Ms basal salts

Agrobacterium tumefaciens C58 expressing split-GFP constructs were grown in liquid LB with appropriate antibiotics for 2 days at 28°C. The bacteria were collected by centrifugation, resuspended in MMi medium [10 g/l sucrose, 5 g/l MS basal salts (Duchefa), 2 g/l MES, 200 μM acetosyringone, pH 5.6] to an OD₆₀₀ of 0.1 and incubated for 1 h at room temperature. Different combinations of split-GFP constructs were made by mixing the appropriate bacterial suspensions in a 1:1 ratio. The suspensions were then injected into the leaves of Nicotiana benthamiana plants which were then grown in a greenhouse at 21°C. Three days post-infiltration, the infiltrated parts were analyzed by confocal microscopy.

The Calbio potassium humate (KH60) product used in this study was provided by Caldic Ibérica S.L.U (Barcelona, Spain). A working solution was prepared at a concentration of 10 mg/mL (w/v) and sterilized by tyndalization.
Wild-type Arabidopsis thaliana seeds (Columbia-0 ecotype) were surface sterilized with commercial bleach diluted 1:1 (v/v) for 15 min and rinsed with sterile water. Stratification was carried out for three days at 4 °C. The plant growth medium used in the tests was MS medium containing a mixture of Murashige and Skoog (MS) basal salts (0.22%; Duchefa Biochemie B V, Haarlem, The Netherlands), sucrose (1%), and 2.6 mM MES (2-(N-morpholino) ethanesulfonic acid buffer), adjusted to pH 5.9 with potassium hydroxide. In all assays, plants were grown under long-day chamber conditions (16 h light/8 h dark, 23 °C, 130 μE m⁻² s⁻¹, 70% relative humidity). When specified, the medium was supplemented with 140 mM NaCl or 24 mM LiCl for saline stress and 280 mM mannitol for osmotic stress, and/or the biostimulant KH60 depending on the assay.

A. thaliana dark grown suspension-culture cell line was maintained in 50 mL of A. thaliana (AT) medium. The AT medium contained 4.3 g/L MS basal salts (Duchefa), 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 4 mL vitamin B5 mixture (Sigma-Aldrich) and 30 g/L sucrose (pH 5.8). Cells were gently agitated at 160 rpm in the dark at 22°C.
To generate cells transiently overexpressing SLIM1, the full length coding sequence of SLIM1 was amplified from the cDNA and cloned into the Gateway pDONR207 vector (Life Technologies) using primers containing attB1 and attB2 sequences (SLIM1_attB1: gggacaagtttgtacaaaaaagcaggcttcATGGGCGATCTTGCTATGTCCGTAGC and SLIM1_attB2: gggaccactttgtacaagaaagctgggtcAGCTCCAAACCATGAGAAATCATCAC). The obtained clone was recombined with the pGWB2 to obtain Pro35S-SLIM1-pGWB2, which was used for transient expression assay.
Transformation of dark-grown cultured Arabidopsis cells was performed using the supervirulent Agrobacteria strains LBA4404.pBBR1MCS.virGN54D containing Pro35S-SLIM1-pGWB2 as described by Koprivova et al. (2000 (link)).