Transformation of the moss ( Ceratodon purpureus ) with plasmid DNA delivered by novel block-copolymers of the dimethylaminoethyl methacrylate

Aim. To investigate the potential of poly(2-dimethylamino)ethyl methacrylate (DMAEMA)-based block-like polymers to serve as gene delivery carriers in moss Ceratodon purpureus (Hedw.) Brid. protoplasts, and to evaluate the level of their phytotoxicity. Methods. Organic synthesis; DNA gel retardation assay; adapted PEG-mediated transformation protocol; PCR; light microscopy. Results. The formation of pDNA complex with DMAEMA-based carriers took place at 0.01-0.1 % concentrations of the polymer. The poly-DMAEMA carriers F8-DM1, F8-DM2 (fluorine-containing), LAcr-DM1, LAcr-DM2 (lauryl acrylate-containing), BAcr-DM1, and BAcr-DM2 (butyl acrylate-containing) were effective as carriers of plasmid DNA pSF3 at C. purpureus transformation . PCR analysis confirmed that the transformants of C. purpureus moss contain GFP as a gene of interest after the protoplast transformation by polymers LAcr-DM2, LAcr-DM1, BAcr-DM2, BAcr-DM1 and F8-DM2. The poly-DMAEMA carriers at working concentration (0.0025 %) were relatively non-toxic for protoplasts of C. purpureus moss. 83.1-93.9 % of viable protoplasts of C. purpureus moss were found after the treatment with studied carriers at that dose. However, at 0.25 % i.e. 100 times higher concentration than that used for moss transformation, the poly-DMAEMA carriers reached their IC 50 level. Conclusion. The novel block-like poly-DMAEMA carriers were effective in transformation of C. purpureus moss protoplasts and demonstrated low toxicity.


Introduction
Genetic engineering of plants is crucial at crop engineering to achieve higher yields and resistance to different environmental stresses and diseases [1,2], as well as to meet the energy demands [3] and to provide pharmaceutical manufacturing of recombinant proteins and small-molecular drugs [4,5]. The Agro bacterium-mediated delivery is the most commonly used tool for gene delivery into plants [6]. The biolistic particle delivery with "gene gun", PEG-mediated, chemical-based methods are also used for plant transformation [7][8][9]. The plant viral vectors (e.g. tobacco mosaic virusbased) could be applied for the plant transformation [5].
The plant cell wall is a main barrier for exogenous gene delivery to plants. Current delivery methods are limited by low transformation efficiencies, cell/tissue damage or unavoidable DNA integration into the host genome [5,10]. The viral vectors are effective only for selected plant species and limited by the content and size of the expression cassette [5,11].
Genetic engineering of mosses might serve multiple purposes, such as identifying molecular mechanisms of environmental adaptation of mosses and formation of biologically active products. Ceratodon purpureus is a moss used as a model system in genetic and developmental studies [29,30].
Present study was aimed at investigation of the potential of DMAEMA-based block polymers to serve as gene delivery carriers in the protoplasts of the model species Ceratodon purpureus moss and at evaluation of cytotoxicity.
Novel DMAEMA-containing block-copolymers were synthesized at the Department of Organic chemistry of Lviv Polytechnic National University (Lviv, Ukraine). The block-copolymers were synthesized in two stages: 1) synthesis of polyfunctional macroinitiators (telechelic oligoperoxides -polyalcyl acrylate or fluorinated poly(meth)acrylate with terminal peroxide groups), and 2) synthesis by grafting radical polymerization of the polycationic polymer chains [24,31]. The structures and compositions of polymers are shown in Figure 1 and Table 1.
Transformation of moss protoplasts. Moss Ceratodon purpureus (obtained from the collection at the Institute of Ecology of the Carpathians, National Academy of Sciences of Ukraine, Lviv, Ukraine) was cultured at 24-26 °С in solid Knop medium with light intensity of 5-20 W/m 2 with alternate 16/8 h day and night cycle. The culture of C. purpureus moss was initiated from the spores. Protoplasts of C. purpureus were isolated by a digestion of cell wall of 100 mg of protonemal tissue  with 1 % Driselase (Sigma-Aldrich, USA) for 1 h in darkness at room temperature with slight continuous shaking. The digested moss tissue was transferred to the wet with 8 % D-mannitol (Sigma-Aldrich, USA) nylon filter. The tube with filtrated protoplasts was centrifuged at 200×g for 5 min. The pellet was gently resuspended in 10 mL of 8 % D-mannitol. The number of protoplasts was counted using Neubauer chamber [9]. The protoplasts were recentrifuged at 200×g for 5 min. The pellet was gently resuspended in the MMM solution (9 % D-mannitol, 0.015 M MgCl 2 , 0.1 % MES-KOH, pH 5.6) to obtain the protoplasts suspension at 1.6 ×10 6 /mL concentration. Plasmid DNA pSF3 [28] that contained the gene of green fluorescent protein and the gene of Hygromycin B resistance, was used to transform the protonema of C. purpureus moss. The modified PEG-mediated transformation method, developed for moss P. patens, was used to transform the protoplasts of C. purpureus [32]. The pDNA complexes with poly-DMAEMA carrier (1 µL of pDNA and 2.5 µL of 0.1 % polymer) or with PEG-6000 (1 µL of pDNA and 2.5 µL of 40 % PEG-6000, LobaChemie, Austria) were used. The сomplexes were ad ded to 0.1 mL of protoplasts suspension (1.6 ×10 6 /mL) in 8 % of D-mannitol (Sigma-Aldrich, USA) (the final concentration of polymeric carriers was 0.0025 %). The tubes with transformed protoplasts were transferred in a light-tight cardboard box and incubated for 24 h at 24-26 °С. The transformed protoplasts were mixed with 2 mL of PRMT medium containing 0.6 % agar (approximately 37-42 °C) and placed on Petri dishes with solid PRMB medium containing 0.5 % glucose. The regenerants were trans-ferred on a selective medium containing Hygromycin B (50 µg/mL, Sigma-Aldrich, USA) for 9 days at 24-26 °С with light intensity of 5-20 W/m 2 with alternate 16/8 h day and night cycle. The regenerants were cultivated for the next 14 days on the medium without Hygromycin B antibiotic. After that, they were sub-cultivated for next 8 weeks on the medium with or without Hygromycin B antibiotic in order to obtain stable transformants of C. purpureus moss [28].

