained in liquid MS medium with sucrose and the antibiotic. After the fourth subculture under these conditions, the eventual presence of Agrobacterium and the transformation of the cultures were analyzed by PCR. At ninety days after transformation, a sufficient number of roots of increased length (Figure 5d) were obtained for the quantification analyses. The success of transformation depends on several factors, such as the type of explant, its age and the species, as well as the Agrobacterium strain [33], since some are more virulent than others. The transformation capacity is partially related to the variety of Ri (root inducing) plasmids within each bacterial strain. In a previous trial, poor results were obtained with the A. rhizogenes LBA9402 strain (data not shown). In contrast, the A4 strain successfully produced hairy roots. Transformation efficiency in Fagopyrum tataricum with the same strain was 100% [34], being somewhat lower in the H. quinquenervis explants, at 66.7%. 2.4. PCR for the Confirmation of Root Transformation The transformation of H. quinquenervis hairy roots was confirmed by PCR. Amplification of rolC, aux1 and virG primers showed that the transformation system was complete and stable (Figure 6). The PCR results revealed that TL -DNA (rolC) and TR -DNA (aux1) fragments were successfully inserted in the three assays (prick, sonication and co-culture). Successful elimination of A. rhizogenes infection in the three cultures was confirmed by the absence of the band representing the virG amplified product, which was only observed in the sample corresponding to the A. rhizogenes A4 strain. The absence of infection by A. rhizogenes was also confirmed in leaves (used as explants), as well as in H. quinquenervis untransformed roots. The complete and stable transformation of H. quinquenervis hairy roots was confirmed by identifying the bacterial genes rolC and aux1 using PCR analysis. Likewise, contamination by A. rhizogenes was discarded in both nontransformed and transformed roots, when no band was observed for the virG gene. The amplification of rolC (534 bp) in the roots produced by the different techniques indicated that the TL -DNA fragment was integrated into the plant genome, which is essential for hairy root induction [32]. Amplification of aux1 confirmed that the TR -DNA was also integrated. The segment, containing aux1, aux2 and ags genes, controls opine and auxin biosynthesis [35], the latter being indispensable for the growth and differentiation of the roots, which did not need auxins in the culture medium. The complete integration of TR -DNA and TL -DNA was confirmed in the H. quinquenervis transformed roots, but not in the nontransformed roots of L. album (L-), used as a negative control, nor in the nontransformed roots of H. quinquenervis, as expected. The confirmed presence of the rol and aux genes in the transformed roots of H. quinquenervis is valuable for the role they would play in the synthesis of secondary or specialized metabolites. Such is the case of Artemisia carvifolia and its role in enhancing the production of antioxidants [36]. This relationship has been well documented in a wide number of plant species [37]. The PCR results revealed that TL‐DNA (rolC) and TR‐DNA (aux1) fragments were successfully inserted in the three assays (prick, sonication and co‐culture). Successful elimination of A. rhizogenes infection in the three cultures was confirmed by the absence of the band representing the virG amplified product, which was only observed in the sample corresponding to the A. rhizogenes A4 MoleculesThe strain. 25, 3231 of infection by A. rhizogenes was also confirmed in leaves (used as explants), 2020,absence 9 of as 20 well as in H. quinquenervis untransformed roots. Molecules 2020, 25, x 9 of 21 Figure 6. Polymerase chain reaction (PCR) analysis of aux1 (350 bp), rolC (534 bp) and virG (350 bp) Figure 6. Polymerase chain reaction (PCR) analysis of aux1 (350 bp), rolC (534 bp) and virG (350 bp) from T-DNA from T‐DNA of of the the Ri Ri of of A. A.rhizogenes. rhizogenes. Molecular Molecular marker marker (M); (M); Linum Linum album album L(−) L(−) ** (negative (negative control); control); Linum album L(+)* (positive control); roots obtained by prick (P), sonication Linum album L(+)* (positive control); roots obtained by prick (P), sonication (S) (S) and co‐culture (Co‐C); and co-culture strain A4 of A. rhizogenes (A4); H. quinquenervis leaves (Hl) (negative control); wild (Co-C); strain A4 of A. rhizogenes (A4); H. quinquenervis leaves (Hl) (negative control); wild roots roots of H. of quinquenervis (negative control) (Hq). * Once nontransformed H. quinquenervis material was H. quinquenervis (negative control) (Hq). * Once nontransformed H. quinquenervis material was obtained,obtained, it was it was preferred preferred asas control. control. 