Cloning GA20ox2 for GFP Fusion

The full-length CDS of GA20ox2 was cloned into a pCambia2300–green fluorescent protein (GFP) to form a translation fusion with the N-terminus of the GFP. The vector was kindly provided by Professor Xu Yan, Northwest A&F University. Agrobacterium tumefaciens strain EHA105, containing either a pCambia 2300 vector with 35S::GFP or the GA20ox2—35S::GA20ox2-GFP, was grown at 28°C in Luria–Bertani medium containing 50 mg L⁻¹ kanamycin and 25 mg L⁻¹ rifampicin. After 24 h, the Agrobacterium cells were harvested and resuspended in infiltration buffer [10 mM MgCl₂, 10 mM MES (pH 5.6) and 150 mM acetosyringone] to a final OD₆₀₀ of 0.8. The resuspended cells were shaken for 4 h at room temperature and then subjected to infiltration by a syringe. The methods of infection were based on Hellens et al. (2005) (link). The leaves were incubated in the dark at 22°C for 12 h and then placed in a growth chamber (25°C, 16-h/8-h day/night) for 4 to 5 days. Fluorescence Microscopic (BX51 + PD72 + IX71, OLYMPUS, Japan) imaging system was used to observe the anatomical images. Excitation and emission wavelengths were 498 nm and 516 nm respectively.