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To understand the mechanical behavior and failure mechanism of the surrounding layered rock masses during the excavation of underground caverns for a hydropower station, an enhanced equivalent continuum model based on the ubiquitous-joint concept is developed and compiled as a plugin DLL file in FLAC3D. This model is then applied to analyze two engineering geological issues arisen during the excavations of a large underground powerhouse. Both cases are the typical responses controlled primarily by the internal structure of layered rock mass. The first case is mainly concerned with the continuous increase in the displacement of the upstream sidewall after removal of an auxiliary tunnel crown. Numerical simulation reveals the mixed shear-tensile fractures developed along bedding planes. The preserved crown thickness can affect the maximum displacement of sidewall. Failure region will progressively deepen into the interior if no effective reinforcements are adopted. The second case exhibits as the gradual cracking or slabbing of the shotcrete at downstream roof. Stress concentration is the main cause of shotcrete cracking as shown by numerical simulation. The degree of stress concentration can be influenced largely by the angle between rock strata and cavern axis. This degree also varies with the initial stress level and with excavation process, which is confirmed by field monitoring data. The validity and capability of the newly developed model are thus verified by the actual engineering issues.

The severity and harmfulness of a rockburst event are significantly correlated with the scale of rock mass ejection, especially when the rock mass are not supported. This paper presents a microseismicity-based method for the early estimation of rockburst occurrence and its potential scale, which is graded according to the volume of the rockburst pit (Rv). The establishment of the estimation method involves a rockburst database, a grading scheme of the rockburst scale, selection and clustering analysis of rockburst samples, training of an artificial neural network (ANN) model, and dynamic updating. Firstly, a rockburst database is established from cases that were collected from the tunnels at depths of 1900–2525 m in the Jinping II hydropower station, located in southwest China. A grading scheme regarding the rockburst scale is preliminarily proposed on the basis of statistical analysis. Next, seventy-four rockburst cases, collected in tunnels with microseismic (MS) monitoring from October 2010 to March 2011, are selected as typical rockburst samples by using cluster analysis, and the relationships between the microseismicity and rockburst scale are deeply revealed. Then, three MS parameters, namely, the cumulative number of events, the cumulative energy, and the cumulative apparent volume, are determined and used together as input indicators for the identification of the rockburst scale. The estimation model is trained and cross-validated by the ANN optimized through genetic algorithm (GA). Finally, the performance of this microseismicity-based method has been validated by thirty-one cases that occurred in the tunnels with a cumulative length of 1.85 km, excavated from April 2011 to November 2011. The result indicates that approximately 83.9% of the rockburst cases could be reliably estimated. This study provides a new and feasible method for rockburst scale estimation, which can be used separately or applied as a complementary approach to current prediction methods for risk assessment and management of rockbursts in drill-and-blast tunneling.

In order to represent the spatial variability of layered rock mass mechanical parameters in Finite Difference Method (FDM) grids, an approach based on the numerical simulation of cross-correlated non-Gaussian random fields was proposed in this paper. First, the discretization of a Gaussian random field was performed using the Karhunen-Loeve (K-L) expansion. Then, the Gaussian random field for layered rock mass parameter was built by geostatistics and the random field size calculation. Next, the cross-correlated non-Gaussian random fields were simulated using Nataf transformation and isoprobabilistic transformation methods. Furthermore, a grid model coupled to the random fields was finally built using regroupment and element traversal methods. The engineering application showed that the spatial variability, autocorrelation, cross-correlation and non-Gaussianity described by the random fields agreed closely with the statistical characteristics. The correspondence between the random fields and grid model verified that the random parameters in the grid model can represent the anisotropy of layered rock mass parameters. This study provides a computational basis for quantitative risk assessment during tunnel excavation in layered rock mass.

Terpenes constitute the largest class of secondary metabolites in plants. Some terpenes are essential for plant growth and development, membrane components, and photosynthesis. Terpenes are also economically useful for industry, agriculture, and pharmaceuticals. However, there is very low content of most terpenes in microbes and plants. Chemical or microbial synthesis of terpenes are often costly. Plants have the elaborate and economic biosynthetic way of producing high-value terpenes through photosynthesis. Here we engineered the heterogenous sesquiterpenoid patchoulol production in A. annua. When using a strong promoter such as 35S to over express the avian farnesyl diphosphate synthase gene and patchoulol synthase gene, the highest content of patchoulol was 52.58 μg/g DW in transgenic plants. When altering the subcellular location of the introduced sesquiterpene synthetase via a signal peptide, the accumulation of patchoulol was observably increased to 273 μg/g DW. This case demonstrates that A. annua plant with glandular trichomes is a useful platform for synthetic biology studies.

