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Double-stranded RNA (dsRNA)-degrading enzyme (dsRNase) is one of the negative factors influencing the RNA interference (RNAi) effect in Colorado potato beetle (CPB). We have previously shown that plastid-mediated RNAi (PM-RNAi) can be utilized to control CPB. In this study, we aimed to test whether silencing dsRNase could increase the RNAi effect in CPB via PM-RNAi. We generated transplastomic potato (Solanum tuberosum) plants expressing dsRNA targeting shibire (dsSHI) of CPB. St-dsSHI plants (transplastomic plants expressing dsSHI) showed much less potent RNAi response than transplastomic potato plants expressing dsRNAs targeting CPB Shrub (St-dsSHR) or β-Actin (St-dsACT), two CPB-resistant transplastomic lines generated previously. The three types of transplastomic lines have weak (St-dsSHI), moderate (St-dsSHR) and strong (St-dsACT) resistant levels to CPB. Treatment with in vitro-synthesized dsdsRNase1/2 (a fusion dsRNA targeting dsRNase1 and dsRNase2 of CPB) led to enhanced suppression of dsRNase1 and dsRNase2 in CPB larvae fed with St-dsSHI plants and dsRNase2 in CPB larvae fed with St-dsSHR plants. St-dsSHR or St-dsSHI plants showed significantly reduced CPB larva weight or damaged area by CPB feeding. These results demonstrate that dsRNase-silencing approach could improve the effect of PM-RNAi when the silencing of target genes has weak or moderate strength of the insecticidal effects. The findings provide a new proof-of-concept to enhance crop resistance to insect herbivores to via an “RNAi-of-RNAi” approach. [ABSTRACT FROM AUTHOR]

Plastid-mediated RNA interference (PM-RNAi) has emerged as a promising strategy for pest control. Expression from the plastid genome of stable double-stranded RNAs (dsRNAs) targeted against essential insect genes can effectively control some herbivorous beetles, but little is known about the efficacy of the transplastomic approach in other groups of pest insects, especially nonchewing insects that do not consume large amounts of leaf material. Here we have investigated the susceptibility of the western flower thrip (WFT, Frankliniella occidentalis), a notorious pest in greenhouses and open fields, to PM-RNAi. We show that WFTs ingest chloroplasts and take up plastidexpressed dsRNAs. We generated a series of transplastomic tobacco plants expressing dsRNAs and hairpin RNAs (hpRNAs) targeted against four essential WFT genes. Unexpectedly, we discovered plastid genome instability in transplastomic plants expressing hpRNAs, suggesting that dsRNA cassettes are preferable over hpRNA cassettes when designing PM-RNAi strategies. Feeding studies revealed that, unlike nuclear transgenic plants, transplastomic plants induced a potent RNAi response in WFTs, causing efficient suppression of the targeted genes and high insect mortality. Our study extends the application range of PM-RNAi technology to an important group of nonchewing insects, reveals design principles for the construction of dsRNAexpressing transplastomic plants, and provides an efficient approach to control one of the toughest insect pests in agriculture and horticulture. [ABSTRACT FROM AUTHOR]

Plastid transformation for the expression of recombinant proteins and entire metabolic pathways has become a promising tool for plant biotechnology. However, large-scale application of this technology has been hindered by some technical bottlenecks, including lack of routine transformation protocols for agronomically important crop plants like rice or maize. Currently, there are no standard or commercial plastid transformation vectors available for the scientific community. Construction of a plastid transformation vector usually requires tedious and time-consuming cloning steps. In this study, we describe the adoption of an in vivo Escherichia coli cloning (iVEC) technology to quickly assemble a plastid transformation vector. The method enables simple and seamless build-up of a complete plastid transformation vector from five DNA fragments in a single step. The vector assembled for demonstration purposes contains an enhanced green fluorescent protein (GFP) expression cassette, in which the gfp transgene is driven by the tobacco plastid ribosomal RNA operon promoter fused to the 5' untranslated region (UTR) from gene10 of bacteriophage T7 and the transcript-stabilizing 3'UTR from the E. coli ribosomal RNA operon rrnB. Successful transformation of the tobacco plastid genome was verified by Southern blot analysis and seed assays. High-level expression of the GFP reporter in the transplastomic plants was visualized by confocal microscopy and Coomassie staining, and GFP accumulation was ~9% of the total soluble protein. The iVEC method represents a simple and efficient approach for construction of plastid transformation vector, and offers great potential for the assembly of increasingly complex vectors for synthetic biology applications in plastids. [ABSTRACT FROM AUTHOR]

Structure and crystalline behavior of the ternary system ZnO-BO-PO glasses were investigated by means of X-ray diffraction (XRD) and infrared Raman spectra. The research showed that number of the planar [BO] units increases with the increase of BO content. When the BO content is above ≥10 mol %, the relative content of planar [BO] units increases rapidly and causes weakening of the glass structure and decrease in the chemical stability. In the crystallized glasses the predominant crystal phase ZnPO decreases with the increase of BO content, while the crystal phase BPO increases with it, which cause the declining of chemical stability and the decrease of thermal coefficients of expansion. [ABSTRACT FROM AUTHOR]

To establish a successful in vitro plant regeneration system in Alternanthera philoxeroides (Mart.) Griseb, an orthogonal design was used to investigate the effects of three factors (plant growth regulators, explant types and dark treatment in initial-stage), each having three levels. The effects of these factors and levels on callus induction and shoot regeneration were quantitatively evaluated by analysis of variance. The experimental results showed that the callus induction was significantly affected by 2, 4-dichlorophenoxyacetic acid (2, 4-D), and shoot differentiation from subcultured pieces of callus was enhanced mostly by dark treatment in initial-stage. The optimal conditions for callus induction are obtained from the stem explants cultured on semi-solid Murashige and Skoog (MS) medium plus 2.2 μM BA and 2.2 μM 2, 4-D, with 20 days dark treatment in initial-stage. The highest frequency of shoot regeneration is obtained from the calli cultured on semi-solid MS medium plus 8.8 μM BA, without dark treatment in initial-stage. [ABSTRACT FROM AUTHOR]

Abstract  Immature embryos have been used frequently as target tissues in the genetical transformation of wheat. However, obtaining a large number of high quality immature embryos throughout the year is a laborious and delicate process, because of the need to cultivate the plants under controlled conditions. To circumvent this, we have employed mature embryos rather than immature ones as starter explants for Agrobacterium-mediated transformation of an elite wheat (Triticum aestivum L.) cultivar EM12. The neomycin phosphotransferase ІІ (npt ІІ) and β-glucuronidase (gus) genes were used as selectable and screenable marker genes, respectively, to assess and optimize the performance of T-DNA delivery. With the aid of an orthogonal design, the effect of four factors in combination on transfer DNA (T-DNA) delivery was studied. These factors were preculture duration, different kinds of inoculation, length of inoculation and co-culture condition. Optimal conditions for T-DNA delivery were obtained for mature embryos precultured for 14 days, followed by immersing in inoculation suspension with full strength Murashige and Skoog (MS) salts in darkness at 23–25°C for 3 h, and then co-culturing with Agrobacterium under desiccating condition in the dark at 23–24°C for 2–3 days. Complete analysis of transgene insertion demonstrated that the optimized method for Agrobacterium-mediated transformation of mature embryos of wheat was efficient and practicable. [ABSTRACT FROM AUTHOR]