Location: Grain, Forage & Bioenergy Research
2013 Annual Report
It has been demonstrated that a transgene with separate short hairpins (200-300 nucleotides) derived from different viruses can provide resistance to multiple viruses. Hence, we proposed to develop hairpin constructs from the conserved regions of WSMV, TriMV and HPV genomic RNA sequences between the maize ubiquitin promoter and NOS-terminator sequences in a binary vector. Similarly, putative microRNA precursor sequences from the genomic RNAs of WSMV, TriMV and HPV will be identified and cloned in a binary vector between the maize ubiquitin promoter and NOS-terminator. The binary plasmids containing expression cassettes comprising the hairpin or microRNA precursor sequences from WSMV, TriMV and HPV will be used to transform wheat. The transformants containing hairpins or microRNA precursor sequences will be screened for resistance against WSMV, TriMV and HPV.
A hairpin construct of WSMV and TriMV, each with ~200 nucleotide RNA sequence, was engineered under the ubiquitin promoter in a plant transformation vector. This construct was used to transform wheat cv. CB 037, and 10 independent events of transgenic wheat lines were obtained. Transgenic wheat lines were screened for the presence of transgene by NPTII assay and by Northern blot hybridization for the presence of small interfering RNAs generated from the transgene as the result of host defense mechanisms. Transgenic wheat lines from the T1 generation were screened for resistance against WSMV and TriMV. Wheat lines from four independent transgenic events provided mild to moderate resistance to WSMV but not to TriMV. Seeds from moderately resistant T2 wheat lines were collected for further screening against WSMV and TriMV resistance.
Additional RNAi constructs with a single hairpin sequence comprising WSMV and TriMV with an intron loop and another construct with two independent cassettes of WSMV and TriMV hairpins with intron loops were developed in a plant transformation vector. These two constructs will be used for wheat transformation to obtain additional transgenic wheat lines.