M5519

Seeds were sown on ½ MS media (M5519, Sigma-Aldrich, St Lois, MO, USA) supplemented with 1% sucrose and 0.8% plant agar (Duchefa Biochemie, Haarlem, Netherlands), stratified for 2 d, and allowed to grow under 16 h light/8 h dark cycles. In order to prepare a stock solution of 0.5 mg/mL, cisplatin (cis-diamminedichloroplatinum (II), Sigma-Aldrich, St Lois, MO, USA) was dissolved primarily in 1 mL of dimethylformamide and mixed with 19 mL of 0.9% saline solution. In order to evaluate genotoxicity in control and mutant plants, 3 d old seedlings were transferred to media supplemented with 0–8 mg L^-1 cisplatin and Petri dishes were placed horizontally for 12 d, or vertically for 3 d when investigating the effect on shoot and primary root development, respectively. Root measurements were accomplished using Image J (https://imagej.nih.gov/ij/index.html).

Two hundred tomato seeds (Solanum lycopersicum “Ailsa Craig”) were disinfected using 100% ethanol for 10 sec and positioned on solid MS substrate (Murashige and Skoog, 1962 (link)) (Sigma, M5519) supplemented with 10 g/L sucrose and 0.8% agar. The petri dishes were placed vertically in a plant growth chamber for 10 days at 24°C temperature and photoperiod of 14 h light/10 h dark. PDCA (P63395; Sigma-Aldrich, St. Louis, MO) was dissolved in ddH₂O sterilized and added at the appropriate concentrations (0–100–250 μM) in the MS. The root and hypocotyl length of a total of 30 seedlings was determined and the experiment was repeated 3 times.

To prepare the culture media, MS culture medium (Sigma-Aldrich M5519, St. Louis, MO, USA) was used, supplemented with 3% sucrose (Macron Fine Chemicals^TM) [71 (link)], and 0.15% Phytagel^®. The pH of the culture medium was adjusted to 5.8. Then, it was distributed in the amount of 20 mL in 25 × 150 mm test tubes. Subsequently, the test tubes containing the culture medium were sterilized in an autoclave at a temperature of 121 °C, under a pressure of 1.5 atm, for a period of 20 min. A total of 154 seeds were disinfected with an ethanol solution (70%) for 1 min, followed by immersion in a commercial solution of sodium hypochlorite (2–2.5%) for 8 min and, subsequently, washed three times with sterile distilled water. After this, the seeds were inoculated and kept in a growth room with a lighting intensity of 50 µmol.m⁻²s⁻¹, for a photoperiod of 12 h, for 120 days. The beginning of germination was observed from root emergence.

Tomato (Solanum lycopersicum) seeds were acquired from the “El semillero” shop in Mexico City. Seeds were immersed in 10% sodium hypochlorite solution for 5 min and then rinsed three times with deionized water to ensure surface sterility. Then the seeds were put into a magenta vessel with MS medium (Sigma Aldrich, M5519, St. Louis, MO, USA), with and without 20 mg/L of TiO₂ NPs respectively. All experiments were triplicated, and 60 plants were grown and used in this study.
TiO₂ NPs were suspended directly in deionized water and dispersed by ultrasonic vibration (100 W, 60 kHz) for 30 min. The TiO₂ NPs were sterilized, added to the MS media at either 0 or 20 mg/L, mixed and put into magenta vessels for the tomato plant culture.
An environmental chamber (LAB-LINE Biotronette mark III, Burlington, VT, USA) was employed to cultivate the tomato plants in a photoperiod of 12/8 h light/dark at 24 °C, with a relative humidity of 70 ± 25%. The absorption root zone was used for experimental conditions at 21 days of growth.

B. subtilis strains and Pseudomonas species were washed in PBS, and their OD₆₀₀ was adjusted at 0.6. Ten µL of cell suspension was then spotted at a 0.7 cm distance onto Murashige and Skoog (MS; Sigma – M5519; 20.61 mM NH₄NO₃, 100 µM H₃BO₃, 2.99 mM CaCl₂, 0.11 µM CoCl₂·6H₂O, 0.1 µM CuSO₄·5H₂O, 100 µM Na₂-EDTA, 100 µM FeSO₄·7H₂O, 1.5 mM MgSO₄, 100 µM MnSO₄·H₂O, 1.03 µM Na₂MoO₄·2H₂O, 5 µM KI, 18.79 mM KNO₃, 1.25 mM KH₂PO₄, 29.91 µM ZnSO₄·7H₂O, 26.64 µM glycine, 0.56 µM myo-inositol, 4.06 µM nicotinic acid, 2.43 µM pyridoxine·HCl, 0.30 µM thiamine·HCl, 0.5% v/v glycerol, and 0.5% v/v glutamate) or MSgg (5 mM potassium phosphate and 100 mM MOPS (3-(N-morpholino) propanesulfonic acid) at pH 7.0 with 2 mM MgCl₂, 700 μM CaCl₂, 50 μM MnCl₂, 50 μM FeCl₃, 1 μM ZnCl₂, 2 μM thiamine, 0.5% v/v glycerol, and 0.5% v/v glutamate) supplemented with 1.5 % w/v agar, and incubated at 30 °C. When needed, X-Gal (5-Bromo-4-Chloro-3-Indolyl β-D-Galactopyranoside) was added at a final concentration of 120 µg·mL⁻¹. To assess biomass and CFUs, B. subtilis biofilms were removed using a sterile P200 tip and transferred into an Eppendorf tube containing 1 mL PBS. Biofilms were sonicated for 30 s to 60 s (until homogeneity) without pause at 30% amplitude. Cells were diluted and plated on LB for CFUs counting. Optical density at 600 nm (Biomass) was evaluated with a Genesys 30 S UV-Vis spectrophotometer.

The plasmids pHGWFS7 and pH7WG2D containing the correctly cloned fragments of interest were used to transform A. tumefaciens GV3101 strains by electroporation at 2,2 kV, 25 μF, with 1 wrist controller at 200 or 400 Ω. Plates containing YEP medium with gentamicin and hygromycin were incubated overnight at 28 °C. For the confirmation of positive bacterial clones, PCR was performed using the primer set PGmHsp22.4-F and PGmHsp22.4-R for the promoter and the primer set pH7WG2D-F and pH7WG2D-R for the coding region (Additional file 5). The recombinant bacteria were used to transform the A. thaliana Columbia (Col-0) ecotype, using the floral dip method [39 (link)]. The selection of transformed seeds in T0 and the subsequent T2, T3 and T4 generations was performed in medium containing 1 / 2x MS medium (Sigma Chemicals n°m-5519), 0.8% agar (Sigma Chemicals n° A-1296), and 15 μg / ml-1 of hygromycin. Transformants were identified as hygromycin-resistant seedlings when they did not present growth retardation. Positive events were confirmed via PCR (Additional file 4) and then propagated until the T4 lineage to be used in subsequent experiments.