A series of chimeric promoters for higher-level expression of foreign genes in plants was constructed as fusions of a gene for beta-glucuronidase (GUS) with the terminator of a gene for nopaline synthase (nos) or of the cauliflower mosaic virus (CaMV) 35S transcript, and the strength of these promoters was assayed in transient and stable expression systems in tobacco and rice. As parts of these promoters, the CaMV 35S core promoter, three different 5'-upstream sequences of the 35S promoter, the first intron of a gene for phaseolin, and a 5'-untranslated sequence (omega sequence) of tobacco mosaic virus were used in various combinations. In tobacco and rice protoplasts, all three fragments of the 35S promoter (-419 to -90, -390 to -90 and -290 to -90, relative to the site of initiation of transcription), the intron, and the omega sequence effectively enhanced GUS activity. Some chimeric promoters allowed levels of GUS activity that were 20- to 70-fold higher than those obtained with the 35S promoter in pBI221. In tobacco protoplasts, the two longer fragments of the 35S promoter were more effective than the shortest fragment. In rice cells, by contrast, the shortest fragment was as effective as the two longer ones. The terminator of the 35S transcript was more effective than that of the nos gene for gene expression. In transgenic tobacco plants, a representative powerful promoter, as compared to the 35S promoter, allowed 10- and 50-fold higher levels of expression on average and at most, respectively, with no clear qualitative differences in tissue- and organ-specific patterns of expression. When the representative promoter was introduced into tobacco with a gene for luciferase, the autofluorescence of detached leaves after a supply of luciferin to petioles was great and was easily detectable by the naked eye in a dark room.

Gas plasma generated and applied under two different systems, atmospheric pressure plasma and low pressure plasma, was used to investigate the inactivation efficacy on the seedborne pathogenic fungus, Rhizoctonia solani, which had been artificially introduced to brassicaceous seeds. Treatment with atmospheric plasma for 10 min markedly reduced the R. solani survival rate from 100% to 3% but delayed seed germination. The low pressure plasma treatment reduced the fungal survival rate from 83% to 1.7% after 10 min and the inactivation effect was dependent on the treatment time. The seed germination rate after treatment with the low pressure plasma was not significantly different from that of untreated seeds. The air temperature around the seeds in the low pressure system was lower than that of the atmospheric system. These results suggested that gas plasma treatment under low pressure could be effective in disinfecting the seeds without damaging them.

The aim of this study was to investigate the effect of low-pressure plasma treatment on seed disinfection and the possible mechanisms underlying this effect. Seed-borne disease refers to plant diseases that are transmitted by seeds; seed disinfection is an important technique for prevention of such diseases. In this study, the effectiveness of low-pressure plasma treatment in the inactivation of the seed-borne plant pathogenic bacterium, Xanthomonas campestris, inoculated on cruciferous seeds, was evaluated. The highest inactivation effect was observed when the treatment voltage and argon gas flow rate were 5.5 kV and 0.5 L/min, respectively. The viable cell number of X. campestris was 6.6 log cfu/seed before plasma treatment, and decreased by 3.9 log after 5 min of treatment and by 6.6 log after 40 min. Ethidium monoazide treatment and quantitative real-time PCR results indicated that both the cell membrane and target DNA region were damaged following 5 min of plasma treatment. Although both heat and ozone were generated during the plasma treatment, the contribution of both factors to the inactivation of X. campestris was small by itself in our low-pressure plasma system. Overall, we have shown that our low-pressure plasma system has great applicability to controlling plant pathogenic bacterium contamination of seeds.

Potential crop diseases and yield loss occur due to seed-borne plant pathogens; therefore, the control of these pathogens is an important issue in food production. Seed disinfection is one of the basic control measures for these diseases. Plasma treatment has been widely used for the sterilization of medical instruments and treatment of material surfaces, but its application to agricultural products has just started in recent years. In this study, authors evaluated the effect of atmospheric gas plasma on the inactivation of two seed-borne pathogens, Rhizoctonia solani, which causes damping-off of Japanese hornwort, and Pyricularia oryzae, which causes rice blast disease. Atmospheric argon plasma was generated by AC high voltage discharge, and applied voltage to electrodes and gas flow rate were 10 kV and 3 L/min, respectively. The mycelium of R. solani and the conidia of P. oryzae were treated with atmospheric gas plasma: Post treated mycelial growth and conidial germination were measured after an incubation period of 1 to 4 days. Under these conditions, when the distance of the plasma source to the sample surface was 5 cm, no effect was found on the mycelial growth of R. solani compared with the non-treated one, even when the treatment time was as long as 15 min. However, when the distance was 3 cm, the inactivation effect of mycelial growthwas observed in time depended manner. The 5 min treatment time at a 3 cm distance decreased the growth rate, and the longer treatment time (10 min) markedly inhibited the growth of R. solani. The plasma treatment at a distance of 3 cm for 10 min strongly inhibited the conidial germination of P. oryzae, as well. These results indicated that gas plasma treatment from an appropriate distance is effective in inactivating the two plant-pathogenic fungi. Therefore, this technology could be useful in seed disinfection.