Plant Cell, Tissue and Organ Culture (PCTOC) https://doi.org/10.1007/s11240-019-01615-5 ORIGINAL ARTICLE Establishment of callus‑cultures of the Argentinean mistletoe, Ligaria cuneifolia (R. et P.) Tiegh (Loranthaceae) and screening of their polyphenolic content M. V. Ricco1,2 · M. L. Bari1 · F. Bagnato1 · C. Cornacchioli1 · M. Laguia‑Becher2,5 · L. U. Spairani1,6 · A. Posadaz4 · C. Dobrecky3,7 · R. A. Ricco3 · M. L. Wagner3 · M. A. Álvarez1,2 Received: 25 November 2018 / Accepted: 27 April 2019 © Springer Nature B.V. 2019 Abstract Ligaria cuneifolia (R. et P.) Tiegh (Loranthaceae), known as liga, muérdago criollo, or Argentinean mistletoe, is a hemiparasitic plant with a broad distribution in central and northern Argentina. Pharmacological studies showed that L. cuneifolia extracts have hypolipemic, antioxidant, antibacterial, and immunomodulatory effects. We have established callus cultures from embryo and haustoria fragments. The highest frequency of callus formation from embryos (85%) was obtained on White medium with 4% (w/v) sucrose and 2.5 µM 1-naphtalene acetic acid and 9.2 µM kinetin as plant growth regulators (PGRs). From haustoria, the best result (35%) was obtained on Gamborg medium with 3% (w/v) sucrose and 0.45 µM 2,4-dichlorephenoxyacetic acid and 0.47 µM zeatin as PGRs. Thin layer chromatography showed that callus methanolic extract (2.5% w/v) had a lower content of flavonoids and proanthocyanins as compared to the wild plant (5% w/v for leaves, stems, and flowers), but a higher content of hydroxycinnamic acids. High performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) showed the presence of quercetin glycosides and phenolic acids in the methanolic extracts both from the parent plant and the callus obtained from embryo. Key message Callus cultures were established from embryo and haustorium explants of Ligaria cuneifolia. Leaves, stems, and meristems were recalcitrant to in vitro culture. Callus tissues contained quercetin glycosides and phenolic acids. Keywords Medicinal plants · Liga · Hemiparasitic plant · Callus culture · Flavonoids Communicated by Sergio J. Ochatt. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11240-019-01615-5) contains supplementary material, which is available to authorized users. * M. A. Álvarez alvarez.mariaalejandra@maimonides.edu 1 Carreras de Farmacia y Bioquímica, Cátedra de Farmacobotánica y Farmacognosia, Facultad de Ciencias de la Salud, Universidad Maimónides, Hidalgo 775, Ciudad de Buenos Aires, Argentina 2 Consejo Nacional de Investigaciones Científicas y Tecnicas, Godoy Cruz 2290, Ciudad de Buenos Aires, Argentina 3 Departamento de Farmacología, Cátedra de Farmacobotánica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, Ciudad de Buenos Aires, Argentina 4 Facultad de Turismo y Urbanismo, Universidad Nacional de San Luis, Av del Libertador s/n, Barranca Colorada, Villa de Merlo, San Luis, Argentina 5 Centro de Estudios Biotecnológicos, Biológicos, Ambientales y Diagnóstico (CEBBAD), Universidad Maimónides, Hidalgo 775, Ciudad de Buenos Aires, Argentina 6 Instituto Antártico Argentino, Dirección Nacional del Antártico, Av. 25 de Mayo 1143, San Martín, Buenos Aires, Argentina 7 Cátedra de Tecnología Farmacéutica I, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, Ciudad de Buenos Aires, Argentina 13 Vol.:(0123456789) Plant Cell, Tissue and Organ Culture (PCTOC) Introduction Ligaria cuneifolia (R. et P.) Tiegh (Loranthaceae), known as liga, muérdago criollo, or Argentinean mistletoe, is an aerial-photosynthesizing hemiparasitic plant widely distributed in Latin America, from Perú to Central Argentina (Abiatti 1946; Amico and Nickrent 2007). Its hosts are mostly members of the Fabaceae family such as Prosopis caldenia Burkart P, P. torquata (Lag.) DC, Geoffroea decorticans (Hook & Arn.), and Acacia caven (Molina) Molina. Also, it can be found growing on species from the Anacardiaceae (e.g. Schinus fasciculata I.M. Johnst., S. areira L.), Ulmaceae (e.g. Ulmus pumila L.), and Verbenaceae [e.g.: Aloysia gratisima (Gillies & Hook) Tronc.] families. L. cuneifolia flowers are hexamershaped, arranged in clusters, and colored from orange to red, according to the region where they grow. Fruits are dark violet or black berries that are dispersed by birds (Varela et al. 2001; Amuchástegui et al. 2003). It has a haustorium (a modified root structure) that extracts water and minerals from the host xylem producing a vascular continuity (Rustán et al. 2003). Preparations of L. cuneifolia are widely used in popular (folk and indigenous) medicine in the northern and central provinces of