Improved Transformation Method Research Articles

Gallic acid as biofilm inhibitor can improve transformation efficiency of Ruminiclostridium papyrosolvens.

Ruminiclostridium papyrosolvens is an anaerobic, mesophilic, and cellulolytic clostridia, promising consolidated bioprocessing (CBP) candidate for producing renewable green chemicals from cellulose, but its genetic transformation has been severely impeded by extracellular biofilm. Here, we analyzed the effects of five different inhibitors with gradient concentrations on R. papyrosolvens growth and biofilm formation. Gallic acid was proved to be a potent inhibitor of biofilm synthesis of R. papyrosolvens. Furthermore, the transformation efficiency of R. papyrosolvens was significantly increased when the cells were treated by the gallic acid, and the mutant strain was successfully obtained by the improved transformation method. Thus, inhibition of biofilm formation of R. papyrosolvens by using gallic acid will contribute to its genetic transformation and efficient metabolic engineering.

An improved multi-attribute group decision-making method for selecting the green supplier of community elderly healthcare service

With rapid social and economic development, the process of population aging has increased the demand for community elderly healthcare service (CEHS) in China. However, the traditional government-oriented service supply cannot meet the various needs of CEHS, and it is critical to select a suitable supplier of CEHS to provide high-quality green services in the community. Therefore, this study focuses on the issue of green supplier selection of CEHS, explores an improved transformation method for processing multi-type data, and proposes an integrated method of multi-attribute group decision-making (MAGDM) which innovatively applies the degree of overall deviation measure (ODM) to determine expert weight. Finally, the effectiveness and accuracy of the new method are verified by experimental analysis. The results show that H2 is the top choice in the green supplier selection of CEHS, followed by H1, H4, H8, H5, H6, H3, H7, H12, H11, H9, and H10. In addition, the authors apply the traditional ED method to calculate expert weights and compare the results of ODM and ED. It is a fact that the improved ODM method should be more efficient and accurate than the traditional ED method.

An improved method for transformation of Actinidia arguta utilized to demonstrate a central role for MYB110 in regulating anthocyanin accumulation in kiwiberry

Actinidia arguta and related species produce small edible kiwifruit (kiwiberry) with attractive consumer features and high nutritional value. As a recently developed crop, kiwiberry can be improved further by classical breeding or genetic manipulation. The currently available Agrobacterium-mediated transformation protocol is only applicable to a limited number of kiwiberry genotypes such as the female cultivar ‘Hortgem Tahi’. We developed protocols for regeneration and Agrobacterium-mediated transformation of two A. arguta genotypes, female AA06-01 and male AA05-06, also applicable to ‘Hortgem Tahi’. Altered composition of basal salts in the callus-induction media was sufficient to prevent callus browning in AA06-01 and ‘Hortgem Tahi’, but not in AA05-06. Altering the hormone composition to avoid a prolonged callus stage was successfully used to prevent browning and induce shoot development in all three genotypes. Using our improved protocols, Agrobacterium-mediated transformation of a binary vector carrying the neomycin phosphotransferase II (nptII) expression cassette gave rise to kanamycin-resistant plants of both AA06-01 and AA05-06 and the presence of the nptII transgene was confirmed by genomic PCR. The improved protocols were also used to ectopically overexpress the anthocyanin-related transcription factor AcMYB110 in ‘Hortgem Tahi’ giving rise to purple callus with elevated anthocyanin accumulation. AcMYB110 expression and increased levels of anthocyanins were detected in mature leaves of established transgenic plants compared to controls, clearly demonstrating the important role for MYB110 in regulation of anthocyanin accumulation in kiwiberry. The protocols developed during this study provide tools for further functional analyses and genetic manipulation of kiwiberry genotypes. Using a newly developed transformation method we were able to generate transgenic lines of three Actinidia arguta genotypes and demonstrate an important role for MYB110 in anthocyanin accumulation in kiwiberry.

An improved transformation based probabilistic load flow analysis using appropriate reference variable

Power systems are faced with a huge number of uncertainties, which should be addressed in the operational analyses. In this paper, an improved transformation method is used to consider the uncertainties of random variables in probabilistic load flow (PLF) problems. This method transforms non-normal input random variables with non-linear dependence to appropriate independent reference variables. The proposed transformation overcomes the point estimation method (PEM) limitations in estimating high statistical moments of PLF outputs. The proposed PLF method is examined on IEEE 14-bus and 118-bus test systems, consisting of uncertain loads, wind farms, and PV power plant generation. The results of Monte Carlo Simulation (MCS) are used as a benchmark. The results are compared with Nataf transformation, Rotational transformation, and particle swarm optimization clustering methods to prove the accuracy and efficiency of the proposed method.