2.5. Detection and Quantification The complete and stable oftransformation Chromenes In Vitro of H.Cultured Cells of H. quinquenervis quinquenervis hairy roots was confirmed by identifying the bacterial genes rolC and aux1 using PCR analysis. Likewise, contamination by A. In its wild form, H. quinquenervis produces chromenes and benzofuranes, which have been rhizogenes was discarded in both nontransformed and transformed roots, when no band was observed identified in aerial parts and roots [7]. In order to determine if H. quinquenervis in vitro cultures for the virG gene. The amplification of rolC (534 bp) in the roots produced by the different techniques produce secondary metabolites of interest, an assay was developed to produce calli in sufficient indicated that the TL‐DNA fragment was integrated into the plant genome, which is essential for hairy quantities for phytochemical analysis. A frequent drawback of undifferentiated cell masses is that root induction [32]. Amplification of aux1 confirmed that the TR‐DNA was also integrated. The they do not produce significant amounts of target compounds, as secondary metabolism usually relies segment, containing aux1, aux2 and ags genes, controls opine and auxin biosynthesis [35], the latter on cell differentiation [38]. However, secondary metabolite production has also been described in being indispensable for the growth and differentiation of the roots, which did not need auxins in the undifferentiated cells of other Asteraceae species, such as Piqueria trinervia, whose calli produced culture medium. The complete integration of TR‐DNA and TL‐DNA was confirmed in the H. piquerol A, a monoterpene with antifungal and allelopathic activity [39], and Rudbeckia hirta, whose calli quinquenervis transformed roots, but not in the nontransformed roots of L. album (L‐), used as a and cell suspension cultures produced pulchelin E, a sesquiterpene lactone with immunostimulating negative control, nor in the nontransformed roots of H. quinquenervis, as expected. The confirmed properties [40]. presence of the rol and aux genes in the transformed roots of H. quinquenervis is valuable for the role GC-MS analysis of chloroform extracts from H. quinquenervis calli revealed the presence of they would play in the synthesis of secondary or specialized metabolites. Such is the case of Artemisia encecalin (Table 2 and Figure 7). carvifolia and its role in enhancing the production of antioxidants [36]. This relationship has been well Because the synthesis pathways of chromenes and benzofuranes are not well determined, and there documented in a wide number of plant species [37]. are compounds such as terpenes that depend on the incidence of light for their synthesis [41], it was interesting to assess whether light affected synthesis in any way. In this respect, it was found that 2.5. Detection and Quantification of Chromenes in vitro Cultured Cells of H. quinquenervis lighting was not a prerequisite for encecalin biosynthesis as both light and dark-grown calli produced In its(Table encecalin wild form, 2). In H. quinquenervis addition, encecalin produces chromenes production by calliand benzofuranes, revealed that calliwhich kept inhave been the dark identified 1.7 produced in times aerial more partsencecalin and rootsthan[7]. those In order to photoperiodic under determine if H. quinquenervis conditions (Tablein2).vitro cultures produce secondary Encecalin metabolites was also detected inofsuspension-cultured interest, an assay was developed cells of to produce H. quinquenervis (Tablecalli in produced 2) that sufficient quantities almost for(1.98 twice phytochemical analysis. times) as much A frequent of encecalin thandrawback of aundifferentiated calli kept in cell 2). photoperiod (Table masses is that The higher they do of amount not produce encecalin significant produced amounts by cell of target suspension compounds, cultures as secondary may be explained by themetabolism usually stress provoked by relies on cell differentiation [38]. However, secondary metabolite production the immersion and shaking of the cells, which could elicit their synthesis [42]. has also been described in undifferentiated cells of other Asteraceae species, such as Piqueria trinervia, whose calli produced piquerol A, a monoterpene with antifungal and allelopathic activity [39], and Rudbeckia hirta, whose calli and cell suspension cultures produced pulchelin E, a sesquiterpene lactone with immunostimulating properties [40]. GC‐MS analysis of chloroform extracts from H. quinquenervis calli revealed the presence of Molecules 2020, 25, x 10 of 21 Molecules 2020, 25, 3231 10 of 20 The higher amount of encecalin produced by cell suspension cultures may