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Mowlick S.,Yamagata University | Yasukawa H.,Nara Prefectural Agricultural Experiment Station | Inoue T.,Yamaguchi Prefectural Technology Center for Agriculture and Forestry | Takehara T.,Japan National Agriculture and Food Research Organization | And 3 more authors.
Crop Protection | Year: 2013

Biological soil disinfestation (BSD) is a method of controlling soil-borne pests and diseases through anaerobic decomposition of plant biomass incorporated in field soil with temporary irrigation and covering with sheets. In this study, effects of BSD on suppression of spinach wilt disease were investigated in two different field experiments using mainly Brassica juncea plants as plant biomass. Soil bacterial community compositions were analyzed with clone library analysis based on 16S rRNA gene sequences to determine the relationship between the bacterial composition in the treated soil and suppression of the disease. For the BSD-treated soils, oxidation-reduction potential dropped, and acetate was usually detected at high concentrations. Although the control treatment (irrigation and polythene covering without biomass) decreased the wilt disease incidence in spinach plants cultivated in the treated plot as compared with those for the non-treated plot, BSD-treatments suppressed the disease more effectively. The clone library results showed that both non-treated and control soils contained diversified bacterial communities of various phylogenetic groups, while members of the Firmicutes mainly from the class Clostridia dominated in the BSD-treated soils. The clostridial groups detected were diverse and the major clone groups were closely related to strictly anaerobic fermentative bacteria such as Clostridium saccharobutylicum, Clostridium cylindrosporum, Clostridium sufflavum, and Clostridium xylanovorans. These clostridial groups were almost eliminated from the soil bacterial community when the BSD-treated soil was treated again with irrigation and covering without biomass before the next cropping, in which the wilt disease was hardly suppressed. © 2013. Source


Okamura K.-I.,NISSHOKU Group Inc. | Okamura K.-I.,Osaka Prefecture University | Matsuda Y.,NISSHOKU Group Inc. | Igari K.,NISSHOKU Group Inc. | And 7 more authors.
Environmental Control in Biology | Year: 2013

Production technologies using closed-type plant production systems have been studied to assess their suitability for stable and uniform expression of biopharmaceutical materials in transgenic plants. We have developed a production system for a veterinary vaccine candidate against swine edema disease, using transgenic plants. In this paper, we report the combined effects of plant cultivation density and light intensity on the production levels of a vaccine candidate, the double repeated B subunit of Shiga toxin 2e (2-Stx2eB), in transgenic lettuce cultivated in a closed-type plant factory. Leaf drymatter yield and total soluble protein (TSP) yield increased at higher plant cultivation densities, but in contrast, the 2- Stx2eB concentration in the plants tended to decrease with an increase in plant cultivation density, so that the 2-Stx2eB yield per unit area at lower plant cultivation density (44.4 plants m-2) was similar to or even higher than that obtained at the highest plant density (222.2 plants m-2). In addition, at the cultivation density (44.4 plants m-2), a photosynthesis photon flux density (PPFD) 200 (200-50 -mol m-2 s-1) was optimal in terms of maximizing the 2-Stx2eB yield and minimizing the electrical consumption of lighting. These results show that an optimal combination of plant cultivation density and light intensity is important in improving the productivity of recombinant protein expression systems in transgenic lettuce leaves when grown in a plant factory. Source


Takahashi N.,Ehime University | Okamura K.-I.,NISSHOKU Group Inc. | Matsuda Y.,NISSHOKU Group Inc. | Igari K.,NISSHOKU Group Inc. | And 8 more authors.
Environmental Control in Biology | Year: 2012