Argentina for its attributed therapeutic properties (Scarpa and Montani 2011). The infusion prepared from the aerial parts is taken as a substitute of Viscum album L. (Santalaceae) in cases of high blood pressure (Martínez 2010). In addition, the infusion or the decoctions are used as antihemorrhagic, abortive, emmenagogue, oxytocic, in case of fractures, and sore throat. Also, it is used in veterinary and as forage for goats and cattle (Scarpa and Montani 2011). Pharmacological studies support the use of L. cuneifolia in popular medicine as antioxidant, antibacterial and as anti-hypertensive or anti-hypotensive depending on the host (Soberón et al. 2014; Gonzálvez et al. 2017). It was also reported the inhibition of the proliferation of murine mitogen-activated lymphocytes, murine T cell leukemia (LB) and breast tumor cells (MMT) by L. cuneifolia whole extract and the ethyl acetate flavonoid fraction (Cerdá Zolezzi et al. 2005). Some of the pharmacological activities of L. cuneifolia are attributed to its content in lectins and polyphenolic compounds. The main identified polyphenolic compounds in L. cuneifolia extracts are quercetin-3-O-glycosides, such as quercetin-3-O-glucoside, quercetin-3-O-xyloside, quercetin-3-O-arabinopyranoside, quercetin-3-Oarabinofuranoside, quercetin-3-O-rhamnoside, and four novel quercetin-galloyl-glycosides: quercetin-3-O-(2″-Ogalloyl) rhamnoside, quercetin-3-O-(3″-O-galloyl) rhamnoside, quercetin-3-O-(2″galloyl)-arabinofuranoside, and 13 quercetin-3-O-(2″-O-galloyl)-arabinopyranoside. Catechin, the main flavan-3-ol, and other proanthocyanidins with different degree of polymerization were also identified (Varela et al. 2001; Dobrecky et al. 2017). Depending on the geographical area, the presence of tyramine was shown (Vázquez y Novo et al. 1989). L. cuneifolia cannot be cultured in the field; moreover, its exploitation in order to extract the active metabolites could imply a threat to the conservation of the species. In this context, in vitro cultures appear as an attractive alternative production platform (Espinosa-Leal et al. 2018). Studies on in vitro cultures are scarce in the Loranthaceae family. In the case of L. cuneifolia they are non-existent, possibly due to the difficulty in culturing a hemiparasitic plant and because of the explant exudation of phenolics leading to medium browning and necrosis (Ishrad et al. 2018). The aim of this work was the establishment of callus cultures of L. cuneifolia and the screening of their polyphenolic content. Materials and methods Plant material Plant material was collected between January 2015 and December 2017 from the region (30 km) comprised between La Población, Córdoba (32°03′34.4″S 65°00′36.6″W) and Villa de Merlo, San Luis (32°21′22.5″S 65°00′20.5″W), Argentina. Samples were placed in plastic bags and stored at 4 °C until being processed. A voucher was deposited in the Museo de Farmacobotánica Juan Aníbal Dominguez herbarium (Buenos Aires, Argentina) BAF 9018. Chemicals and reagents Gamborg et al. (1968), White (1963), and Murashige and Skoog (1962) media, sucrose, casein hydrolysate and agar (plant tissue micropropagation culture grade) were from PhytoTechnology Laboratories (Lenexa, KS). LiChrosolv® Methanol was supplied from Merck (Darmstadt, Germany). Formic acid was purchased from Baker (New Jersey, USA). Ultrapure water was generated with a Barnstead Thermo Scientific™ (Waltham, Massachussets). Quercetin-3-rhamnoside (Q-3-O-Rh) was from Extrasynthese (Lyon, France); catechin (C), quercetin-3-O-glucoside (Q-3-O-G), quercetin-3-O-xyloside (Q-3-O-X), quercetin-3-O-arabinofuranoside (Q-3-O-AF), quercetin-3-O-arabinopyranoside (Q-3-OAP) and chlorogenic acid (CA) were from Sigma (St. Louis, MO, USA). The other chemical, standards, and solvents were purchased from Sigma-Aldrich® (Saint Louis, MO). Plant Cell, Tissue and Organ Culture (PCTOC) Initiation of in vitro callus cultures Surface sterilization Plant material was washed with tap water to remove dust, insects, etc. Then, it was immersed in a 0.2% v/v Tween 20 solution for 60 s and stirred. Young leaves, stems and meristems were dipped in a sodium hypochlorite solution (NaClO, 4% active chloride) for 15 min or in mercuric chloride (HgCl2) at different concentrations (0.05, 0.1, or 0.2% w/v) with a previous treatment with ethanol (70%) (Majid et al. 2014) during 30 or 60 s. Fruits were sterilized with NaClO as described above and used to obtain seeds and embryos. In all cases, after the treatment, explants were washed three times with sterile distilled water under laminar flow cabinet. Oxidative browning