Downregulation of Glucan Synthase-Like (TaGSL) genes in wheat leads to inhibition of transgenic plant regeneration

Abstract

RNA interference (RNAi) cassettes for gene silencing in plants consist of an inverted repeat sequence of the targeted gene and a spacer region separating sense and antisense fragments. The sequences are generally placed under the control of a strong promoter and terminator and produce hairpin RNA structures that generate small interfering RNAs (siRNAs). These siRNAs destroy the mRNA of the matching gene in the cytoplasm. The number and efficiency of siRNAs depend upon the sequence length of inverted repeats and the region in the gene. In the present study, our objective was to downregulate three members of the Glucan Synthase-Like family in wheat. Initially, a 230-nucleotide fragment was cloned under the maize ubiquitin-1 promoter and nos terminator in both sense and antisense directions, and separated by a spacer including the Escherichia coli uidA (GUS) gene. RNAi constructs were prepared for TaGSL8 and TaGSL10. Biolistic delivery of RNAi constructs for TaGSL8 and TaGSL10 into immature zygotic embryos (IZEs) of wheat did not yield any transgenic plants. The experiment was repeated and comparisons were made with a control bar gene construct. IZEs bombarded with a bar selection cassette alone generated healthy calli on selection medium and transgenic plants were recovered. IZEs co-bombarded with the combination of RNAi and bar gene constructs produced only unhealthy calli when cultured on selection medium and no transgenic plants were recovered. This indicated that the RNAi constructs with 230-bp long inverted repeats of TaGSL8 or TaGSL10 were inhibiting regeneration. In the second experiment, the length of the inverted repeats was reduced to 150 and 122 nucleotides for TaGSL3 and TaGSL8, respectively. Two independent transgenic plants were recovered for each of the TaGSL3 and TaGSL8 target genes. These transgenic plants showed transcription of the introduced RNAi constructs, and the transcript levels of the corresponding endogenous genes were reduced. RNAi transgenic lines of TaGSL3 and TaGSL8 showed slightly reduced resistance against Fusarium graminearum compared to nontransgenic control plants.