Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We investigated the possibility of obtaining a suspension cell culture of Arabidopsis thaliana carrying a site-specific integration of a target gene encoding modified human interferon (dIFN) using endonuclease Cas9. For the targeted insertion, we selected the region of the histone H3.3 gene (HTR5) with a high constitutive level of expression. Our results indicated that Cas9-induced DNA integration occurred with the highest frequency with the construction with donor DNA surrounded by homology arms and Cas9 endonuclease recognition sites. Among the monoclones of the four cell lines with knock-in studied, there is high heterogeneity in the level of expression and accumulation of the target protein. The accumulation of dIFN protein in cell lines with targeted insertions into the target region of the HTR5 gene does not statistically differ from the level of accumulation of dIFN protein in the group of lines with random integration of the transgene. However, one among the monoclonal lines with knock-in has a dIFN accumulation level above 2% of TSP, which is very high.

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The objective of this study was to investigate the effects of modification by heat-moisture treatment (HMT) combined with microwave preand post-treatment (MW) on the physicochemical, structural and digestive properties of potato starch. The light microscopy, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD) were used to determine the structural properties of starch. FT-IR and XRD spectra implied that MW and HMT destroyed the double helices and crystalline structure of potato starch. The relative crystallinity of modified starch granules (15.17-18.17%) was lower than that of native starch (19.39%). In the case of physicochemical properties, the modified starches had higher pasting temperature (68.8-93.0°C) and setback viscosity (807-3168 cP), but lower peak viscosity (1315-3662 cP) and breakdown viscosity (17.3-78.3 cP) than that of native potato starch, which were 68.5°C, 496 cP, 6598 cP and 2526 cP, respectively. The HMT and MW modifications significantly increased the content of slowly digestible starch and resistant starch. The resistant starch content of starch obtained by HMT combined with MW post-treatment was significantly higher than that of starch obtained by HMT combined with MW pre-treatment and single HMT. These results may promote good understanding of the effects of HMT combined with MW pre-and post-treatment on physicochemical properties and digestibility of potato starch, and wide utilization of microwave and heat-moisture techniques in starch modification.

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