be explained by the stress provoked by the immersion and shaking of the cells, which could elicit their synthesis [42]. Table 2. Amount of encecalin quantified in Helianthella quinquenervis cultures. Table 2. Amount of encecalin quantified in Helianthella quinquenervis cultures. Encecalin Culture Encecalin Culture (µg/g FW) (μg/g FW) Calli 1 (photoperiod) 165 ± 7 Calli 1 (photoperiod) 165 ± 7 Calli Calli (darkness) (darkness) ± 3± 3 290290 CellCell suspension suspension culture culture ± 14 327327 ± 14 Hairy roots Hairy (co‐culture) roots (co-culture) 1086 1086 ± 31 ± 31 Callikept 11 Calli keptunder undera a1616h/light h/light 8 h/darkness 8 h/darkness photoperiod. photoperiod. Hairy Hairy rootsroots werewere obtained obtained by theby the co‐culture co-culture method. GC-MS-MS: gas chromatography method. GC‐MS‐MS: with mass detection gas chromatography withinmass masses-masses detectionmethod. The cultures were in masses‐masses kept for method. Thesix months under the conditions described here before making the extractions. cultures were kept for six months under the conditions described here before making the extractions. a b Figure7.7. (a) Figure (a) Chromatogram Chromatogram of a sample sample of of callus callus where wherethe thepeak peakofofencecalin encecalincan canbe beseen, seen,asaswell wellasas (b)mass (b) massspectrum spectrum obtained obtained by by fragmentation fragmentation of the precursor precursor ion ion of of mass mass 217. 217. 2.6. 2.6. Hairy Hairy Root Root Growth Growth and and Encecalin Quantitative Analysis Encecalin Quantitative Analysis GC-MS GC‐MS analysis analysis of chloroform extracts extracts from from two‐month‐old two-month-oldrootsrootsfrom fromininvitro vitroplants plantsrevealed revealed the thepresence presenceofof some someof the mostmost of the important secondary important metabolites secondary of H. quinquenervis, metabolites such as encecalin of H. quinquenervis, such as and the structurally encecalin related compound and the structurally euparin (Figure related compound 1), (Figure euparin although1),inalthough low concentrations. In the three in low concentrations. transformed root lines obtained In the three transformed root linesby prick, sonication obtained and co-culture by prick, sonication techniques, and co‐culture after three techniques, aftermonths three encecalin was produced months encecalin in sufficient was produced quantities in sufficient to perform quantities quantification to perform (Figure (Figure quantification 8). 8). Encecalin production significantly varied in relation to the method used for H. quinquenervis transformation as determined by one-way ANOVA (F(2,6) = 457.9; p < 0.0001). Although co-culture resulted in the highest transformation frequency, it provided roots with a slightly lower concentration of encecalin (110 ± 5 µg/g FW) than the sonication assay (120 ± 6 µg/g FW); whereas the prick assay resulted in a low transformation frequency and an encecalin concentration of 19 ± 1 µg/g FW. Even though the production of encecalin was highest when using the sonication technique, the fact that the percentage of infection was much higher for the co-culture technique, and that the encecalin production difference, although significant, was only slightly higher for the sonication method, the co-culture method of transformation was chosen to continue analyzing the production of encecalin for a longer period. After six months, a concentration of 1086 ± 31 µg/g FW was achieved (Table 2). This result confirmed that in H. quinquenervis, the synthesis of encecalin was more abundant in organized structures (roots) than in disorganized tissues such as calli or cell suspension; also, the higher the level of differentiation of roots, the greater the production of encecalin. Molecules 2020, 25, 3231 11 of 20 Molecules 2020, 25, x 11 of 21 (a) (b) Figure8.8.(a)(a) Figure Encecalin Encecalin quantification quantification in hairyinroots hairy roots (three (three months old)months old) and and percentage percentage of of transformation transformation in in three three assays: prick (P), assays: sonication prick (S) (P), and sonication (S) and co-culture (CC). co‐culture Data (CC). Data of transformation of transformation frequency represent frequency the averagerepresent the measurements. ± SD of five average ± SD of Data five measurements. Data of encecalin of encecalin quantification quantification represent the averagerepresent ± SD of the average three ± SD injections. of three Different letters indicateDifferent injections. significantletters indicate differences significant in encecalin differences production afterina encecalin one-way production ANOVA after a one‐way following test (p < 0.05); by TukeyANOVA following by Tukey test of (b) chromatogram (p