An improved protocol for efficient transformation and regeneration of Setaria italica.

An efficient and improved transformation method for functional genetics studies in S. italica, being a boon for the Setaria scientific community and for crop improvement. Foxtail millet (Setaria italica) is a short life cycle C4 plant, with sequenced genome, and a potential model plant for C4 species. S. italica is also important on a global food security and healthiness context due to its importance in arid and semi-arid areas. However, despite its importance, there are just few transformation protocols directed to this species. The current protocols reached about 5.5-9% of efficiency, which do not make it a valuable model organism. Different types of explants were used in the above mentioned methods, such as immature and mature inflorescence and shoot apex. However, these techniques have many limitations, such as unavailability of explants throughout the year and a crucial, laborious and considerable time-consuming selection. Aiming a simplified and efficient methodology, we adopted dry mature seeds as explants, which are available in abundance, are constant along the year and well responsive to tissue culture, in addition to a differentiated approach that reaches on an average 19.2% transformation efficiency of S. italica. Thus, we propose a protocol that optimizes the transformation efficiency of this cereal crop allowing a high increase on transformation and regeneration rates. Our transformation protocol provides an interesting tool for Setaria community research as well as enables new strategies for breeding enhanced productivity in the species.

Development of a shuttle plasmid without host restriction sites for efficient transformation and heterologous gene expression in Clostridium cellulovorans.

Clostridium cellulovorans capable of producing large amounts of acetate and butyrate from cellulose is a promising candidate for biofuels and biochemicals production from lignocellulosic biomass. However, the restriction modification (RM) systems of C. cellulovorans hindered the application of existing shuttle plasmids for metabolic engineering of this organism. To overcome the hurdle of plasmid digestion by host, a new shuttle plasmid (pYL001) was developed to remove all restriction sites of two major RM systems of C. cellulovorans, Cce743I and Cce743II. The pYL001 plasmid remained intact after challenge by C. cellulovorans cell extract. Post-electroporation treatments and culturing conditions were also modified to improve cell growth and colony formation on agar plates. With the improvements, the pYL001 plasmid, without in vivo methylation, was readily transformed into C. cellulovorans with colonies of recombinant cells formed on agar plates within 24h. Three pYL001-derived recombinant plasmids free of Cce743I/Cce743II restriction sites, after synonymous mutation of the heterologous genes, were constructed and transformed into C. cellulovorans. Functional expression of these genes was confirmed with butanol and ethanol production from glucose in batch fermentations by the transformants. The pYL001 plasmid and improved transformation method can facilitate further metabolic engineering of C. cellulovorans for cellulosic butanol production.

Dose Dependence of Micro-Voids Distributions in Low-Temperature Neutron Irradiated Eurofer97 Steel

The microstructural effects of mixed spectrum neutron irradiation at 250 °C and 300 °C, for 2.7 dpa, 8.4 dpa, and 16.3 dpa doses, have been investigated in standard Eurofer97 (0.12 C, 9 Cr, 0.48 Mn, 0.2 V, 1.08 W, 0.14 Ta wt%) by means of small-angle neutron scattering (SANS) compared with un-irradiated Eurofer97. The observed SANS effects are attributed to the development of micro-voids, also detected by electron microscopy. The micro-voids distributions have been obtained by an improved transformation method of the SANS cross-sections providing consistent results both before and after subtraction of the un-irradiated reference. Mono-disperse micro-voids distributions are found, with average radii increasing with the dose, namely 4.4 Å for irradiation to 2.7 dpa at 300 °C, 6.6 Å for 8.4 dpa at 300 °C, and 12.9 Å for 16.4 dpa at 250 °C; the corresponding volume fractions are 0.001, 0.006, and 0.004, respectively. The differences in such distributions might reflect different damage evolution mechanisms for the different irradiation conditions, as also suggested by the comparison with a Eurofer97 sample irradiated under fast spectrum. A good resistance of Eurofer97 to micro-structural radiation damage, at least under these irradiation conditions, is suggested by the analysis of these experimental results.

