Application of polymeric dimethylaminoethyl methacrylate based carriers of plasmid DNA for genetic transformation of Ceratodon purpureus moss

Introduction. Genetic engineering in plants is of great importance for agriculture, biotechnology and medicine, and nanomaterials are widely used for genetic engineering. The aim of present study was to evaluate the potential of poly(2-dimethylamino)ethyl methacrylate (DMAEMA)-based comb-like polymers as gene delivery systems in moss Ceratodon purpureus (Hedw.) Brid. protoplasts and determine the level of phytotoxicity of these polymers. Materials and Methods. In order to confirm the formation of complex of poly-DMAEMA carrier with plasmid DNA pSF3, gel retardation assay was used. The PEG-mediated transformation protocol was adapted to transform the protoplasts of C. purpureus moss with poly-DMAEMA carriers. Light microscopy was used to study a toxicity of polymers for moss protoplasts. The level of the polymers toxicity was estimated as IC50 value. Results and Discussion. The formation of pDNA complex with DMAEMA-based carriers took place at 0.03% concentration of the polymers BGA-21, BGA-22(2ph), BG-24, BG-25, BG-26 or 0.1% concentration of the BGA-22 polymer. Poly-DMAEMA carriers were able to deliver plasmid DNA pSF3 into protoplasts of C. purpureus moss. Three stable transformants of C. purpureus were obtained at using BGA-22 polymer, 2 clones – at using BGA-21 carrier, and 1 clone – at using BGA-22(2ph), BG-24, BG-25, BG-26 polymers. The poly-DMAEMA carriers at the working 0.0025% dose were relatively non-toxic for protoplasts of C. purpureus moss. 83.1-88.4% of viable protoplasts of C. purpureus moss were detected after treatment with studied carriers at 0.0025% dose. A survival ratio of protoplasts reached 66.7-72.9% under the effect of these polymers at 0.025% dose, which is 10 times higher than their working concentration. The IC50 value of poly-DMAEMA carriers was in the range of 0.113-0.164%, that was approximately 10 times higher than that of the PEG-6000 used for gene delivery in plants. Conclusion. Novel synthetic poly-DMAEMA carriers delivered the gene of interest into moss C. purpureus protoplasts and possessed a low phytotoxicity. Thus, these carriers can be useful for gene delivery into plant cells.