Generation of transgenic barley lines

The full-length 1068bp HvNAC005 coding sequence was PCR-amplified from barley ‘Golden Promise’ cDNA and cloned into the pUCE_UBI:USER:NOS vector (Hebelstrup et al., 2010) that was used for Agrobacterium-mediated transformation of barley ‘Golden Promise’ (Holme et al., 2012). Primers used during cloning are listed in Supplementary Table S1. Regenerated T₀ lines were screened by qRT-PCR for presence of the transgene, using primer pairs spanning the promoter/coding sequence junction and the coding sequence/NOS terminator junction. Primers are listed in SupplementaryTable S1. Regenerated plants that were negative in tests for the transgene were designated null-transgenic and some of these were used as controls in the microarray and qRT-PCR experiments.