Molecular-genetic analysis of transformants of C. purpureus moss with PCR.
To verify the presence or absence of the transferred gene of interest in the obtained transformants, PCR analysis was performed. DNA from transgenic plants was isolated after 8 weeks of cultivation according to the described method [32]. The primers VN10 (forward) and VN11 (reverse) (Genomed, Poland) were used to detect the transgenesis of the GFP-PTS1 chimeric gene (Table 2).  [33].
Toxicity of polymers for moss protoplasts. Protoplasts of C. purpureus moss were isolated, as described previously. The effects of polymeric carriers and PEG-6000 on C. purpureus protoplasts were studied in the following range of the final concentrations of carriers: 0.0025 (effective concentration), 0.025 and 0.25 %. Polymers were added to 0.1 mL aliquots of protoplasts suspension (1.6 ×10 6 /mL) in 8 % of D-mannitol and gently mixed. Protoplasts were co-cultivated with carriers for 24 h at 24-26 °С with light intensity of 5-20 W/m 2 with alternate 16/8 h day and night cycle. The cytotoxicity of polymers was examined via calculating the amount of normal and damaged protoplasts using Neubauer chamber. The le vel of polymer toxicity was evaluated using IC 50 value (the concentration of polymer that reduced protoplasts viability by 50 %).
The obtained data are presented as Mean (M) ± Standard deviation (SD) from three replications. Statistical analysis was performed using one-way ANOVA test at GraphPad Prism 6 software. P value < 0.05 was considered as statistically significant.

