6 benzylaminopurine

The aseptic culture of S. betulifolia ssp. aemiliana obtained in the Laboratory of Biotechnology in the CSBG SB RAS (Novosibirsk) was used in this study [12 ,13 (link)]. The explants were axillary buds of a generative plant introduced into the experimental field of the Laboratory of Phytochemistry (CSBG SB RAS, Russia) from Kunashir Island (the caldera of Golovin’s volcano). Microshoots were incubated under a 16 h photoperiod at 40 μmol m⁻² s⁻¹ light intensity provided by cool white fluorescent lamps at 23 ± 2 °C in the MS [14 (link)] solid medium (supplemented with 0.6% of agar; PanReac, Barcelona, Spain) containing 3% of sucrose (Shostka Chemica l Reagents Plant, Shostka, Ukraine). Micropropagation of S. betulifolia ssp. aemiliana was carried out in the medium containing growth regulators: 5 µM 6-benzylaminopurine (Sigma-Aldrich, St. Louis, MO, USA) and 1 µM α-naphthylacetic acid (Sigma-Aldrich, St. Louis, MO, USA); elongation was performed in hormone-free media of the same mineral composition. Passage duration was 4–6 weeks.

LaCl₃ (purity >99.99%) was purchased from Aladdin Bio-Chem Technology Co. Ltd (Shanghai, China). FM4-64 was purchased from Thermo Fisher Scientific Co. Ltd (Shanghai, China). DPI, TyrA23, Murashige and Skoog medium (MS), methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate (Benomyl), 6-benzylaminopurine, indole acetic acid (IAA), and JA were purchased from Sigma-Aldrich Co. Ltd (Shanghai, China). Flg22 was purchased from MedChemExpress Co. Ltd (Shanghai, China). Anti-plant actin mouse monoclonal antibody (A01050) was purchased from Abbkine Scientific Co. Ltd (Wuhan, China). Goat anti-mouse IgG H&L [horseradish peroxidase (HRP)] (ab205719) and HRP anti-GFP antibody (ab6663) were purchased from Abcam Co. Ltd (Shanghai, China). Other chemicals used in this study were analytical reagents and were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China).

When green fluorescence was visible in all areas of the upper systemic young leaves, leaves were cut off, washed with a washing soap, and rinsed thoroughly 2–3 min in running water, repeated two-three times. Then, leaves were transferred in a laminar flow cabinet, and the following procedures were carried out under sterile conditions: sterilized in 70% ethanol about 60 s, two times rinsed thoroughly in sterile distilled water, cut to two to three pieces (explants), dried on a sterile filter paper for a few minutes, and put on the regeneration medium (MSR). MSR was the nutrition agar Murashige and Skoog (MS) medium [38 (link)] supplemented with 0.1 mg/L of 1-Naphthaleneacetic acid (Merck, Kenilworth, NJ, USA) and 1 mg/L of 6-Benzylaminopurine (Sigma-Aldrich, St. Louis, MO, USA) [36 (link),37 (link)], and with 700 mg/L cefotaxime (or without antibiotic for tissue culture “rejuvenation”). Petry dishes with the explants were placed in a growth chamber at 25–26 °C and 16 h light photoperiod. The obtained regenerants were maintained in vitro on MS medium without any supplements. The regenerants with roots were transferred from in vitro to soil, adapted to the greenhouse conditions (2–3 days), and then grew normally for 1–1.5 months. In total, 10 regenerants of different ages (2–5.5 months old) were used for the greenhouse experiments to assess recombinant protein production.

Root explants from two-week-old in vitro seedlings were used as the explant source for callus initiation. The roots were cut into small segments (10–15 mm in length) and aseptically cultured on MS medium with agar (0.8%), sucrose (3%), 2,4-dichlorophenoxyacetic acid (2,4-D; 1 mg/L), 6-benzylaminopurine (BAP; 0.5 mg/L), and kinetin (KN; 0.5 mg/L) (Sigma-Aldrich, St. Louis, MO, USA). Callus cultures were incubated for three weeks in growth chambers at 25 ± 1 °C and a 16 h photoperiod (30 µmol·m⁻²·s⁻¹) provided by 40 W white fluorescent lamps. Cell suspension cultures were initiated by using friable calli in liquid MS medium supplemented with 2,4-D (1 mg/L) and thidiazuron (TDZ; 0.5 mg/L; Sigma-Aldrich) in 250-mL Erlenmeyer flasks. The cultures were maintained under continuous shaking at 110 rpm in an orbital shaker and incubated at 25 ± 1 °C and a 16 h photoperiod (30 µmol·m⁻²·s⁻¹) provided by cool white fluorescent lamps.