removal As leaves, meristems, and stems produced browning exudation when cultured in vitro in MSRT medium (Nigra et al. 1987), different procedures were performed in order to avoid it. A first experiment was made culturing the HgCl2-sterilized explants in minimal White medium or White medium with the addition of activated charcoal and polyvinylpyrrolidone (PVP) (Trigiano 2011). A second experiment involved soaking the explants in filter sterilized (0.22 μm Millipore) antioxidant solutions (citric acid, ascorbic acid, L-cysteine HCl, AgNO3) at different concentrations during 5, 15, or 30 min after sterilization with HgCl2 (on line resource 1) (Karunaratne et al. 2014; Ndakidemi et al. 2014; Ahmad et al. 2016). Then, explants were transferred to sterile Petri dishes containing White medium supplemented with 8 g/L agar and 4.5 µM 2,4-dichlorophenoxyacetic acid (2,4-D), pH 5.6–5.8. The test was performed in complete darkness or under a 16-h light photoperiod as described below. A third experiment was performed subculturing the explants to fresh media for five times daily as was described by other authors (Ahmad et al. 2013). Presence or absence of oxidative browning exudation was visually assessed. Influence of explant source, culture media, and PGRs For callus induction, 1 cm2-young leaf fragments, 1 cmlength stem pieces, or 5 mm meristem pieces were separated from the whole plant. To obtain embryos and haustoria, the exocarp was removed from the fruits. Embryos were separated from the naked fruit with a scalpel under a laminar flow cabinet. As for haustorium, they developed after transferring the seeds to culture vessels with half strength MS medium with RT vitamins (Khana and Staba 1968), 8 g/L agar and 3% w/v sucrose, pH 5.6–5.8. After 15 days in culture, 1 cm-long pieces from the elongated haustoria were taken for callus initiation. In the case of leaves, stems, and meristems explants, after the sterilization and pre-treatment with the most effective antioxidant solution, they were transferred to glass tubes containing approximately 10 mL of culture medium. The culture medias assayed were MSRT, half-strength MSRT, and White all at pH 5.6–5.8, and Gamborg B5 at pH 5.5. All media included 8 g/L of agar and 30 g/L sucrose. PGRs were added as detailed in Table 1. In the case of embryos, after sterilization they were transferred to glass tubes with 10 mL of White medium with the addition of 500 mg/L casein hydrolysate (Johri and Bajaj 1962, 1964; Bajaj 1967; Ohofeghara 1971). The sucrose concentration and PGRs relationships were set according to a full factorial design. The pH was adjusted to 5.6–5.8. Four central points were used to analyze the effect of sucrose, 1-naphtalene acetic acid (NAA) and kinetin (Kin) concentrations on the initiation of in vitro cultures from embryos. This particular design was chosen to assess a possible third order interaction between the variables. The central points were included to evaluate the curvature of the resulting model. The response variable was the percentage of callus formation. Each experimental unit was composed of 10 embryos and was run twice. The 20 randomized runs where divided into two blocks, each of 10 runs, to split the experiment into 2 days due to the high workload (Table 2). The results were Table 1 Plant growth relationships used for initiating Ligaria cuneifolia in vitro cultures from leaves, stems, and meristems Treatment Auxin (µM) Cytokinin (µM) 1 2 3 4 5 6 7 8 9 10 11 12 13 2,4-D 2.25 2,4-D 4.50 2,4-D 10.0 IAA 2.25 IAA 4.50 IAA 10.0 2,4-D 2.25 2,4-D 2.25 2,4-D 4.50 2,4-D 4.50 2,4-D 10.0 2,4-D 10.0 NAA 5.40 – – – – – Kin 0.46 Kin 2.30 Kin 0.46 Kin 2.30 Kin 0.46 Kin 2.30 Kin 0.46 All treatments were made with n = 10 in MSRT, MS/2, Gamborg B5 or White culture media, in a growth chamber at 24 ± 2 °C, a 16-h light photoperiod with an irradiance 1.8 w/m2/s 2,4-D 2,4-dichlorophenoxyacetic acid, NAA 1-naphthaleneacetic acid, IAA indole-3-acetic acid, KIN kinetin 13 Plant Cell, Tissue and Organ Culture (PCTOC) Table 2 23 full factorial design matrix used for the in vitro cultures initiation from Ligaria cuneifolia embryo Standard order 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Block Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 1 Day 2 Day 2 Run order 9 17 3 19 10 13 5 15 6 14 2 12 4 11 8 20 1 7 16 18 Factor 1 Factor 2 Factor 3 Response 1 A:sucrose B:NAA C:kin Callus formation % v/v µM µM % 2 2 4 4 2 2 4 4 2 2 4 4 2 2 4 4 3 3 3 3 2.50 2.50 2.50 2.50 10.70 10.70 10.70 10.70 2.50 2.50 2.50 2.50 10.70 10.70 10.70 10.70 6.60 6.60 6.60 6.60 2.30 2.30 2.30 2.30 2.30 2.30 2.30 2.30 9.20 