Aim: To explore the patterns of gene expression and functionally characterize the differentially expressed genes (DEGs) in thyroid cancer. Methods: DEGs were determined between 57 paired thyroid cancer and noncancerous tissues using DESeq2. Subsequently, the main functions of the DEGs were studied by a variety of analyses. Results: We identified a cohort of 752 upregulated and 309 downregulated DEGs in thyroid cancer. Several hub DEGs were found in the protein–protein interaction networks. We also revealed a set of DEGs that were dysmethylated, involved in copy number variations and associated with clinical features in thyroid cancer. Conclusion: These results provide some novel findings on DEGs in thyroid cancer, which will be useful to guide further investigation and target therapy for this disease. [Formula: see text]

In this study, a layered rock mass was regarded as a composite material, composed of interlayered intact rocks and bedding planes. Based on this assumption, we developed a transversely isotropic elastic–plastic model to describe the elastic response and post-peak failure behavior based on the Mohr–Coulomb and maximum tensile-stress criteria. Then, we proposed a damage index to estimate the damage degree of layered rock mass during excavation based on the developed model. The numerical simulation of the conventional uniaxial tests indicated that the failure mode and strength predictions of the model were in well agreement with the physical model test results. The index distribution was also shown to be more effective than the plastic zone distribution in identifying layered rock mass failure. The combined application of the mode and the index to assess the stability of the rock mass around the diversion tunnels at the Wudongde hydropower station in China showed that they could accurately estimate the locations and the depth of the potential collapses. Therefore, they are of significant practical value in the stability evaluation of layered rock mass surrounding underground openings.

The purpose of this paper is to research talents education mode reform in the general classification enrollment. Using the method of document study and comparison method obtained necessary data in order to understand general classification enrollment. The students’ educating model through general classification enrollment has many advantages, which can educate students in need, integrate the internal resources of higher education institutions as well as promote the quality of students. This paper analyzes the related issues in general classification enrollment of higher education institutions, puts forward reasonable solutions, and improve in general classification enrollment mode.

Focusing on the problems caused by rockburst hazards in deep tunnels, such as casualties, damage to construction equipment and facilities, construction schedule delays, and project cost increase, this research attempts to present a methodology for dynamic risk assessment and management of rockbursts in D&B tunnels. The basic idea of dynamic risk assessment and management of rockbursts is determined, and methods associated with each step in the rockburst risk assessment and management process are given, respectively. Among them, the main parts include a microseismic method for early warning the occurrence probability of rockburst risk, an estimation method that aims to assess potential consequences of rockburst risk, an evaluation method that utilizes a new quantitative index considering both occurrence probability and consequences for determining the level of rockburst risk, and the dynamic updating. Specifically, this research briefly describes the referenced microseismic method of warning rockburst, but focuses on the analysis of consequences and associated risk assessment and management of rockburst. Using the proposed method of risk assessment and management of rockburst, the occurrence probability, potential consequences, and the level of rockburst risk can be obtained in real-time during tunnel excavation, which contributes to the dynamic optimisation of risk mitigation measures and their application. The applicability of the proposed method has been verified by those cases from the Jinping II deep headrace and water drainage tunnels at depths of 1900–2525 m (with a length of 11.6 km in total for D&B tunnels).

Numerous collapses have occurred during the excavation of diversion tunnels in the thin and extremely thin layered rock strata at Wudongde Hydropower Station in China. Hence, a reliable method is required to predict the risk and the depth of collapse. However, both theory and practice indicate that one single criterion methods cannot satisfactorily predict the collapse depth accurately. In this study, using an artificial neural network (ANN), an intelligent prediction method has been investigated. Through theoretical and statistical analyses, six input parameters (i.e., cover depth, minor–major principal stress ratio, geological strength index, excavation method, support strength and attitude of rock), have been selected and used in the model. Obtained from three diversion tunnels at Wudongde Hydropower Station, forty-five learning samples and six testing samples were used in the training of the model. The structural parameters and the initial weights of the ANN have been optimized by a genetic algorithm (GA). The trained model was then used to predict the collapse depth of another six excavation sites. The predictions show good agreement with the measurements at the sites. The absolute errors between the predicted and the measured collapse depths are all less than 0.35 m, and the relative errors are all less than 15%. Application of the improved ANN method to the tunnel collapse analysis at Wudongde Hydropower Station confirms its effectiveness in predicting collapse depth during tunnelling.

