Development of virus-induced genome editing methods in Solanaceous crops.

Genome editing (GE) using CRISPR/Cas systems has revolutionized plant mutagenesis. However, conventional transgene-mediated GE methods have limitations due to the time-consuming generation of stable transgenic lines expressing the Cas9/single guide RNA (sgRNA) module through tissue cultures. Virus-induced genome editing (VIGE) systems have been successfully employed in model plants, such as Arabidopsis thaliana and Nicotiana spp. In this study, we developed two VIGE methods for Solanaceous plants. First, we used the tobacco rattle virus (TRV) vector to deliver sgRNAs into a transgenic tomato (Solanum lycopersicum) line of cultivar Micro-Tom expressing Cas9. Second, we devised a transgene-free GE method based on a potato virus X (PVX) vector to deliver Cas9 and sgRNAs. We designed and cloned sgRNAs targeting Phytoene desaturase in the VIGE vectors and determined optimal conditions for VIGE. We evaluated VIGE efficiency through deep sequencing of the target gene after viral vector inoculation, detecting 40.3% and 36.5% mutation rates for TRV- and PVX-mediated GE, respectively. To improve editing efficiency, we applied a 37°C heat treatment, which increased the editing efficiency by 33% to 46% and 56% to 76% for TRV- and PVX-mediated VIGE, respectively. To obtain edited plants, we subjected inoculated cotyledons to tissue culture, yielding successful editing events. We also demonstrated that PVX-mediated GE can be applied to other Solanaceous crops, such as potato (Solanum tuberosum) and eggplant (Solanum melongena). These simple and highly efficient VIGE methods have great potential for generating genome-edited plants in Solanaceous crops.

Development of selective mercury recovery technology by using iron iodide from waste sludge of non-ferrous metal smelting process.

The waste sludge from non-ferrous metal smelter contains high concentrations of mercury (Hg), arsenic (As) and sulfur (S). The Article 11 of the Minamata Convention on Mercury mandates the recovery of Hg before the disposal stage of Hg waste. However, As compounds have similar boiling points with Hg compounds, and they are considered interfering substances in the recovery of Hg. Moreover, a high concentration of S requires significant energy to volatilize Hg. This study examined the optimal conditions for selective recovery of Hg and energy reduction by introducing FeI2 as an additive during thermal treatment. Thermogravimetric analysis was utilized to evaluate the conversion of HgS to HgI2 under the influence of FeI2. The optimal conditions for thermal treatment such as temperature, treatment time, and molar ratio of [Hg]:[As]:[FeI2] were explored. The simulated waste indicated that the maximum separation efficiency of Hg was ∼95%, thereby allowing a selective separation of 81.5% of Hg from waste sludge with an Hg content of 0.33%, As content of 23.8%, and S content of 30.7%. Sequential extraction procedure was applied to evaluate the stability of Hg and As for residues. As a result, most Hg was vaporized and As was stabilized in sulfide, crystalline, and amorphous forms.