RUDN Journal of Agronomy and Animal Industries (Apr 2022)
Agrobacterium-mediated transformation of potato Solanum tuberosum L. with constructs carrying the strong plant-derived promoter pro-SmAMP1 from Stellaria media L.
Abstract
The effectiveness of plant genetic transformation is determined by the choice of genetic structures and their regulatory sequences that cause a high and stable expression level of heterologous genes. In this regard, the actual task of biotechnology is the use of highly effective plant promoters. The choice of promoter determines not only the level of the expression gene, but also the effectiveness of genetic transformation. The purpose of our study was to evaluate the influence of explant type and 5-deletion variants of the plant strong pro-SmAMP1 promoter, on the Agrobacterium -mediated transformation efficiency of potato ( Solanum tuberosum L.) cv. Udacha. To analyze the regenerative capacity of potato stem and leaf explants, AGL0 strain carrying constructs containing the 5-deletion variants of the promoter fragment of gene encoding antimicrobial peptide from Stellaria media L. ( pro-SmAMP1 ) was carried out. Four genetic constructs based on the plant expression vector pCAMBIA1381Z were used in this work, containing the selectable gene hptII and reporter gene uidA under different 5-deletion variants of the pro-SmAMP1 promoter (-442, -675, -732 and -1196 bp relative to the transcription initiation site); as well as two binary vectors based on the expression vector pCAMBIA1302 with 5-deletion pro-SmAMP1 promoter variants (-442 and -1196 bp), controlling the expression of gfp reporter gene. It was found that the effectiveness of Agrobacterium -mediated transformation depended on the type of genetic construction used, but not on the type of explant being cultivated. The insertion of the promoter region pro-SmAMP1 gene, hptII , as well as the absence of the bacterial Vir E gene was confirmed by PCR. Depending on the type of genetic construct, the transformation efficiency for the reporter gene varied from 2.0 to 7.2 %. The results are compared with previously conducted few studies, according to which the choice of promoter determines not only the expression level of marker genes, but also has a significant influence on the genetic transformation efficiency.
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