Overexpression of heat stress transcription factors in orchids

Both full-length FaTHSFA2a and FaTHSFB1a cDNAs were PCR-amplified with a pair of primers with attB1/B2 sites (Table S4) and cloned into the pDONR221 vector by using Gateway BP Clonase II Enzyme Mix (Invitrogen, Carlsbad, CA, USA). Each construct was subsequently recombined as an N-terminal fusion of GFP into the Gateway destination binary vector pK7WGF2 (Functional Genomics Division of the Department of Plant Systems Biology, Gent, Belgium), yielding 35S::GFP–FaTHSFA2a and 35S::GFP–FaTHSFB1a by an attL× attR recombination reaction (Invitrogen, Carlsbad, CA, USA). These GFP fusion constructs were isolated and transformed into floral lips of orchids by bombardment transformation [71 (link)]. A Zeiss LSM 510 META laser-scanning confocal microscope using an LD C-Apochromat 409/1.1 W objective lens was subsequently used to observe the fluorescence emitted in the transformed cells, as previously described [71 (link)].