WDVGuide4Guard barley lines were generated by transformation of Hordeum vulgare L. cv. Golden promise.
Aim of the modification was the creation of Wheat dwarf virus (WDV) resistant barley lines.
To produce transgenic plants the WDVGuide4Guard binary vector with four different WDV genome-specific sgRNA (1-4) sequences and the maize codon-optimized Cas9 gene controlled by the constitutive maize Ubi1 promoter was used. Immature barley embryos were transformed with Agrobacterium tumefaciens AGL-1 harboring the WDVGuide4Guard vector.
Four T0 lines were checked for the presence of the transgene cassette by PCR analyses and sequencing.
After infection with WDV, the WDVGuide4Guard lines 1, 3 and 4 exhibited no viral symptoms, and the presence of the virus was detected neither by northern blot nor PCR analysis.
Expression of sgRNA-WDV2 in barley plants induced a three-nucleotide insertion in the WDV genome after infection with WDV. The WDV genome specific sgRNA-WDV2 targets the Rep/RepA coding sequence at the N-terminus of the proteins.
Seven of ten T1 lines showed no phenotypic signs of virus infection and no WDV-derived products were detected by PCR or northern blot analyses.
Source: Kis et al., (2019), Creating highly efficient resistance against Wheat dwarf virus in barley by employing CRISPR/Cas9 system. Plant Biotech J 17:1004-1006