9.20 9.20 9.20 9.20 9.20 9.20 9.20 5.75 5.75 5.75 5.75 0 20 60 66 40 55 60 30 55 90 100 70 20 20 60 60 88 40 80 66 All cultures were performed on basal White medium with casein hydrolysate (500 mg/L), pH 5.6–5.8, in a growth chamber at 24 ± 2 °C, a 16-h light photoperiod and an irradiance 1.8 w/m2/s NAA 1-naphthaleneacetic acid, Kin kinetin analyzed using the software Design Expert 11 Trial version (Stat-Ease and 2018). Subcultures were performed every 4 weeks. In the case of haustoria, explants were directly transferred to tubes containing 10 mL Gamborg media with 3% w/v sucrose and PGRs at different relationships. The PGRs tested were 2,4-D, NAA, indole-3- acetic acid (IAA), 6-benzylaminopurine (BAP), indole butyric acid (IBA), zeatin, and Kin. The pH was adjusted to 5.5. The results were analyzed through a contingency table, a chi square test of independence and the Fisher’s Exact Test for Count Data (Table 3). This last test was used since more than 20% of the contingency table cells had expected count values lower than five (McHugh 2013). The chi squared was calculated using the function chisq.test and the Fisher’s Exact Test using the function fisher.test, both from the R base stats package in the R studio software (R Core Team 2018; RStudio Team 2018). Post hoc analysis was done using the adjusted standardized residuals (Adj Res) method following the recommendations of Sharpe (2015). 13 The 0.05 critical value (α) and its z critical = ± 1.96 were corrected using the Bonferroni method, as recommended in the case of large contingency tables by Sharpe (2015), resulting in α = 0.000757 and a z = ± 3.37. Subcultures were performed every 4 weeks. Growth index (GI): it was calculated as the ratio of the final fresh weight (FW) to the initial FW (Payne et al. 1991). Culture conditions: all the experiments were performed in a growing chamber at 24 ± 2 °C, under a 16-h light photoperiod using fluorescent daylight lamps (Narva T8 LT 18 W/760-010 daylight, Germany) with an irradiance intensity of 13.5 µmol/m2/s. In the case of oxidative browning removal, a set of the experiment was also performed in continuous darkness. Qualitative and quantitative analysis of polyphenolic compounds Thin layer chromatography (TLC): methanolic extracts from leaves, stems, and flowers (5% w/v) and calli Plant Cell, Tissue and Organ Culture (PCTOC) Table 3 Contingency table and chi square test of independence results for the experiment on the effect of plant growth regulators on the initiation of callus from L. cuneifolia haustoria Treatment 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Callus viability Observed Expected Adj Res Observed Expected Adj Res Observed Expected Adj Res Observed Expected Adj Res Observed Expected Adj Res Observed Expected Adj Res Observed Expected Adj Res Observed Expected Adj Res Observed Expected Adj Res Observed Expected Adj Res Observed Expected Adj Res Observed Expected Adj Res Observed Expected Adj Res Observed Expected Adj Res Observed Expected Adj Res Yes No 0 3.28 − 1.95 1 1.21 − 0.19 0 1.34 − 1.18 1 1.34 − 0.30 0 1.21 − 1.12 0 1.34 − 1.18 1 1.34 − 0.30 0 1.27 − 1.14 0 1.27 − 1.14 0 1.27 − 1.14 1 1.21 − 0.19 3 1.34 1.46 2 1.34 0.58 4 1.34 2.33 4 1.34 2.33 49 45.7 1.96 17 16.8 0.05 20 18.7 0.31 19 18.7 0.07 18 16.8 0.30 20 18.7 0.31 19 18.7 0.07 19 17.7 0.31 19 17.7 0.31 19 17.7 0.31 17 16.8 0.05 17 18.7 − 0.40 18 18.7 − 0.16 16 18.7 − 0.63 16 18.7 − 0.63 Marginals Plant growth regulator 49 2,4-D (2.25 µM) 18 2.4-D (4.5 µM) 20 2.4-D (10 µM) 20 2.4-D + KIN (2.25 µM:2.3 µM) 18 2,4-D + KIN (4.5 µM:2.3 µM) 20 2,4-D + KIN (10 µM:2.3 µM) 20 2,4-D + KIN (2.25 µM:4.65 µM) 19 2,4-D + KIN (4.5 µM:4.65 µM) 19 2,4-D + KIN (10 µM:4.65 µM) 19 2,4-D + KIN (0.45 µM:0.46 µM) 18 IAA (2.25 µM) 20 IAA (4.5 µM) 20 IAA (10 µM) 20 NAA + KIN (2.25 µM:2.30 µM) 20 NAA + KIN (4.5 µM:2.30 µM) 13 Plant Cell, Tissue and Organ Culture (PCTOC) Table 3 (continued) Treatment 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 13 Callus viability Observed Expected Adj Res Observed Expected Adj Res Observed Expected Adj Res Observed Expected Adj Res Observed Expected Adj Res Observed Expected Adj Res Observed Expected Adj Res Observed Expected Adj Res Observed Expected Adj Res Observed Expected Adj Res Observed Expected Adj Res Observed Expected Adj Res Observed Expected Adj Res Observed Expected Adj Res Observed Expected Adj Res Observed Expected Adj Res Yes No 2 1.27 0.66 4 1.27 2.46 0 1.27 − 1.14 1 1.21 − 0.19 1 1.27 − 0.24 0 1.14 − 1.08 0 1.21 − 1.12 0 1.27 − 1.14 1 1.07 − 0.07 1 1.14 − 0.13 2 1.34 0.58 7 1.34 4.97 4 1.21 2.57 1 1.34 − 0.30 2 1.34 0.58 1 