The moss Physcomitrella patens was engineered to produce the diterpenoid sclareol, an important precursor for the synthesis of ambergris substitutes for the perfume industry. The best total yield of sclareol was 2.84 mg/g dry weight (2.28 mg/l culture) obtained after 18 days of cultivation in liquid media (extracted from both media and cell pellet). The two active sclareol synthase genes were integrated in a random fashion, and linked with the ribosomal skip 2A under the control of the CaMV 35S promoter. We conclude that moss can produce sclareol and utilize the ribosomal skip 2A. In addition, we observed growth impairment in all our sclareol-producing lines and moss lines knocked out in the endogenous diterpene synthase (copalyl/kaurene synthase—PpCPS/KS). A RT-PCR study, with ubiquitin as the best reference gene, showed that there was a down-regulation of the transcription of the terpenoid biosynthetic genes in the PpCPS/KS knock out moss. This down-regulation was recovered by the introduction of the two sclareol synthases, suggesting that the regulation of the general terpenoid biosynthesis is very flexible and can be amended in future biotechnological engineering.

The mechanical behaviours of layered rock mass exhibit significant differences in the directions parallel and vertical to the bedding planes. The deformation and failure of a layered rock mass has remarkable weak-plane dependence, which brings a major challenge to the control of the stability of the surrounding rock mass in underground openings. In this study, a layered rock mass is firstly regarded as a composite material composed of interlayered rocks and bedding planes. Then, based on the Mohr–Coulomb and maximum tensile stress criteria, an index of point safety factor for a layered rock mass is established considering the mechanical properties of interlayered rocks and bedding planes. The safety of the artificial layered rock mass specimens in the triaxial test is evaluated using this index. The results show that the distribution of this index is in good agreement with the macroscopic failed zone of the rock specimen, indicating that this index is feasible for characterizing the macroscopic failure of rock masses. Finally, the index is adopted to evaluate the stability of the midpartition between the #3 and #4 diversion tunnels at the right bank of the Wudongde hydropower station before and after its reinforcement. The results indicate that there is a yielded zone where the point safety factor is less than 1.0 in the unreinforced midpartition of the collapsed tunnel section, and it is nearly connected. If it is not reinforced in time, collapse cut-through of the entire midpartition may occur and then endanger the overall stability of the tunnel. After the emergency reinforcement measure with two-ended anchored piles and concrete backfill, the safety of the midpartition is significantly improved. In this case, the safety factor is much larger than 1.0, indicating that the adoption of this emergency reinforcement measure is effective.

Romp algorithm can reconstruct the signal fast and accurate as long as the signal satisfy Restricted Isometry Property. However, the number of iterations will affect the quality of the image reconstruction directly while the sparsity of the signal is unknown. A new solution will be put forward in this paper which can get the reasonable iteration times of the ROMP algorithm base on some knowledge of each iteration process, Experiments show that this solution can get good quality reconstruction for the image of different features.

This paper focuses on the evolution processes of different types of rockbursts occurring in deep tunnels. A series of laboratory tests and in-situ monitoring in deep tunnels excavated by tunnel boring machine (TBM) and drill-and-blast (D&B) method have been conducted to understand the mechanisms and processes of the evolution of different types of rockbursts, including strain rockburst, strain-structure slip rockburst, immediate rockburst and time-delayed rockburst. Three different risk assessment methods are proposed to evaluate the intensity and potential failure depth of rockbursts. These methods can be applied before excavation and the results can be updated according to the real-time information during excavation. Two micro-seismicity based real-time warning systems have been established for predicting various intensities of rockbursts, such as slight, moderate, intensive and extremely intensive rockbursts. Meanwhile, the probability and intensity of the rockburst are also given. The strategy for excavation and support design has been suggested for various intensities of rockbursts before excavation. The strategy for dynamic control of the rockburst evolution process is also proposed according to the monitoring results. The methodology has been successfully applied to rockburst risk reduction for deep tunnels at Jinping II hydropower project. The results have illustrated the applicability of the proposed methodology and techniques concerning rockbursts.

A highly sensitive and simple spectrofluorimetric method for the determination of reduced glutathione based on the fluorescence quenching effect of glutathione on the hemoglobin-catalyzed reaction of H2O2 with L-tyrosine was developed. The concentration of glutathione is linear with the fluorescence quenching (ΔF) of system under the optimal experimental conditions. The calibration graph is linear in the range 6.25 × 10−9 to 3.75 × 10−6 M with the detection limit of 2.23 × 10−9 M. This method can be used for the determination of glutathione in vegetables with satisfactory results.