Gentic overexpression increases production of hypocrellin A in Shiraia bambusicola S4201

Hypocrellin A (HA) is a perylenequinone (PQ) isolated from Shiraia bambusicola that shows antiviral and antitumor activities, but its application is limited by the low production from wild fruiting body. A gene overexpressing method was expected to augment the production rate of HA in S. bambusicola. However, the application of this molecular biology technology in S. bambusicola was impeded by a low genetic transformation efficiency and little genomic information. To enhance the plasmid transformant ratio, the Polyethylene Glycol-mediated transformation system was established and optimized. The following green fluorescent protein (GFP) analysis showed that the gene fusion expression system we constructed with a GAPDH promoter Pgpd1 and a rapid 2A peptide was successfully expressed in the S. bambusicola S4201 strain. We successfully obtained the HA high-producing strains by overexpressing O-methyltransferase/FAD-dependent monooxygenase gene (mono) and the hydroxylase gene (hyd), which were the essential genes involved in our putative HA biosynthetic pathway. The overexpression of these two genes increased the production of HA by about 200% and 100%, respectively. In general, this study will provide a basis to identify the genes involved in the hypocrellin A biosynthesis. This improved transformation method can also be used in genetic transformation studies of other fungi.

Polyethylene glycol (PEG)-mediated transformation of the fused egfp-hph gene into Pleurotus ostreatus

A transformation system for the basidiomycete Pleurotus ostreatus was established using PEG-mediated transformation. The homologous glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter and terminator regions of P. ostreatus were amplified and cloned onto the restriction site of the pUC19 vector. Enhanced green fluorescent protein-encoding gene (egfp) and a selection marker, the hygromycin phosphotransferase gene (hph), were then cloned and an expression vector, pUEGFP-hph, was constructed. Protoplasts of P. ostreatus were prepared and used as the recipients in transformation procedures. An improved transformation method was established in the present study and about 100 to 200 resistant colonies per microgram DNA per 107 viable protoplasts were obtained. After subculturing, putative transformants were screened by PCR and verified by Southern blot and RT-PCR analyses showing the correct insertion into the genomic DNA and transcription of the transformed DNA. Moreover, the quantitative PCR and green fluorescence observation were carried out, thus demonstrating expression of the egfp gene driven by the homologous gpd promoter. This was the first report of transformation of P. ostreatus using homologous gpd promoter by PEG-mediated methods. These data will be helpful in future investigations using PEG-mediated transformation for functional characterisation of genes in the edible fungi basidiomycete P. ostreatus. Key words : Pleurotus ostreatus, PEG-mediated transformation, EGFP.

Effects of antioxidants on Agrobacterium-mediated transformation and accelerated production of transgenic plants of Mexican lime (Citrus aurantifolia Swingle)

Four antioxidants including glycine betaine, glutathione, lipoic acid, and polyvinylpyrrolidone were evaluated to improve transformation efficiency of Mexican lime, a precocious but recalcitrant citrus cultivar to Agrobacterium mediated transformation. Lipoic acid substantially improved the transformation efficiency of Mexican lime by aiding in callus development and improving shoot growth from cut ends of epicotyl segments co-cultivated with Agrobacterium. Glycine betaine was moderately beneficial while glutathione and polyvinylpyrrolidone did not have the ability to improve the transformation efficiency. A bi-functional gus-egfp fusion gene used in the study enabled visual identification of transformants and quantitative analysis of gene expression. We describe an improved protocol that allows regenerated transgenic plants to flower within 20–22 months after in vitro regeneration. This enabled the rapid evaluation of transgenic flowers and fruits. Gene expression levels could not be correlated to copy number as determined using Southern blot analysis. Our improved transformation method facilitates the rapid production and evaluation of transgenic plants, especially regarding the functional analysis of transgenes in citrus.

An improved method for Beauveria bassiana transformation using phosphinothricin acetlytransferase and green fluorescent protein fusion gene as a selectable and visible marker

An improved transformation method for the biocontrol agent, Beauveria bassiana, was developed. For convenience of transformation selection and detection, the coding regions of the genes for phosphinothricin acetyltransferase and green fluorescent protein were fused and an expression vector, pBFT, carrying this fusion was constructed. Under optimum conditions, over 60 transformants microg(-1) plasmid DNA were obtained. B. bassiana conidia frozen 1 month at -80 degrees C were fully competent for transformation. The method was significantly less laborious and more rapid than current methods for B. bassiana. The bar::egfp provides a selectable and visible marker which may expedite future genetic engineering of this fungus.