All the chemicals used were analytical grade. Clorox (5.25% of sodium hypochlorite), sulfuric acid, sucrose, agar, sodium hydroxide (NaOH), hydrochloric acid (HCl), indole-3-acetic acid (IAA), picloram, α-Naphthalene acetic acid (NAA), 6-Benzylaminopurine (BAP), kinetin (Kin), Naphthalene acetic acid (NAA), ethanol, distilled water, 2, 2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), quercetin, ascorbic acid, sodium nitrate, aluminum chloride, potassium persulfate, Folin–Ciocalteu reagent and 2,3,5-triphenyl-tetrazolium chloride (TZ), were purchased from Sigma-Aldrich GmbH (Munich, Germany). Free peat (peat moss Holland) soil media were purchased from local nursery. UV-Vis spectrophotometer were used to measure absorbance (UV-Vis. Cary 4000, Agilent, UK). Rotary evaporator (Rotavapor R-200, Switzerland).

Korean pine megametophyte was used as an explant and put in EC induction medium. The EC induction medium was mLV [42 (link)] basic medium supplemented with L-glutamine (500 mg·L⁻¹), sucrose (30 g·L⁻¹), acid hydrolyzed casein (500 mg·L⁻¹), 1-Naphthylacetic acid (2 mg·L⁻¹), 6-benzylaminopurine (1.5 mg·L⁻¹), and gelrite (4 g·L⁻¹) (gelrite, Sigma Aldrich, St Louis, MO, USA). Five explants were inoculated into each cultural dish (90 mm diameter, 20 mm depth). After adjusting pH to 5.8, we carried out high-temperature and high-pressure sterilization (121 °C, 20 min), cooled the cultural medium to 55–60 °C, and added L-glutamine by filtration sterilization (0.22 μm filter). After inoculating, the cultural dishes with explant were cultured in the dark at 23 ± 2 °C.
When the EC cluster grew to a diameter of 1–2 cm, it was transferred to the proliferation medium. The medium was mLV basic medium supplemented with sucrose (30 g·L⁻¹), 2,4-dichlorophenoxyacetic acid (2.26 μmol·L⁻¹), 6-benzylaminopurine (0.44 μmol·L⁻¹), acid hydrolyzed casein (500 mg·L⁻¹), L-glutamine (500 mg·L⁻¹), and gelrite (4 g·L⁻¹). The EC was sub-cultured every two weeks. The initial inoculation amount of EC was 0.2 g, 3 replicates per cell line, and the cultural conditions were the same as those of induction culture.

To study how nutrient availability affects growth of B. cinerea in the presence of CK, 6-benzylaminopurine (6-BAP; 100 μM, Sigma-Aldrich B3408) was dissolved in 10 mM NaOH and added to the different media detailed in Table 1. To study how B. cinerea responds to metabolic inhibitors in the presence of CK, 2-deoxy-d-glucose (2-DG; Sigma-Aldrich) and oligomycin (OM; Sigma-Aldrich) were used. 2-DG was added to the different media detailed in Table 1 above at a final concentration of 2.5 mM. oligomycin (OM), which proved too inhibitory in defined media, was assayed in solid PDA alone at 0.1 μg/mL. For liquid medium, B. cinerea spores (10⁶/mL) were grown in defined medium or PDB for 72 h, after which the fungal mass was dried, and the dry weight was measured. For PDA plates, mycelial plugs (5 mm) taken at ~1 cm from the edge of a fresh plate were placed at the center of PDA plates and incubated at 22 ± 2°C in the dark for 5 days.