Plant material
Fruits of different mangabeira plants (H. speciosa) from the Gameleira Farm in the georeferenced region of the municipality of Montes Claros de Goiás, state of Goiás (GO), Brazil (16° 0′ 28″ S, 51° 23′ 49″ W) were transported to the Plant Tissue Culture Laboratory of the Goiano Federal Institute, Rio Verde Campus, GO. The collection of biological material complied with legal aspects in accordance with federal law No. 12.651 of May 25, 2012. Considering the provisions of Article 21, the collection of fruits in small volumes fulfilled the ripening period, and in accordance with the mentioned guidelines, for the study it was not necessary institutional authorization to carry out the collection.
After manual pulping, the seeds were coated with gauze and immersed in running water for 30 min, followed by 70% alcohol for 1 min, and then immersed in sodium hypochlorite (NaClO) solution (20% of the commercial solution) with a drop of polysorbate (Tween®) for 15 min. Then three rinses were performed in a laminar flow hood using autoclaved distilled water to remove residues of the disinfecting solutions.
Disinfected seeds were inoculated aseptically in test tubes (25 × 150 mm) containing 10 mL of MS medium11, with 50% concentrations of salts and vitamins, 30 g L−1 of sucrose, 3.5 g L−1 of agar (Dinâmica®), and pH adjusted to 5.7 ± 0.3 (Fig. 1a). The medium had been autoclaved at 121 °C and 1.05 kg cm−2 pressure for 20 min. The test tubes were sealed with a plastic (polypropylene) lid and kept in a growth room at a temperature of 25 ± 3 °C and relative humidity of 45 ± 2%, and the medium was changed every 30 days. They were kept under a photoperiod of 16 h under photosynthetically active radiation (45–55 μmol m−2 s−1) provided by 20-W LED lamps (Lanao Tubes series, China) whose spectral composition (400–700 nm) was verified using a USB2000 spectroradiometer (OceanOptics). Explants pre-established in vitro (Fig. 1b) were kept under these conditions.
Process of the in vitro establishment and culture of H. speciosa (Gomes) cell culture. Seeds disinfected and inoculated in a tube (a). Explants obtained after 60 days of culture (b). Friable calli 120 days after the beginning of induction from leaves (c). Stable cell suspension (d). Orbital shaker used to culture the cell suspension under light support with 50 μmol m−2 s−1 irradiance (e). Bar = 1 cm.
Callus induction and cell suspension
Leaf fragments (1 cm2) of previously established seedlings were inoculated in glass flasks containing 40 mL of 50% MS medium and 30 g L−1 of sucrose supplemented with 3.5 g L−1 of agar. To the growth medium was added a combination of growth regulators at a concentration of 2.5 mg L−1 of naphthalene acetic acid (NAA) and 1 mg L−1 of 6-benzylaminopurine (BAP), and the pH was adjusted to 5.7 ± 0.3. The flasks containing the cultures were kept in a room with the same environmental conditions under which the explants were obtained.
To establish the cell-suspension culture, friable calli (Fig. 1c) were selected and transferred to 250-mL glass flasks containing 40 mL of medium with the respective concentrations of salts and growth regulators in which the calli had been cultured but without agar. The cultures were placed under agitation (shaker LS-183, Solab™) at 110 rpm under the same light and temperature conditions described above (Fig. 1d,e). After 3 months, stable and contamination-free cultures were selected to begin the elicitation process.
Carbon source in the culture medium
To initiate the elicitation process, approximately 20 mL of inoculum and 20 mL of 50% MS medium containing 1 mg L−1 BAP and 2.5 mg L−1 NAA were added, supplemented with different carbon sources (sucrose, glucose, fructose, sorbitol, and mannitol) at different concentrations (1, 2, 3, 4, and 5%) and with the pH adjusted to 5.7 ± 0.03 before autoclaving. The flasks were kept on an orbital shaker at 110 rpm for 30 days in a growth room under the same environmental conditions mentioned above.
Evaluations
Biometric characteristics
At the end of the culture period after the addition of eliciting agents, the pH, electrical conductivity, and osmotic potential were measured in the suspensions, which were then filtered through 0.45-µm filter paper to remove the culture medium. After filtration, the fresh cell weight was measured, and the samples were then placed in a forced-air oven at 35 °C ± 2 for 24 h to measure the dry weight.
Electrical conductivity, pH, and osmotic potential
A portable conductivity meter and pH meter were used to measure the electrical conductivity and pH of the cell suspensions, respectively. The Vapro® osmometer (model 5600) was used to evaluate the osmotic potential.
Cell viability and cell area
For cell viability analysis, a 50-µL aliquot was added to the cell-suspension culture sample in a 1.5-mL microtube along with 50 µL of 0.2% trypan blue dye (Sigma-Aldrich, Brazil). Next, 50-µL aliquots were transferred to slides and counted under an optical microscope (BX61, Olympus). The plant cells were photomicrographed using a U-photo system with a DP73 camera. These images were analyzed using ImageJ® software, version 1.52, to measure the cell areas.
Phytochemical analysis
The extracts were prepared using 0.1 g of dry sample with 2 mL of methanol-HPLC (Neon) in an ultrasound bath for 30 min. The extracts were filtered through cotton and a membrane filter (Advantec HP020AN—20 µm). Next, the chromatographic analysis was performed in a Shimadzu HPLC with a SPD-M20A photodiode detector (λ = 254 nm) and an LC18 column (25 cm × 4.6 mm, 5 µm, Supelcosil) coupled to a 2-cm LC18 precolumn (Supelguard, Supelco) in an oven set at 30 °C.
Ten microliters of each extract was injected in triplicate, and the phenolic compounds were detected. The elution flow rate was set to 1 mL/min, with 0.1% acetic acid as mobile phase A and HPLC-grade methanol (Neon) as mobile phase B. The concentration of mobile phase B increased from 10 to 66% over 32 min, decreased from 66 to 10% from 32 to 35 min, and stayed at 10% for 5 min, for a total run time of 40 min.
The compounds present in the samples were detected by comparison with the peaks of known phenolic and flavonoid standards, and quantification was performed using the standard-curve equations for the following standards: gallic acid, epicatechin, caffeic acid, chlorogenic acid, ferulic acid, orientin, vitexin, rosmarinic acid, myricitrin, isovitexin, hesperidin, rutin, quercetin-3-glucoside, kaempferol-3-galactoside, quercitrin, kaempferol-3-glucoside, kaempferol-3-rutinoside, quercetin, kaempferol, luteolin, and apigenin.
Statistical analysis
The experimental design was completely randomized, in a 5 × 5 factorial arrangement, with five carbon sources (sucrose, glucose, fructose, sorbitol, and mannitol) at five different concentrations (1, 2, 3, 4, and 5%), with five replicates (40-mL flask). After analysis of variance with the F test (5%), the means of the quantitative variables were analyzed by regression and the means of the qualitative factors by Tukey’s test. These tests were run in SISVAR software at 5% probability12.
Pearson’s correlation was used to quantify the relationships between the variables analyzed. To obtain the estimates, the software R version 3.5.2 (R Development Core Team, 2018) was used, and the heatmap graph was prepared using the Corrplot package.