Expression of a major house dust mite allergen gene from Dermatophagoides farinae in Lotus japonicus accession miyakojima MG-20

Transformation of a model legume Lotus japonicus accession Miyakojima MG-20 was examined using Agrobacterium tumefaciens with a binary expression vector. Using the improved transformation method, we introduced a major allergen gene from a house dust mite, Der f 1, into MG-20. Analyses by Southern hybridization, reverse transcription (RT)-PCR, and Western blotting showed that the Der f 1 gene was integrated into the genome of L. japonicus, expressing the gene product in the T1 lines. Our results imply future application of oral allergen-specific immunotherapy using legume plants.

Method for transforming HMMS for speaker-independent recognition in a noisy environment

On improved transformation method uses an initial set of Hidden Markov Models (HMMs) trained on a large amount of speech recorded in a low noise environment R to provide rich information on co-articulation and speaker variation and a smaller database in a more noisy target environment T. A set H of HMMs is trained with data provided in the low noise environment R and the utterances in the noisy environment T are transcribed phonetically using set H of HMMs. The transcribed segments are grouped into a set of Classes C. For each subclass c of Classes C, the transformation Φ c is found to maximize likelihood utterances in T, given H. The HMMs are transformed and steps repeated until likelihood stabilizes.

An improved method for transformation of lettuce by Agrobacterium tumefaciens with a gene that confers freezing resistance

An efficient method for constructing transgenic lettuce cultivars by Agrobacterium tumefaciens was described by Torres et al., 1993. In the present work, an improvement of the above procedure is described and applied to transform the cultivar Grand Rapids with a mutated P5CS gene. The major modifications were concerned with turning more practical the transformation and regeneration protocols. Also we tried to improve transformation steps by increasing injured area in explants and prolonging co-cultivation with Agrobacteria (in larger concentration). A more significant selective pressure was used against non-transformed plants and bacteria. In these work we were concerned to obtain T1 and T2 seeds. The P5CS gene codes for a delta¹-pyrroline-5-carboxylate synthetase, a bifunctional enzyme that catalyzes two steps of proline biosynthesis in plants (Zhang et al., 1995; Peng et al., 1996), while the mutated gene is insensitive to feedback inhibition by proline. The potential benefit of this gene is to confer water stress resistance (drought, salt, cold) due to increased intracellular levels of proline that works like an osmoprotectant. In this work could obtain and characterize transgenic lettuce lineages which are resistant to freezing temperature.

Genetic transformation of Populus genotypes with different chimaeric gene constructs: transformation efficiency and molecular analysis

Aspen (Populus tremula) and hybrid aspen (P. tremula × P. tremuloides) were transformed with different gene constructs using two types of promoter. The aim was to determine the influence of the reporter gene rolC, controlled by promoters of viral or plant origin, on genetic and morphologic expression of different transgenic aspen clones. An improved transformation method using leaf discs was developed, by which putative transgenic plantlets were regenerated at high efficiencies (up to 34%) on kanamycin-containing medium. Transgenic aspen carrying the rolC gene from Agrobacterium rhizogenes under control of the cauliflower-35S-promoter are reduced in size with smaller leaves, whereas aspen transgenic for the same rolC gene, but under control of the light inducible rbcS promoter from potato, are only slightly reduced in size compared to untransformed controls. However, all clones carrying 35S-rolC and rbcS-rolC genes revealed light-green colouration of leaves when compared to untransformed aspen. Owing to this special feature, constructs were used in which expression of the rolC gene was inhibited by insertion of a transposable element, Ac, from maize. Transgenic aspen transformed with the 35S-Ac-rolC and rbcS-Ac-rolC genes were morphologically similar to untransformed aspen, but out of 54 independently regenerated 35S-Ac-rolC transgenic aspen clones, 30 clones showed light-green/dark green variegated leaves. In contrast, out of 19 independently transformed rbcS-Ac-rolC aspen clones, only two clones revealed light-green/dark green variegated leaves. The role of bacterial strains in transformation, and molecular genetics of transgenic aspen plants (including the function of the transposable element, Ac, in the aspen genome) are discussed

Improved transformation method for Acetobacter with plasmid DNA.

An improved method for transformation of derivative strains of A. aceti subsp. aceti No. 1023 with plasmid DNA has been developed. Addition of polyethylene glycol or dimethylsulfoxide increased the transformation efficiency by a factor of about ten. In the presence of PEG 4,000, various transformation conditions were examined. Cells were also made transformation competent by treatment with other divalent cations than Ca2+ . The pH of the buffer did not affect the efficiency significantly. The growth phase influenced the efficiency. Mutants showing high competence were derived by treatment with N-methyl-N′-nitro-N-nitrosoguanidine. By the improved method using the highly transformable mutants, a transformation efficiency of approximately 105 transformants per γg plasmid DNA was achieved.