基于K 2 ZrF 6 在碱性溶液中形成负电 Zr(OH) 4 颗粒的作用机理, 在添加K 2 ZrF 6 的Na 2 SiO 3 -KOH溶液中, 对LY12铝合金表面进行微弧氧化处理. 利用扫描电镜(SEM)、能谱(EDX)和X射线衍射仪(XRD)探讨了K 2 ZrF 6 添加剂对成膜速率、膜层形貌及结构的调制作用, 并借助热阻隔性能测试评价了不同溶液中所形成膜层的热阻隔效果. 结果表明, 溶液中添加K 2 ZrF 6 增大了微弧氧化的成膜速率, 使膜层的内外表面较为平整, 且膜层中大尺寸缺陷减少. EDX线扫描分析表明, 添加K 2 ZrF 6 后形成的膜层中出现大量Zr元素, 且其含量随距膜/基界面距离的增加而增加. XRD结果显示, 未添加K2ZrF6溶液制得的膜层主要由γ-Al 2 O 3 和α-Al 2 O 3 相组成; 添加K 2 ZrF 6 后, 膜层中晶态氧化物含量降低、非晶态物质增多. 隔热性能测试显示, 经K 2 ZrF 6 调制后的膜层具有更加优良的热阻隔性能.

Funding: J.F.S. acknowledges the financial support of the Strategic Priority Research Program of the Chinese Academy of Sciences (grant number XDB07030200). Erasable electrical conductive domain walls in an insulating ferroelectric matrix provide novel functionalities for applications in logic and memory devices. The crux of such success requires sufficiently high wall currents to drive high‐speed and high‐power nanodevices. This work provides an appealing strategy to increase the current by two orders of magnitude through the careful selection of current flowing paths along the charged walls. The dense walls come into form through the hierarchical evolution of the 71°, 109°, and 180° domains of epitaxial BiFeO3 films in a planar‐geometry ferroelectric resistance‐switching memory cell. The engineered films grown on SrTiO3 and GdScO3 substrates allow the observation of detailed local configurations and the evolution of the different domain types using vector piezo‐force microscopy. The higher local electrical conductivity near the charged domain walls is identified by conductive atomic‐force microscopy. It is shown that 180° domain reversal proceeds by three‐step 71° rotations of the pristine domains. Surprisingly, a maximum current of ≈300 nA is observed for current paths along charge‐uncompensated head‐to‐head hierarchical domain walls connecting the two electrodes on the film surface. Furthermore, the achievable current level can be conveniently controlled by varying the relative directions of the initial polarization and the applied field. Postprint

Sugar beet (Beta vulgaris) is an important arable crop, traditionally used for sugar extraction, but more recently, for biofuel production. A wide range of pests, including beet cyst nematode (Heterodera schachtii), root-knot nematodes (Meloidogyne spp.), green peach aphids (Myzus persicae) and beet root maggot (Tetanops myopaeformis), infest the roots or leaves of sugar beet, which leads to yield loss directly or through transmission of beet pathogens such as viruses. Conventional pest control approaches based on chemical application have led to high economic costs. Development of pest-resistant sugar beet varieties could play an important role towards sustainable crop production while minimising environmental impact. Intensive Beta germplasm screening has been fruitful, and genetic lines resistant to nematodes, aphids and root maggot have been identified and integrated into sugar beet breeding programmes. A small number of genes responding to pest attack have been cloned from sugar beet and wild Beta species. This trend will continue towards a detailed understanding of the molecular mechanism of insect-host plant interactions and host resistance. Molecular biotechnological techniques have shown promise in developing transgenic pest resistance varieties at an accelerated speed with high accuracy. The use of transgenic technology is discussed with regard to biodiversity and food safety.

Efficient regeneration via somatic embryogenesis (SE) would be a valuable system for the micropropagation and genetic transformation of sugar beet. This study evaluated the effects of basic culture media (MS and PGo), plant growth regulators, sugars and the starting plant material on somatic embryogenesis in nine sugar beet breeding lines. Somatic embryos were induced from seedlings of several genotypes via an intervening callus phase on PGo medium containing N6-benzylaminopurine (BAP). Calli were mainly induced from cotyledons. Maltose was more effective for the induction of somatic embryogenesis than was sucrose. There were significant differences between genotypes. HB 526 and SDM 3, which produced embryogenic calli at frequencies of 25–50%, performed better than SDM 2, 8, 9 and 11. The embryogenic calli and embryos produced by this method were multiplied by repeated subculture. Histological analysis of embryogenic callus cultures indicated that somatic embryos were derived from single- or a small number of cells. 2,4-dichlorophenoxyacetic acid (2,4-D) was ineffective for the induction of somatic embryogenesis from seedlings but induced direct somatic embryogenesis from immature zygotic embryos (IEs). Somatic embryos were mainly initiated from hypocotyls derived from the cultured IEs in line HB 526. Rapid and efficient regeneration of plants via somatic embryogenesis may provide a system for studying the molecular mechanism of SE and a route for the genetic transformation of sugar beet.

