Mlr 351

Experiments were performed using the M. polymorpha ecotype BoGa, which was obtained from the Botanical Garden of Osnabrück, Germany. Agrobacterium tumefaciens mediated transformation of sporelings with two constructs was carried out as previously described by Ishizaki et al. [10 (link)] with minor modifications. Differing from Ishizaki et al. [10 (link)], the separation of sporelings from A. tumefaciens after three days of co-cultivation was conducted by a 15 min sedimentation followed by decantation of the added washing solution (½ Gamborg B5 medium with vitamins, Duchefa). Sedimentation and decantation were repeated four times and the sporelings were then plated on agar plates with ½ Gamborg B5 medium with vitamins supplemented with 10 µg/ml hygromycin (Sigma-Aldrich), 5 µg/ml G418 (Sigma-Aldrich) and 100 µg/ml cefotaxime (Duchefa). Gemmae were investigated from the G1 up to the G4 generation. Plants were grown under sterile conditions on solid medium (1% agar medium and ½ Gamborg B5 medium with vitamins, Duchefa) in 10 × 10 × 2 cm petri dishes (Sarstedt, Nümbrecht, Germany) under a 16 h/8 h day/night regimen at 22 °C in climate cabinets (Sanyo MLR351).

Seeds were surface-sterilized with 1% (w/v) NaClO and 0.05% (v/v) Tween-20 for 8 min and washed 4–5 times with sterile water. Surface-sterilized seeds were sown on a Petri plate made with one-half strength Murashige and Skoog (MS) medium, 0.8% (w/v) agar, and 3% (w/v) sucrose. After stratification for 2d at 4°C in the dark, plates were transferred to controlled condition in a Plant Growth Chamber (MLR-351, Sanyo, Japan) set at 16 h/8 h light dark cycle and a light intensity of ∼100 μmol photons m^-2 s^-1. Salt stress tolerance was analyzed using two homozygous transgenic lines (OE3 and OE5) overexpressing OsSHMT3 in T3 generation of Arabidopsis. Seedlings of the wild-type and transgenics after 7 d of germination were transferred to MS agar plate (90 × 18 mm) supplemented with different concentrations of NaCl (0, 100, 150, and 200 mM). After 10 days growth, seedlings of the wild-type and transgenics were harvested and analyzed for their morphophysiological responses. ImageJ software was used for measuring the root length from the scanned images. For documenting the physiological data, 7-day-old seedlings after germination on MS agar plates were transferred to vermiculite culture in pots (70 mm diameter) with one-half strength MS solution supplemented with different concentrations of NaCl (0, 100, 150, and 200 mM) in three replicates with 3 plants in each pot.

Arabidopsis seeds were imbibed and stratified at 4 °C for 4 d before planting on John Innes Centre Arabidopsis Soil Mix (Levington F2 compost with Intercept and 4-mm grit at a 6:1 ratio) in growth cabinets (Sanyo MLR-351), with ∼100 µE/m² light from LED arrays (NVC Lighting; NL/18/LED/T8/4/840, 800 lm, 4,000 K), with cycles of short days (8-h light/16-h dark) or long days (16-h light/8-h dark). EOD-FR used short-day cycles, with a 15-min pulse of FR light (Rapid LED; CREE LED array, peak wavelength 730 nm, 250 µE/m²) at the end of an 8-h light period.

Escherichia coli strain DH5α was used for plasmid propagation and selection in the preparative molecular biology steps. The cells were grown in Luria–Bertani (LB) medium at 37 °C in a shaker at 150–200 rpm or on the solid LB plates containing 1.5% (w/v) agar. When necessary, LB medium was supplemented with appropriate antibiotics at concentrations 50 µg/ml spectinomycin (Sp) and 34 µg/ml chloramphenicol (Cm).
A glucose tolerant substrain of Synechocystis sp. PCC 6803 [27 (link)] obtained originally from Professor Aaron Kaplan (Hebrew University of Jerusalem, IL) was used for all the cyanobacterial experiments. The cells were grown in 25–50 ml Erlenmeyer flasks in liquid BG-11 medium buffered with 20 mM TES-KOH (pH 8.0) [28 ] with supplemented 25 µg/ml Sp and 10 µg/ml Cm to maintain transformant selection pressure throughout all cultivations. The cultures were incubated at 30 °C in ~ 120 rpm orbital shaking under continuous light of 20–50 μmol photons m⁻² s⁻¹ under 1% CO₂ atmosphere (MLR-351 growth chamber Sanyo, Japan) or under ambient CO₂ (Algaetron 230 growth chamber, Photon Systems Instruments, CZ). Solid plate cultivations were conducted on BG-11 plates containing additional 1.5% (w/v) Bactoagar (Difco, USA) and 0.3% (w/v) sodium thiosulfate under corresponding conditions (MLR-351, Sanyo).