L 1 sucrose

Seeds of all species were sterilized in 70% ethanol for 90 s and commercial bleach for 12 min and rinsed three times in sterile distilled water for 3, 7, and 10 min. Seeds were germinated on a solidified half-strength Murashige and Skoog (MS) medium [33 (link)] in sterile conditions for 2 weeks. Sterile leaves of the seedlings were placed on ½ MS media [33 (link)] or ½ MS with various combinations of auxins (2,4-dichlorophenoxyacetic acid; 2,4-D or α-naphthaleneacetic acid; NAA) and cytokinins (6-benzylaminopurine; BAP or kinetin; KIN) as follows: 2 mg L-1 2,4-D + 2 mg L⁻¹ BAP, 2 mg L⁻¹ 2,4-D + 2 mg L⁻¹ KIN, 2 mg L⁻¹ NAA + 2 mg L⁻¹ BAP, 2 mg L⁻¹ NAA + 2 mg L⁻¹ KIN, 0.5 mg L⁻¹ NAA + 5 mg L⁻¹ BAP.
All media were supplemented with 30 g L⁻¹ sucrose (Sigma-Aldrich, St Louis, MO, USA) and solidified with 8 g L⁻¹ agar (Duchefa Biochemie, Amsterdam, The Netherlands). The pH of the medium was adjusted to 5.7–5.8. The cultivation was carried out under stable artificial conditions, in a growth chamber at 25 ± 3 °C with a 16 h photoperiod under cool-white-fluorescent lamps (light intensity 70–100 μmol m⁻² s⁻¹). Culture media and instruments were sterilized in a steam autoclave (121 °C, 1.05 bar; Prestige Medical, Lancashire, UK). The tissue cultures were passaged onto fresh media every 2–3 weeks. The callus tissue was initiated to develop after 1–3 weeks, depending on the species.

To obtain cell suspensions, embryogenic cultures were multiplied and maintained in the basic liquid culture medium MSG [38 ] supplemented with 30 g l^-1 sucrose, 1.4 g l^-1 L-glutamine (Sigma-Aldrich, St. Louis, USA), and 0.1 g l^-1 myo-inositol (Merck KGaA, Darmstadt, Germany), and the pH of the culture medium was adjusted to 5.7 before autoclaving at 121°C for 20 min, 1.5 atm. The embryogenic cell suspension cultures were subcultured every 15 days by adding 10 ml of the old suspension culture to 60 ml of fresh liquid medium. Embryogenic cell suspension cultures were kept on an orbital shaker (Cientec, Minas Gerais, Brazil) at 100 rpm in the dark, at 25 ± 2°C.
To analyze the effect of Mps1 inhibition on cellular growth and the PEM morphology, embryogenic cell suspension cultures were grown in basic MSG culture medium supplemented with 30 g l^-1 sucrose, 1.4 g l^-1 L-glutamine, and 0.1 g l^-1 myo-inositol, and with or without Mps1 inhibitor SP600125 (Sigma-Aldrich). The Mps1 inhibitor was filter-sterilized through a 0.2-μm PVDF membrane (Millipore, São Paulo, Brazil) before being added to the culture medium. After the inoculation of embryogenic cell culture with 15-day-old cell suspensions, the flasks were maintained on an orbital shaker at 100 rpm in the dark, at 25 ± 2°C.

Fruit pericarp was manually removed and the seeds were desinfested according to Oliveira et al. (2013) and germinated in a medium
composed of MS salts (Sigma) PageBreakand vitamins (Murashige and Skoog 1962 ), 30 g L^-1 sucrose (Sigma), 7 g
L^-1 agar and 2.685 µM naphthaleneacetic acid (NAA, Sigma).
Solanumlycopersicum and
Pisumsativum seeds were subjected to the
same disinfestation procedure and inoculated in medium without NAA. Germination was
done at 25 °C under a 16/8 hours (light/dark) regime.

The morphogenic callus formed by shoot apexes and immature inflorescences was transferred under aseptic conditions onto regeneration medium containing different combinations and concentrations of thidiazuron (TDZ), of zeatin (ZEA), and of α-naphthylacetic acid (NAA) (Table 2).
The nutrient media were supplemented with 30 g L^–1 sucrose (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) and 8 g L^–1 Agar-Agar (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). The pH of the media was 5.7 ± 0.1. The sterile culture was grown under controlled conditions: light intensity of 50 µmol m^–2 s^–1., photoperiod of 16/8 h (day/night), ambient temperature of 22 ± 2 °C. The frequency of shoot formation (%) and the number of shoots per explant were determined after 4 weeks.

Sodium citrate, urea, and thiourea were purchased from Chemical Reagent Co. Ltd. (Tianjin, China). Xylose and glucose were purchased from Aladdin Ltd. (Shanghai, China). Nutrient broth (NB) medium (1 g L⁻¹ yeast extract, 3 g L⁻¹ beef extract, 5 g L⁻¹ poly peptone, 10 g L⁻¹ sucrose) was purchased from Sigma-Aldrich. All other chemicals were of analytical grade and used as received. Double distilled water was used in all experiments.

Populus tomentosa clones (TC1521) were grown in culture on a half-strength Murashige–Skoog (MS) medium (Murashige and Skoog, 1962 (link)) (pH = 6.2) containing 20 g L^–1 sucrose (Sigma-Aldrich, St. Louis, MO, United States) and 0.4 mg L^–1 indole-3-butyric acid (IBA) (Sigma-Aldrich) at 25°C under a 16/8 h (day/night) photoperiod. Sixty-day-old plants were transferred into a hydroponic solution with sufficient N level for 5 days, which was changed for fresh solution every 2 days. The plants were then transferred to a solution with or without sufficient N as the control and treatment groups for 3 days as described previously (Ren et al., 2015 (link)). Briefly, plants were grown in modified half-strength mass spectrometry (MS) liquid medium (pH = 6.2) with 2 mM NH₄NO₃ (Sigma-Aldrich) and 1 mM KNO₃ (Sigma-Aldrich) as sufficient N conditions (KK) (control) or with.01 mM NH₄NO₃ and 1 mM KCl (Sigma-Aldrich) instead of KNO₃ for low-N treatment (DN). Whole P. tomentosa plants were harvested in the midmorning, immediately frozen in liquid N, and stored at −80°C.