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Gene silencing by RNA interference is a particularly important tool in the study of gene function in polyploid cereal species for which the collections of natural or induced mutants are very limited. Previously we have been testing small interfering RNA-based approach of gene silencing in wheat and triticale. In this research, artificial microRNAs (amiRs) were studied in the same species and the same target genes to compare effectiveness of both gene silencing pathways. amiR cassettes were designed to silence  Puroindoline a  ( Pina ) and  Puroindoline b  ( Pinb ) hardness genes in wheat and their orthologues  Secaloindoline a  ( Sina ) and  Secaloindoline b  ( Sinb ) genes in triticale. Each of the two cassettes contained 21 nt microRNA (miR) precursor derived from conserved regions of  Pina / Sina  or  Pinb / Sinb  genes, respectively. Transgenic plants were obtained with high efficiency in two cultivars of wheat and one cultivar of triticale after using the  Pinb -derived amiR vector for silencing of  Pinb  or  Sinb , respectively. Lack of transgenic plants in wheat or very low transformation efficiency in triticale was observed using the  Pina -derived amiR cassette, despite large numbers of embryos attempted. Silencing of  Pinb  in wheat and  Sinb  in triticale was highly efficient in the T 1  generation. The transcript level of  Pinb  in wheat was reduced up to 92% and  Sinb  in triticale was reduced up to 98%. Moreover, intended silencing of  Pinb / Sinb  with  Pinb -derived amiR cassette was highly correlated with simultaneous silencing of  Pina / Sina  in the same transgenic plants. High downregulation of  Pinb / Pina  genes in T 1  plants of wheat and  Sinb / Sina  genes in T 1  plants of triticale was associated with strong expression of  Pinb -derived amiR. Silencing of the target genes correlated with increased grain hardness in both species. Total protein content in the grains of transgenic wheat was significantly lower. Although, the  Pinb -derived amiR cassette was stably inherited in the T 2  generation of wheat and triticale the silencing effect including strongly decreased expression of silenced genes as well as strong expression of  Pinb -derived amiR was not transmitted. Advantages and disadvantages of posttranscriptional silencing of target genes by means of amiR and siRNA-based approaches in polyploid cereals are discussed.

frontiers in plant science

Artificial MicroRNA-Based Specific Gene Silencing of Grain Hardness Genes in Polyploid Cereals Appeared to Be Not Stable Over Transgenic Plant Generations