Plant metabolites vary at different stages of their life cycle. Although it is well documented that environmental factors stimulate biosynthesis of secondary metabolites, the regulation by endogenous developmental cues remains poorly understood. The microRNA156 (miR156)-targeted squamosa promoter binding protein-like (SPL) factors function as a major age cue in regulating developmental phase transition and flowering. We show here that the miR156-targeted SPL transcription factor plays an important role in the spatiotemporal regulation of sesquiterpene biosynthesis. In Arabidopsis thaliana, the miR156-SPL module regulates the formation of (E)-β-caryophyllene in the flowering stage through modulating expression of the sesquiterpene synthase gene TPS21. We demonstrated that SPL9 directly binds to TPS21 promoter and activates its expression. In the perennial fragrant herb Pogostemon cablin, the accumulation of patchouli oil, largely composed of sesquiterpenes dominated by (-)-patchoulol, is also age-regulated, and the SPL promotes biosynthesis of sesquiterpenes in elder plants by upregulating patchoulol synthase (PatPTS) gene expression. As miR156-SPLs are highly conserved in plants, our finding not only uncovers a molecular link between developmental timing and sesquiterpene production but also suggests a new strategy to engineer plants for accelerated growth with enhanced production of terpenoids.

Improved in vitro tissue culture systems are needed to facilitate the application of recombinant DNA technology to the improvement of sugar beet germplasm. The effects of N 6-benzyladenine (BA) and thidiazuron (TDZ) pretreatment on adventitious shoot and somatic embryogenesis regeneration were evaluated in a range of sugar beet breeding lines and commercial varieties. Petiole explants showed higher frequencies of direct adventitious shoot formation and produced more shoots per explant than leaf lamina explants. TDZ was more effective than BA for the promotion of shoot formation. The optimal TDZ concentrations were 2.3–4.6 μM for the induction of adventitious shoot regeneration. Direct somatic embryogenesis from intact seedlings could be induced by either BA or TDZ. TDZ-induced somatic embryogenesis occurred on the lower surface of cotyledons at concentrations of 0.5–2μM and was less genotype-dependent than with Ba. A high frequency of callus induction could be obtained from seedlings and leaf explants, but only a few of the calluses derived from leaf explants could regenerate to plants via indirect somatic embryogenesis. These results demonstrated that TDZ could prove to be a more effective cytokinin for in vitro culture of sugar beet than BA. Rapid and efficient regeneration of plants using TDZ may provide a route for the production of transgenic sugar beet following Agrobacterium-mediated transformation.

The co-transformation of a single plant genome with two independent T-DNA regions provides opportunities for genetic separation in subsequent generations. In an effective strategy, co-delivery events must form a high proportion of the total transformed population. In this study, using the model plant species tobacco (Nicotiana tabacum), it was shown that the frequency of co-transformation within a given To population could be as high as 100% and this was found to be dependent, at least in part, on designing the plasmid vectors so that the kbp size of the first selected T-DNA region was >2-fold that of the designated T-DNA region for co-transfer. Overall, 40-50% of To lines demonstrated the capacity for segregational separation of co-transformed T-DNA regions. Hence, the estimate of the required number of total transformants for such an independent strategy may seem to be as little as 2-fold that for a conventional, single T-DNA strategy, but we strongly temper such estimates with indications that high co-transformation frequencies may be associated with a higher incidence of linkage. In this co-transformation study we used a single (Agrobacterium) strain system in which a single binary plasmid contained either two or three T-DNA regions, each with a selectable marker. This arrangement could reveal that 'read-through' events within the Agrobacterium cells, resulting in the co-transfer of adjacent T-DNA regions as a single linked unit, accounted for up to 20% of co-transformed plant lines. Such read-through co-delivery appeared to be more frequent from the 'supervirulent' EHA101 A. tumefaciens strain, compared to the 'ordinary' LBA4404 strain. By using the binary plasmid with three selectable T-DNA regions, we have been able to consider the frequency of co-integration of a third independent T-DNA within a T0 subpopulation of co-transformants. This was found to be higher than expected. These observations were applied to the co-transfer of (unwanted) plasmid backbone sequences and showed that screening against such sequences may add a significant factor in achieving the desired, final genotype.