1.27 − 0.24 17 17.7 − 0.17 15 17.7 − 0.65 19 17.7 0.31 17 16.8 0.05 18 17.7 0.07 17 15.9 0.28 18 16.8 0.30 19 17.7 0.31 15 14.9 0.03 16 15.9 0.03 18 18.7 − 0.16 13 18.7 − 1.34 14 16.8 − 0.69 19 18.7 0.07 18 18.7 − 0.16 18 17.7 0.07 Marginals Plant growth regulator 19 NAA + KIN (10 µM:2.30 µM) 19 NAA + KIN (0.45 µM:0.46 µM) 19 BAP (0.25 µM) 18 BAP (1 µM) 19 BAP (0.5 µM) 17 2,4-D + zeatin (2.25 µM:0.23 µM) 18 2,4-D + zeatin (4.5 µM:0.23 µM) 19 2,4-D + zeatin (10 µM:0.23 µM) 16 2,4-D + zeatin (2.25 µM:0.47 µM) 17 2,4-D + zeatin (4.5 µM:0.47 µM) 20 2,4-D + zeatin (10 µM:0.47 µM) 20 2,4-D + zeatin (0.45 µM:0.47 µM) 18 IBA + BAP (4.90:0.5 µM) 20 IBA + BAP (4.90:0.4 µM) 20 IBA + BAP (4.90:0.2 µM) 19 2,4-D + IAA (2.25:2.25) Plant Cell, Tissue and Organ Culture (PCTOC) Table 3 (continued) Treatment 32 33 Callus viability Observed Expected Adj Res Observed Expected Adj Res Marginals Yes No 0 1.34 − 1.18 0 1.27 − 1.14 44 20 18.7 0.31 19 17.7 0.31 613 Marginals Plant growth regulator 20 2,4-D + IAA (4.5:4.5) 19 2,4-D + IAA (0.46:0.46) 657 Statistics Chi square test of independence results X2 N Fisher’s exact test for count data p = 0.0000007039 Value df p 74.225 657 32 0.00003325 The highlighted value, treatment 27, corresponds to an adjusted standardized residual (Adj Res) superior than 3.37. Cultures were performed in Gamborg media with 3% w/v sucrose, pH 5.5, in a growth chamber at 24 ± 2 °C, a 16-h light photoperiod and an irradiance of 1.8 w/m2/s 2,4-D 2,4-dichlorophenoxyacetic acid, NAA 1-naphthaleneacetic acid, IAA indole-3- acetic acid, BAP 6-benzylaminopurine, IBA indole butyric acid, zeatin, Kin kinetin initiated from embryo (2.5% w/v) were made. Plant material was previously air-dried in darkness (25 °C for 7 days). The qualitative analysis of polyphenols by TLC was made with Silica Gel 60 (Merck) as stationary phase and ethyl acetate: formic acid: acetic acid: water (100:11:11:23) as mobile phase. Flavonoids and hydroxycinnamic acid derivatives were revealed with a 1% v/v methanolic solution of the reagent for natural products (NP, AEDBE: 2-aminoethyl diphenyl borate ester, SigmaAldrich), and proanthocyanidins with an ethanolic solution of vainillin:HCl (4:1). High performance liquid chromatography-tandem mass spectrometry (HPLC MS/MS): dried callus and parental plant material obtained as described above were successively extracted with pure methanol (5% w/v) and combinations of 80% methanol–water and 50% methanol–water with continuous shaking. Then, they were evaporated to dryness under reduced pressure in a rotary evaporator and further resuspended in methanol, filtered and finally diluted 1/5 in 0.1% aq. formic acid. HPLC–MS/MS system consisted of UltiMate 3000 HPLC coupled to a TSQ Quantum Access MAX Triple Quadrupole Mass Spectrometer with electrospray ionization (Massachusetts). Positive mode was employed for quercetin glycosides and negative mode for chlorogenic acid. The column was a C18 Hypersil 150 × 4 mm, 5 µm particle size Thermo Scientific™. HPLC conditions: The mobile phase consisted of a gradient of 0.1% formic acid in methanol and 0.1% aqueous formic acid 15:85 (0 min), 40:60 (25 min), 40:60 (50 min), 85:15 (60 min), return to starting conditions and equilibration in 15 min. Flow rate was 1 mL/min and the temperature of the oven and sampler were set at 40 °C and 20 °C respectively. Injection volume was 25 µL MS/MS conditions: Tuning conditions were as follows: spray voltage 3.5 kV, vaporizer temperature 233 °C, capillary temperature 314 °C, while sheath gas pressure and aux gas pressure were set at 10 and 45 units respectively. The instrument method comprised two scan events involving single ion monitoring (SIM) and single reaction monitoring (SRM). Mix standard solution: Stock standard solutions of 1 mg/mL were prepared in methanol. A mix standard solution was prepared by combining aliquots of each stock standard 13 Plant Cell, Tissue and Organ Culture (PCTOC) solution and diluting with 0.1% aq. formic acid to a final concentration of 1 µg/mL. Results and discussion Initiation of in vitro cultures Surface sterilization As for leaves, stems, and meristems the most efficient treatment (80% sterility) was obtained by dipping the explants in alcohol 70% (30 s exposure) followed by a treatment with HgCl2 (indistinctively 0.05 or 0.1% w/v). In the case of seeds and embryos the efficiency of sterilization was 85% and 92.4% respectively. Fig. 1 Ligaria cuneifolia explants and callus. a Fruit with pedicel. b Seed in half strength MS medium with RT vitamins and 3% w/v sucrose, day 0. c 3 day developed haustorium in B5 medium with 2,4-D (2.25 µM). d Embryo in White medium, day 0. e Embryo in White medium with casein hydrolysate 500 mg/L, NAA KIN (2.70 µM:9.20 µM), and sucrose 4% (w/v), day 9. f Callus developed from embryo in White medium with casein hydrolysate 500 mg/L, ANA: Kin (2.70 µM:9.20 µM), sucrose 4% (w/v), 3 months in culture. Cultures are developed in a growth chamber at 24 ± 2 °C, 16-h light photoperiod, and 13.5 µmol/m2/s irradiance 13 Oxidative browning removal Explants from leaves, meristems, and stems in MSRT medium produced browning exudation within a few hours resulting toxic and leading to the death of the tissues in a few days, as was reported previously with other species (Debergh and Read 1991). The same was observed in explants cultured in White medium or White medium with adsorbing agents (activated charcoal and PVP). Sub-culturing the explants every 24 h to fresh medium strongly reduced oxidative browning from the third transfer, although it resulted a cumbersome procedure (time and labor consuming). Finally, we have succeeded in inhibiting browning by soaking the explants in different antioxidant solutions (online resource 1) either in darkness or under a 160-h light photoperiod, Plant Cell, Tissue and Organ Culture (PCTOC) Table 4 ANOVA table for in vitro cultures initiation from Ligaria cuneifolia embryo Source Sum of squares df Mean square Block Model A-sucrose B-NAA C-Kin AB AC BC ABC Curvature Residual Lack of fit Pure error Cor total 57.80 9060.75 2652.25 841.00 1296.00 196.00 1.00 2352.25 1722.25 1051.25 3116.20 1866.20 1250.00 13286.00 1 7 1 1 1 1 1 1 1 1 10 8 2 19 57.80 1294.39 2652.25 841.00 1296.00 196.00 1.00 2352.25 1722.25 1051.25 311.62 233.27 625.00 F-value p-value 4.150 8.510 2.700 4.160 0.629 0.003 7.550 5.530 3.370 0.021 0.015 0.132 0.069 0.446 0.956 0.021 0.041 0.096 Significant Significant 0.3730 0.871 Non-significant Significant Significant A sucrose, B 1-naphthaleneacetic acid (NAA) and C kinetin (Kin). AB. AC and BC represent second order interactions between the variables. ABC represents a triple order interaction. p values lower than 0.05 were considered significant Table 5 In vitro culture initiation from Ligaria cuneifolia embryo expected values if the optimal conditions for each variable are used Response Predicted mean 85 Callus formation (%) Std dev SE mean 95% CI low for mean 95% CI high for mean 17.6 12.3 57.5 112.5 In this case sucrose = 4% w/v, 1-naphthaleneacetic acid (NAA) = 2.50 μM and kinetin (Kin) = 9.20 μM. The value 112.5% should be disregarded since the maximum value can be 100% Std Dev standard deviation, SE standard error, CI confidence interval except with citric acid (200 mg/L). Unfortunately, we could not induce callus formation after these pre-treatments even though toxic browning was inhibited. Influence of explant source, culture media, and PGRs Calli were successfully initiated from embryo and haustoria (Fig. 1). Moreover, such cultures did not produce browning at the conditions tested; hence, the antioxidant treatment was not required. On the other hand, leaves, meristems, and stems did not produce viable callus at the conditions tested irrespectively of the antioxidant pre-treatment. Conversely, Rousset et al. (2003) working with the obligated parasitic weed Striga hermonthica, obtained callus from young leaves on MS medium with 2% w/v sucrose, 200 mg/L casein hydrolysate and 0.4% Agargel. Establishment of callus from Viscum album was also reported from stems in MS medium plus 5.0 mg/L 2,4-D but at a very low frequency (Lee and Lee 2013). As for the influence of media and PGRs, the highest frequency of callus induction from embryo was 85% in White media with sucrose 4% w/v as carbon source and NAA:Kin (2.5 µM:9.2 µM) as PGRs (GI = 1.16 ± 0.08) (Fig. 1a, d–f). The result of the full factorial design showed some variability between days (Table 2, column response). Analyzing the ANOVA results (Table 4), the sum of squares for the block effect (day variability) was low compared to other factors. The model was significant (p < 0.05 criteria) as well as the factor A (sucrose, p = 0.015), the double interaction BC (NAA and Kin, p = 0.021) and the triple interaction ABC (p = 0.041). A multiple linear equation was built with the information provided by the full factorial experiment allowing us to predict a mean value of callus formation. Callus formation (%) = −123.3 + 45.9 × sucrose + 18.4 × NAA + 22.6 × Kin − 5.1 × sucrose × NAA − 4.8 × sucrose × Kin − 3.1 × NAA × Kin + 0.7 × sucrose × NAA × Kin As an example, using the optimal conditions for this experiment, i.e. sucrose 4% w/v, NAA 2.50 µM, and Kin 