2.4.1. Colonization experiments
Tomato seeds were surface disinfected with 70% ethanol for
5 min followed by one wash with sterile water and immersion in
2% sodium hypochlorite for 10 min followed by three washes with
sterile water. Seeds were germinated for 4 days at 28 ◦C in the dark
on semisolid medium (0.5% agar). Seedlings were immersed in an
appropriate volume of the bacterial inoculum (G. diazotrophicus PAL
5 or UAP 5541/pRGS561) with constant shaking for 10 min. Negative
controls were immersed in an appropriate volume of sterile
pH 6.0 PBS. Inoculated seedlings were placed into flasks containing
100 ml of semi solid Fahräeus medium (0.5% agar). Plants were
grown in a controlled growth chamber under a light/dark cycle of
16 and 8 h, respectively, at 28 ◦C for 25 days. Three independent
experiments with three treatments each (non-inoculated controls,
inoculated with G. diazotrophicus PAL 5 and inoculated with G. diazotrophicus
UAP 5541/pRGS561), and 50 plants/treatment, were
performed.