Electroporation has been used effectively to deliver DNA into the tissue of intact wheat immature embryos. Transformed plantlets have been recovered after electroporation using field strengths of 275 and 750 V/cm, 960-µF capacitor and 50 µg/ml of linear plasmid DNA, containing bar and uidA genes. The field strength of 750 V/cm proved to be more effective for DNA delivery (estimated by transient GUS expression) and for recovery of transformed plants (two transgenic plants were recovered with an efficiency of 0.4%). After application of a field strength of 275 V/cm there was no visual evidence of transient GUS expression, but one transgenic plant was recovered with an efficiency of 0.2%, based on the number of electroporated embryos. This indicates that the amount of DNA delivered into the cells was too low for visual identification of transient GUS expression and that GUS expression may not provide an appropriate assessment of the efficiency of DNA delivery. Southern blot hybridisation has revealed a low copy number of transgene integration with some rearrangements in integrated loci. None of the transgenic plants has shown any visual GUS expression, although we could amplify the transcript of the uidA gene in T(0) progeny using RT-PCR. This may indicate that suppression of uidA expression occurred at the post-transcriptional level. The efficiency of tissue electroporation is still dependent on the quality of the plant material which is used but the transformation events were reproducible from one group of experiments to another. At present, this technique is dependent on a combination of factors including pretreatments of the recipient tissue, quality of tissue culture and optimisation of electroporation conditions.

We have developed a routine system, based on microprojectile bombardment of embryogenic cell clusters for recovery of fertile transgenic rice (Oryza sativa L.) plants. Embryogenic cell clusters were established within 3–4 months from calli derived from mature seeds of nine diverse cultivars. The cell clusters were bombarded with plasmids carrying genes encoding hygromycin phosphotransferase (hph, conferring resistance to hygromycin B) and β-glucuronidase (gus A), both driven by the cauliflower mosaic virus (CaMV)35S promoter. Resistant calli were readily recovered from hygromycin-containing medium. On average 4.6 resistant calli were recovered from each bombarded plate. Transgenic plants were routinely regenerated from such resistant calli. In this system, where the transformation procedure was based on bombardment of embryogenic cell clusters, there were no escapes. Data from Southern blot analysis of T0 and T1 plants confirmed that the transgene was stably integrated into the host genome and inherited in a Mendelian fashion.

Transfer of T-DNA from Agrobacterium tumefaciens and A. rhizogenes to cells of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) is demonstrated following the inoculation of immature embryos and immature embryo-derived callus. Agrobacterium T-DNA vectors containing the C1/Lc anthocyanin-biosynthesis regulatory genes, the gusA gene or a synthetic green fluorescent protein gene (sgfp-S65T) were constructed from original binary vectors. The visual T-DNA markers were used as cell-autonomous reporters of early Agrobacterium-mediated transformation events in the wheat and barley cells. This localization of the transformed cells revealed a non-random distribution throughout each embryo and callus piece.

Barley (Hordeum vulgare L. cvs. Igri and Dissa) cell suspension cultures, which had been initiated from immature embryo-derived (IED) and microspore-derived (MSD) callus, were co-cultivated with various Agrobacterium tumefaciens strains. The T-DNA vectors contained visually-detectable marker genes (C1/Lc orgusA-intron), as reporters of transient T-DNA transfer, and also drug resistance genes (hph or bar) to facilitate selection of stably transformed cell lines. A set of normal binary vectors in a super-virulent Agrobacterium strain [EHA101(pBECKS)] and also a super-binary vector [LBA4404(pTOK233)] were used in this study. Cells of the suspension cultures which received T-DNA were able to proliferate under selection regimes and a number of hygromycin- or phosphinothricin-resistant barley callus lines were isolated which expressed a co-transferred gusA gene. To ensure homogeneity of the cell lines, prolonged tissue culture regimes were used but these resulted in a loss of the capacity to regenerate plants from the transgenic callus lines. The frequency of recovery of transformed callus lines ranged from 0.3% to 2.9%. Southern blot analyses of the transformed callus lines confirmed the presence of the marker genes and demonstrated them to be associated with DNA which was distinct from that of the original Agrobacterium plasmid. Furthermore, independent transgenic lines showed diverse patterns of hybridising bands. These data suggest that the T-DNA fragment was stably maintained through integration into the genomes of the barley cell lines.