pDNA binding properties of studied polymers
The electrostatic interactions between positively charged groups of the carrier and negatively charged phosphate groups of nucleic acid are important for the DNA (RNA or oligonucleotides) condensation and efficient gene delivery [21][22][23]34].
The gel retardation assay was used to confirm the formation of poly-DMAEMA carrier took place at 0.01 % concentration of the polymer ( Fig. 2A-E). Polymer BA-DM2 at 0.1 % concentration fully retarded the electrophoretic mobility of pDNA (lane 1 of Fig. 2F), and this carrier at 0.01 % partly induced retardation of electrophoretic mobility of pDNA (lane 2 of Fig. 2F). BA-DM2 at 0.1 % forms the complex with pDNA.
Thus, the studied poly-DMAEMA carriers are capable of forming the complexes with plasmid DNA.
Thus, the poly-DMAEMA carriers F8-DM1, F8-DM2, LAcr-DM1, LAcr-DM2, BAcr-DM1, and BAcr-DM2 were effective carriers of plasmid DNA at transformation of protoplasts of C. purpureus moss. The PCRanalysis confirmed that transformants of C. purpureus moss contained a gene of interest GFP after protoplast transformation with the studied poly-DMAEMA carriers. Thus, poly-DMAEMA carriers possess the structure providing a possibility of surface adjustment for excellent self-assembly [35,36]. Plasmid DNA delivery to mosses by poly-DMAEMA carriers It was found that poly-DMAEMA carriers with higher amount of DMAEMA blocks (marked as DM2) were more effective for transformation of moss protoplasts. Synatschke et al. reported that the polymers with a branched architecture and an intermediate molecular weight were more prominent for the gene delivery, since they combined low cytotoxicity with acceptable transfection efficiency [37]. Besides, the fluorinated polymers are characterized as hydrophobic and lipophobic carriers with high chemical stability, bio-inertness, and low surface energy [38,39].

Toxicity of polymers towards protoplasts of C. purpureus moss
The poly-DMAEMA carriers under study demonstrated moderate toxicity for the protoplasts of C. purpureus moss (Fig. 5).
The poly-DMAEMA carriers F8MA-DM2, F8MA-DM1, LAcr-DM2, LAcr-DM1, BAcr-DM2, and BAcr-DM1 used in 0.0025 % working concentration were relatively non-toxic for protoplasts of C. purpureus. 87.1-93.9 % of viable protoplasts of C. purpureus moss were detected after treatment. The poly-DMAEMA carriers used at 0.025 % (10 times higher concentration than that used for moss transformation) caused a reduction of alive protoplasts amount to 74.7-76.6 %. At the same time the poly-DMAEMA carriers used at 0.25 % dose reached their IC 50 value and only 46.5-53.5 % of alive protoplasts of C. purpureus moss were found (Fig. 5). The most pronounced cytotoxic effects were demonstrated by the PEG-6000. When used at 0.0025, 0.025 and 0.25 % doses, a decrease in the viability of protoplasts of C. purpureus moss to 74.3, 35.5 and 4.3 %, respectively, was detected (Fig. 5). Several researchers reported that fluorine-containing polymers possessed a reduced toxicity toward targeted cells [40,41]. We did not find a significant difference in the toxicity of the butyl acrylate-, lauryl acrylate-, and fluorine-containing poly-DMAEMA carriers for the protoplasts of C. purpureus moss.

Conclusion
The poly-DMAEMA carriers caused an efficient condensation of plasmid DNA into stable complexes. We found that such carriers noted as F8-DM1, F8-DM2, LAcr-DM1, LAcr-DM2, BAcr-DM1, and BAcr-DM2 were effective in transformation of C. purpureus moss with plasmid DNA. PCR-analysis confirmed that transformants of C. purpureus moss contained the gene of interest, namely GFP, after protoplast transformation with the synthetic polymers LAcr-DM2, LAcr-DM1, BAcr-DM2, BAcr-DM1, F8-DM2, and F8-DM1. The poly-DMAEMA carriers at the effective concentration were relatively non-toxic for protoplasts of C. purpureus moss. However, the poly-DMAEMA carriers used at 0.25 % dose, that is 100 times higher concentration than that used for moss transformation, reached their IC 50 value. Thus, the novel synthetic poly-DMAEMA carriers demonstrated their potential as a promising system for delivering plasmid DNA into plant cells, and the applied approach seems to be beneficial in plant biotechnology.