Idenduction of resistance to barley yellow dwarf virus (pav) in bread wheat using post-transcriptional gene silencing (ptgs)
2013
Yassaie, Mohsen | Nizai, Ali | Afsharifar, Alireza
Barley and cereal yellow dwarf are the most important cereal diseases causing serious damages worldwide. Limited number of resistance and tolerance genes, their inefficiency and complex inheritance justifies the adoption of the pathogen derived resistance approaches such as post transcriptional gene silencing (PTGS). In the present study, Agrobacterium-mediated transformation of mature embryos of bread wheat (Alvand cultivar) using pCambia3301 vector harboring a GUS sequence is optimized. Transformations are done using Agrobacterium tumefacien strain AGLO. Tissue culture steps and their modifications are explained. Screening of GUS tranforamants was done using polymerase chain reaction and Gus histochemical assay. Employing PTGS for induction of resistance against Barley yellow dwarf virus-PAV was followed by its tissue culture optimization. By this mean, an intron hair-pin loop (ihp-loop) silencing construct was designed using a sequence of BYDV-PAV polymerase gene. The sequence of construct and its orientation were confirmed by PCR, restriction enzyme assay and sequencing. Selection of transformed explants was done using appropriate selection marker and then transferred to soil successfully. The transformed explants were evaluated by ELISA, polymerase chain reaction, and dot blot assay using a nonradioactive probe. Artificial inoculation of plants was carried out employing BYDV-PAV viruliferous aphids. Transformed plants remained healthy while control plants developed yellow dwarf symptoms after artificial inoculation of BYDV-PAV.
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