13 Plant Cell, Tissue and Organ Culture (PCTOC) 85 B A 85 100 100 80 60 Calli formation (%) Calli formation (%) 80 40 20 0 2.50 60 40 20 0 2 4.55 9.20 3 6.60 7.47 3 8.65 B: NAA (µM) 4 4 10.70 4 5.75 A: Sucrose (% v/v) 3 4.03 C: Kin (µM) 4 3 2.30 A: Sucrose (% v/v) 2 Calli formation (%) 2 2 60 85 C Prediction 85 D 85 100 60 C+: 9.20 2 2 20 72.5 40 20 4 0 9.20 10.70 7.47 C: Kin (µM) Calli formation (%) 80 2 2 45 63 A+: 4 8.65 A: Sucrose (% v/v) 5.75 C: Kin (µM) 6.60 4.03 4.55 2.30 2.50 Fig. 2 Graphical representations of the results from the 23 full factorial design for initiating Ligaria cuneifolia in vitro cultures from embryos. Subfigures a–c are response surface graphs where the y-axis is the percentage of calli formation and the x-axis and z-axis are the different variables. Subfigure d is the tridimensional representation of the design space where each axis represents a variable. 13 2 2 B: NAA (µM) C-: 2.30 10 B-: 2.50 B: NAA (µM) A-: 2 47.5 B+: 10.70 y = Kin, x = NAA and A = sucrose. The vertices represent the combination of variables. i.e. top back left vertex corresponds to high level of Kin (9.20 µM), low level of NAA (2.50 µM), and high level of sucrose (4% w/v) giving a response of callus formation of 85%. The number 2 in each vertex represent the number of replicates and the 4 in the center of the cube is the number of center points Plant Cell, Tissue and Organ Culture (PCTOC) A B C (+) i ii iii iv i ii iii iv Fig. 3 a Monodimensional analysis of proanthocyanidins from Ligaria cuneifolia methanolic extracts of leaf (i), stem (ii), flower (iii), and callus (iv). The different bands correspond to compounds with different molecular weight (monomers, oligomers and polymers). Catechin [C (+)] was detected in all tissues. Spray reagent: ethanolic solution of vainillin/chlorhydric acid (5% v/v). b Monodimensional analysis of flavonols from the methanolic extract of leaf (i), stem (ii), flower (iii), and callus (iv). Flavonoids (yellow-orange compounds) and hydroxycinnamic acid derivatives (blue-greenish compounds) are present in all cases. Spray reagent: AEDBE: 2-aminoethyl diphenyl borate ester, Sigma-Aldrich). Wavelength: 366 nm 9.20 µM, we can expect a central value of 85% with a confidence interval (95% CI) between 58 and 100% of callus formation (Table 5). The tridimensional representation of the model is shown in Fig. 2. The highest frequency of callus formation from haustoria was 35% in Gamborg medium (GI 1.04 ± 0.07) with the addition of sucrose 3% w/v as carbon source and 2,4-D + zeatin (0.45:0.47 µM) as PGRs (Fig. 1a–c). The p values for both tests resulted significant for p < 0.05, chi square test p = 0.00003325 and Fisher’s Exact Test p = 0.0000007039 (Table 3). Our results differ from those obtained with other parasitic or hemiparasitic species. In the case of the obligate parasitic Orobanche ramosa, calluses were obtained on TB medium with 0.5 and 1 mg/mL IAA, 20 and 25 mg/L GA3, or 5–15 mg/L Kin (Batchbaroiva et al. 1999). The obligate parasite Cuscuta reflexa produced callus from explants of the basal portion of seedlings as well as from shoots (vines) in a modified MS (MMS) medium supplemented with 1 mg/L benzyl adenine (BA) and 3 mg/L NAA (Srivastava and Dwivedi 2001). The Australian mistletoes Amyema and Amylotheca, developed callus and seedlings on modified MS medium with IAA or NAA (Hall et al. 1987). Dendrophthoe, a stem hemiparasitic species produced in vitro cultures in White’s medium (Ram and Singh 1991). Nuytsia, the largest of all mistletoes produced in vitro cultures on White’s medium with casein hydrolysate, IBA, and Kin (Nag and Johri 1976). On the other hand, callus from Viscum album L. var. lutescens M. was induced on a modified half strength MS medium supplemented with 1.0 ppm Kin and 10.0 ppm NAA (Fukui et al. 1990). In V. album L. var. coloratum, callus induction was observed in MS medium containing 5.0 mg/L of 2,4- D, but at a very low frequency (4.8%)(Lee and Lee 2013). Other authors (Kim et al. 2008) referred that the frequency of callus formation was higher (27.3%) when V. album L. var. coloratum flower buds were cultured on B5 medium containing 0.1 mg/L of IAA. Qualitative and quantitative analysis of polyphenolic compounds A preliminary screening of polyphenolic compounds by TLC showed the presence of flavonols, hydroxycinnamic acids, proanthocyanidins of high degree of polymerization, and catechin in callus and plant extracts (Fig. 3a, b). The intensity of the bands corresponding to flavonoids and proanthocyanins from callus was lower than those from the plant, and it was higher in the case of