Background The ability to engineer cereal crops by gene transfer technology is a powerful and informative tool for discovering and studying functions of genes controlling environmental adaptability and nutritional value. Tetraploid wheat species such as emmer wheat and Timopheevi wheat are the oldest cereal crops cultivated in various world areas long before the Christian era. Nowadays, these hulled wheat species are gaining new interest as donors for gene pools responsible for the improved grain yield and quality, tolerance for abiotic and biotic stress, resistance to pests and disease. The establishing of efficient gene transfer techniques for emmer and Timopheevi wheat may help in creation of modern polyploid wheat varieties. Results In the present study, we describe a robust protocol for the production of fertile transgenic plants of cultivated emmer wheat (Russian cv. ‘Runo’) using a biolistic delivery of a plasmid encoding the gene of green fluorescent protein (GFP) and an herbicide resistance gene (BAR). Both the origin of target tissues (mature or immature embryos) and the type of morphogenic calli (white or translucent) influenced the efficiency of stable transgenic plant production in emmer wheat. The bombardment of nodular white compact calluses is a major factor allowed to achieve the highest transformation efficiency of emmer wheat (on average, 12.9%) confirmed by fluorescence, PCR, and Southern blot. In the absence of donor plants for isolation of immature embryos, mature embryo-derived calluses could be used as alternative tissues for recovering transgenic emmer plants with a frequency of 2.1%. The biolistic procedure based on the bombardment of immature embryo-derived calluses was also successful for the generation of transgenic Triticum timopheevii wheat plants (transformation efficiency of 0.5%). Most of the primary events transmitted the transgene expression to the sexual progeny. Conclusion The procedures described here can be further used to study the functional biology and contribute to the agronomic improvement of wheat. We also recommend involving in such research the Russian emmer wheat cv. ‘Runo’, which demonstrates a high capacity for biolistic-mediated transformation, exceeding the previously reported values for different genotypes of polyploid wheat.
Various plant-derived promoters can be used to regulate ectopic gene expression in potato. In the present study, four promoters derived from the potato genome have been characterized by the expression of identical cassettes carrying the fusion with the reporter β-glucuronidase (gusA) gene. The strengths of StUbi, StGBSS, StPat, and StLhca3 promoters were compared with the conventional constitutive CaMV 35S promoter in various organs (leaves, stems, roots, and tubers) of greenhouse-grown plants. The final amount of gene product was determined at the post-transcriptional level using histochemical analysis, fluorometric measurements, and Western blot analysis. The promoter strength comparison demonstrated that the StUbi promoter generally provided a higher level of constitutive β-glucuronidase accumulation than the viral CaMV 35S promoter. Although the StLhca3 promoter was predominantly expressed in a green tissue-specific manner (leaves and stems) while StGBSS and StPat mainly provided tuber-specific activity, a “promoter leakage” was also found. However, the degree of unspecific activity depended on the particular transgenic line and tissue. According to fluorometric data, the functional activity of promoters in leaves could be arranged as follows: StLhca3 > StUbi > CaMV 35S > StPat > StGBSS (from highest to lowest). In tubers, the higher expression was detected in transgenic plants expressing StPat-gusA fusion construct, and the strength order was as follows: StPat > StGBSS > StUbi > CaMV 35S > StLhca3. The observed differences between expression patterns are discussed considering the benefits and limitations for the usage of each promoter to regulate the expression of genes in a particular potato tissue.
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