The transgenic lettuce expressing double repeated Stx2eB (2 × Stx2eB) is considered to be an edible vaccine candidate against swine edema disease. For the efficient vaccine protein production in transgenic lettuce grown in a plant factory, the effects of air flow on vaccine protein production in transgenic lettuce were investigated. Plants were grown hydroponically in a closed-type plant factory for 21 days with four airflow patterns: 1) air flow from the side to leaves at 2.0 m s -1; 2) air flow from the top to the inner developing leaves with flexible transparent tubing at 1.0 m s -1; 3) air flow from the bottom to leaves with transparent tubing at 1.0 m s -1; and 4) no air flow. Our results showed that the total leaf number in air flow from the top and the bottom to leaves was significantly increased compared with that in no air flow. While lettuce growth with inner air flow and bottom air flow was enhanced, tipburn at the developing leaves were eliminated by air flow. 2 × Stx2eB per plant in bottom air flow to leaves was the highest of the four air flow patterns. These results suggest that bottom air flow to the leaves can enhance vaccine protein productivity in transgenic lettuce. Source


Horie T.,Okayama University | Horie T.,Shinshu University | Sugawara M.,Nara Institute of Science and Technology | Okada T.,Nara Institute of Science and Technology | And 5 more authors.
Journal of Bioscience and Bioengineering | Year: 2011

Potassium ion (K+) plays vital roles in many aspects of cellular homeostasis including competing with sodium ion (Na+) during potassium starvation and salt stress. Therefore, one way to engineer plant cells with improved salt tolerance is to enhance K+ uptake activity of the cells, while keeping Na+ out during salt stress. Here, in search for Na+-insensitive K+ transporter for this purpose, bacterial expression system was used to characterize two K+ transporters, OsHAK2 and OsHAK5, isolated from rice (Oryza sativa cv. Nipponbare). The two OsHAK transporters are members of a KT/HAK/KUP transporter family, which is one of the major K+ transporter families in bacteria, fungi and plants. When expressed in an Escherichia coli K+ transport mutant strain LB2003, both OsHAK transporters rescued the growth defect in K+-limiting conditions by significantly increasing the K+ content of the cells. Under the condition with a large amount of extracellular Na+, we found that OsHAK5 functions as a Na+-insensitive K+ transporter, while OsHAK2 is sensitive to extracellular Na+ and exhibits higher Na+ over K+ transport activities. Moreover, constitutive expression of OsHAK5 in cultured-tobacco BY2 (Nicotiana tabacum cv. Bright Yellow 2) cells enhanced the accumulation of K+ but not Na+ in the cells during salt stress and conferred increased salt tolerance to the cells. Transient expression experiment indicated that OsHAK5 is localized to the plant plasma membrane. These results suggest that the plasma-membrane localized Na+ insensitive K+ transporters, similar to OsHAK5 identified here, could be used as a tool to enhance salt tolerance in plant cells. © 2010 The Society for Biotechnology, Japan. Source


Matsui T.,Idemitsu Kosan Co. | Matsui T.,Nara Institute of Science and Technology | Takita E.,Idemitsu Kosan Co. | Sato T.,Idemitsu Kosan Co. | And 16 more authors.
Transgenic Research | Year: 2011

Pig edema disease is a bacterial disease caused by enterohemorrhagic Escherichia coli. E. coli produces Shiga toxin 2e (Stx2e), which is composed of one A subunit (Stx2eA) and five B subunits (Stx2eB). We previously reported production of Stx2eB in lettuce plants as a potential edible vaccine (Matsui et al. in Biosci Biotechnol Biochem 73:1628-1634, 2009). However, the accumulation level was very low, and it was necessary to improve expression of Stx2eB for potential use of this plant-based vaccine. Therefore, in this study, we optimized the Stx2eB expression cassette and found that a double repeated Stx2eB (2× Stx2eB) accumulates to higher levels than a single Stx2eB in cultured tobacco cells. Furthermore, a linker peptide between the two Stx2eB moieties played an important role in maximizing the effects of the double repeat. Finally, we generated transgenic lettuce plants expressing 2× Stx2eB with a suitable linker peptide that accumulate as much as 80 mg per 100 g fresh weight, a level that will allow us to use these transgenic lettuce plants practically to generate vaccine material. © 2010 Springer Science+Business Media B.V. Source

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