A rapid, efficient, routine system has been established for Agrobacterium tumefaciens-mediated production of hundreds of fertile transgenic plants from commercially important rice cultivars, including an indica cultivar, Pusa Basmati 1. Calli induced from embryos of mature rice seeds were cocultivated with A. tumefaciens strain LBA4404 carrying the plasmid pTOK233, then exposed to hygromycin selection followed by an efficient regeneration system. Based on the total number of calli co-cultivated, the transformation frequencies of independent transgenic rice plants including cultivars Pusa Basmati 1, E-yi 105, E-wan 5 and Zhong-shu-wan-geng, were 13.5, 13.0, 9.1, and 9.3%, respectively. T1 seeds were harvested within 7-8 mo of initiation of mature embryo cultures. Data from Southern hybridization analysis proved that foreign genes on T-DNA were stably integrated into the rice genome at low copy/site numbers. Mendelian inheritance of the transgenes was confirmed in T1 progeny.

The pulse-discharge electroporation system HPES-3 has been used to transfer the bialaphos acetyltransferase gene of plasmid pDM302 into three commercial spring wheat cultivars T2003, Huanong No. 400 and Qu an Mai No. 1. These three cultivars are widely farmed in South China. The immature embryos, collected at 12–15 days after anthesis, were electropo- rated and exposed to phosphinothricin-containing medium. Phosphinothricin-resistant embryogenic calli were induced and from these a plantlet of cultivar T2003 was recovered. PCR analysis and DNA dot blot hybridization showed that the bialaphos acetyltransferase gene was present in the cells of the transgenic plant.

Summary A number of stably transformed wheat cell lines were obtained using two different direct gene transfer techniques. When integration of the foreign genes was investigated in DNA samples taken at 10 months post-selection, complicated profiles of transgene bands were observed in Southern blot analysis. Among these, a number of common bands were identified showing similar hybridization patterns between independently transformed cell lines. This type of hybridization pattern has been a common observation and is usually interpreted as concatameric rearrangement of integrated DNA. However, when integration was reinvestigated with DNA samples taken at 30 months postselection, the hybridization pattern changed and most of the common bands had disappeared. Further analysis using a set of methylation-sensitive enzymes revealed that the DNA represented by the common bands was N6-adenine methylated (m6A DNA), and there was even m6A DNA in some 30 month samples. Although the source of m6A DNA in the wheat cultures was not clearly established, the data indicate that transformation of an endophyte (e.g. a mycoplasma-like organism) may have occurred at the same time as the transformation of wheat cells. The integration pattern of undermethylated transgenes in the transformed cells became simpler and clearer after treating the DNA preparations with Dpnl, which only cuts m6A DNA. The implications of these data for other methods of inoculating cereals with DNA are discussed.

The green fluorescent protein (GFP) was used as a visual selectable marker to produce transgenic coffee (Coffea canephora) plants following Agrobacterium-mediated transformation. The binary vector pBECKS 2000.7 containing synthetic gene for GFP (sgfp) S65T and the hygromycin phosphotransferase gene hph both controlled by 35S cauliflower mosaic virus CaMV35S promoters was used for transformation. Embryogenic cultures were initiated from hypocotyls and cotyledon leaves of in vitro grown seedlings and used as target material. Selection of transformed tissue was carried out using GFP visual selection as the sole screen or in combination with a low level of antibiotics (hygromycin 10 mg/L), and the efficiency was compared with antibiotics selection alone (hygromycin 30 mg/L). GFP selection reduced the time for transformed somatic embryos formation from 18 weeks on a hygromycin (30 mg/L) antibiotics containing medium to 8 weeks. Moreover, visual selection of GFP combined with low level of antibiotics selection improved the transformation efficiency and increased the number of transformed coffee plants compared to selection in the presence of antibiotics. Molecular analysis confirmed the presence of the sgfp-S65T coding region in the regenerated plants. Visual screening of transformed cells using GFP by Agrobacterium-mediated transformation techniques was found to be efficient and therefore has the potential for development of selectable marker-free transgenic coffee plants.