hydroxycinnamic acid. We also performed HPLC MS/MS on the callus and parent plant material for metabolite identification by means of retention time, molecular weight and fragmentation pattern (Fig. 4). Following this, quantification was also carried out by comparison with the respective standard. Results are expressed as µg analyte/g dried extract (mean of four replicates ± SEM) (Table 6). Catechin, quercetin-3-O-glucoside, quercetin-3-O-arabinopyranoside, quercetin-3-Oarabinofuranoside, quercetin-3-O-xyloside, quercetin-3-Orhamnoside and chlorogenic acid could be identified. These findings agree with previous reports on the wild plant (Dobrecky et al. 2017). It is noteworthy that there is approximately a 5-fold-increase in the content of the detected metabolites in the parent plant. This is expected, given that the parent plant has its full metabolic machinery in place. Further experiments will be conducted in order to establish cell suspension cultures and to analyze their kinetics of growth and polyphenolics profile and content. Also, analysis of the influence of the host and the phenological state of the wild plant on its polyphenolic content will be performed. 13 Plant Cell, Tissue and Organ Culture (PCTOC) Fig. 4 HPLC–MS/MS chromatogram of the callus extract with identified compounds. Quercetin glycosides were analyzed in positive ionization mode and phenolic acids in negative ionization mode. Catechin (C), quercetin-3-O-glucoside (Q-3-O-Gl), quercetin-3-O-arabi- nopyranoside (Q-3-O-AP), quercetin-3-O-arabinofuranoside (Q-3-OAF), quercetin-3-O-xyloside (Q-3-O-X), quercetin-3-O-rhamnoside (Q-3-O-Rh), chlorogenic acid (CA) Conclusion NAA:Kin (2.5:9.2 µM) as PGRs. In the case of haustoria, the highest frequency of callus induction (35%) was obtained in Gamborg media with sucrose 3% w/v, and 2,4-D and zeatin (0.45:0.47 µM) as PGRs. The HPLC MS/MS analysis unequivocally confirmed the presence of polyphenolic compounds establishing the presence of quercetin glycosides and phenolic acids in callus as was previously seen in the wild plant. The initiation and establishment of L. cuneifolia in vitro cultures was challenging. Leaves, stems, and meristems resulted recalcitrant to in vitro culture in the conditions tested. On the other hand, embryos and haustoria succeeded in producing callus. Embryos gave the highest frequency of callus induction (85%) in White medium with sucrose 4% w/v and 13 Plant Cell, Tissue and Organ Culture (PCTOC) Table 6 Comparative polyphenolic content of calluses and parental plant from Ligaria cuneifolia Analyte Parent plant (µg/g dried extract) Callus (µg/g dried extract) Catechin Q-3-O-glucoside Q-3-O-xyloside Q-3-O-arabinopyranoside Q-3-O-arabinofuranoside Q-3-O-rhamnoside Chlorogenic acid 2225.7 ± 5.6 12.1 ± 4.3 28.3 ± 3.7 24.8 ± 4.2 27.6 ± 4.5 13.4 ± 3.1 31.5 ± 2.9 464.3 ± 2.2 2.2 ± 0.1 5.4 ± 0.5 5.2 ± 0.4 5.3 ± 0.5 2.4 ± 0.3 6.1 ± 0.7 Each value is the mean (4 replicates) ± SEM. quercetin-3-Oglucoside(Q-3-O-glucoside), quercetin-3-O-xyloside(Q-3-O-Xyloside), quercetin-3-O-arabinopyranoside(Q-3-O-arabinopyranoside), quercetin-3-O-arabinofuranoside (Q-3-O-arabinofuranoside), quercetin-3-O-rhamnoside (Q-3-O-rhamnoside) Results are expressed as mean ± SEM Acknowledgements We wish to thank Dr. Sabrina Flor for her assistance in the mass spectrometry analysis (Pharmaceutical Technology Department of the Faculty of Pharmacy and Biochemistry from the University of Buenos Aires), Dr. Javier Calcagno (CONICET/CEBBAD) for his advice regarding the statistical analysis, Dr. Chana Pilberg (Universidad Maimónides) for kindly providing us from plant material from Merlo, and M. Julian Schecter for his advice and careful revision of English. This work was supported by Fondo Nacional de Ciencia y Tecnología (FONCyT), Ministerio de Ciencia, Tecnología e Innovación Productiva from Argentina (PICT2015-2024), Universidad de Buenos Aires, and Universidad Maimónides. M A Alvarez and M Laguia-Becher are researchers from CONICET, MVR has a scholarship from CONICET-Universidad Maimónides, and MLB has a scholarship from FONCyT. Author’s contributions MVR and MLB carried out the experiments and participated in drafting the manuscript; CC and FB carried out experiments; ML-B and CD participated in the analysis of the results; AP participated in selecting, collecting, and classifying the plant material; LUS performed statistical analysis; MLW, RAR and MAA initiated the project and supervised the work throughout, MAA also drafted the manuscript. All authors read and approved the final manuscript. 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