The apical meristems in dry wheat seeds were exposed (dissected seeds) to provide a target for DNA uptake. Using wheat, dwarf virus as the marker DNA, various methods of delivery were compared. Dry dissected seeds imbided in wheat dwarf virus DNA solution gave infection in 16% of the seedlings growing from them. A wheat dwarf virus dimer placed between the T-DNA borders of a vector plasmid inAgrobacterium tumefaciens (disarmed C58) andA. rhizogenes (LBA 9402) gave high levels of infection (79%) when dissected seeds were soaked in theAgrobacterium inoculum (agroinfection). Bombardment of dry dissected seeds with tungsten particles coated in wheat dwarf virus DNA did not give infection, but when softened by presoaking in water for 14 h, infection was observed at a low level (3%). The exposure of the apical meristem in all three methods gave a higher frequency of infection compared with treating intact seeds and in some cases the difference was substantial. The significance of the approach for DNA uptake studies is discussed along with its relevance to achieving stable transformation with non-viral constructs.

Experiments were performed to investigate the flow structure and boiling heat transfer characteristics of water spray cooling on flat and microgrooved surfaces using a high-speed camera and a microscope. The heaters were made of cooper, with surface size of 2.0cm×7.4cm. Three orientations of the heater surfaces were selected: horizontal upward-facing, vertical, and horizontal downward-facing. A full-cone spray nozzle was placed normal to these heated surfaces. The heat transfer was directly measured using thermocouples within the heater. The experimental results show the bubble’s growth, coalescence along/between microgrooves, and break-up as wall heat flux reaches some higher values. It was found that the heat transfer for microgrooved surface is generally higher than that of flat surface at a given flow rate with the same surface orientation. The thermal performance of vertical microgrooved surface was highest at low temperatures; the thermal performance of the horizontal upward-facing was highest at higher wall temperature. The heat transfer performance for the horizontal downward-facing microgrooved surface had the highest critical heat flux (CHF).Copyright © 2009 by ASME

Improved in vitro tissue culture systems are needed to facilitate the application of transgene technology to the improvement of sugar beet germplasms. Several commercially important sugar beet breeding lines (SDM 3, 5, 8, 9, 10, 11, HB 526, and CMS 22003) and commercial varieties (Roberta and Gala) were tested for their regeneration capacity through adventitious shoot organogenesis from cotyledons, hypocotyls, root/hypocotyl/shoot transition zone tissues, and leaf lamina and petiole via an intervening callus phase. Callus induction and adventitious shoot regeneration was dependent on genotype and combinations of plant growth regulators. With cotyledon or hypocotyl explants, SDM 3 and 10 showed a better response on adventitious shoot regeneration in medium containing benzyladenine (BA) and 2,3,5-triiodobenzoic acid or 1-naphthaleneacetic acid (NAA) than SDM 11, 5, and 9. Shoot regeneration was obtained from hypocotyl–root or hypocotyl–shoot transition zone tissue in SDM 9, 10, and HB 526 grown on ...

A highly regenerable, isolated microspore system for barley, Hordeum vulgare L. cv. Igri, has been developed which is amenable to transformation studies using particle bombardment. The system allows DNA to be delivered to microspores at the single cell stage and both transient and stable transformation events have been demonstrated. The potential advantages of using isolated microspores as the target tissue in routine transformation systems are discussed.

Background The development of robust microbes with tolerance to the combined lignocellulose-derived inhibitors is critical for the efficient cellulosic ethanol production. However, the lack of understanding on the inhibition mechanism limited the rational engineering of tolerant strain. Here, through the metabolomic analysis of an adaptation process of Saccharomyces cerevisiae to representative inhibitors, i.e., furfural, acetic acid and phenol (FAP), we figured out the new candidates for improving inhibitor tolerance. Results After metabolomic analysis, proline and myo-inositol were identified as the potential metabolites responsible for strain tolerance to inhibitors. The deletion of genes involved in proline or myo-inositol synthesis weakened strain tolerance against FAP stress. On the contrary, the addition of proline or myo-inositol in medium exerted a protective effect on cell growth under FAP stress. Furthermore, the enhancement of proline or myo-inositol synthesis by overexpressing key gene PRO1 or INO1 conferred yeast strain significantly increased FAP tolerance. All the recombinant strains finished the fermentation within 60 h under FAP stress, while the control strain was still in the lag phase. Meanwhile, it was found that the intracellular level of reactive oxygen species (ROS) under FAP condition was decreased with the increase of proline content, suggesting the function of proline as a ROS scavenger to protect strains from inhibitor damage. Conclusion Increasing proline and myo-inositol were uncovered as the new determinants for improving strain tolerance to FAP under the guidance of metabolomics. Meanwhile, this study displayed the powerful application of metabolomics to develop rational strategies to increase stress tolerance and provided valuable insights into the design of recombinant microbes for the complex traits. Electronic supplementary material The online version of this article (doi:10.1186/s13068-015-0329-5) contains supplementary material, which is available to authorized users.