Efficient Transformation Method Research Articles

Agrobacterium rhizogenes-mediated RNAi of Tripterygium wilfordii and application for functional study of terpenoid biosynthesis pathway genes

Tripterygium wilfordii is an important medical plants which is rich in several valuable bioactive terpenoids, such as triptolide and wilforine. Developing a fast and efficient transformation method is imperative for exploring these metabolites biosynthesis pathway by using RNA interference (RNAi) technology. In the present study, an efficient transformation system using Agrobacterium rhizogenes was established by optimizing four parameters including vacuum infection time, pre-culture duration, appropriate concentration of Agrobacterium, and co-cultivation duration. The transformation efficiency could reach to 62.22% by using leaf sections pre-cultured 5 days as explants, vacuum infection with Agrobacterium at optical density (OD600) of 0.6 for 3 min and co-cultivation for 3 d. The T. wilfordii 3-hydroxy-3-methylglutaryl- coenzyme A reductase (TwHMGR) RNAi hairy root was induced to validate the applicability of this system. The identification of transgenic hairy root was monitored by fluorescence microscopy and verified by PCR analysis. Real-time quantitative PCR analysis showed that the expression of TwHMGR was significantly decreased in transgenic hairy roots compared with control lines. The detection of metabolites showed that the silence of TwHMGR could highly decrease the content of triptolide and wilforine. These results indicated that this A. rhizogenes-mediated RNAi system is an effective protocol for characterizing the function of genes involved in the terpenoid biosynthetic pathway in Tripterygium wilfordii.

Methane to Methanol Conversion Facilitated by Transition-Metal Methyl and Methoxy Units: The Cases of FeCH3+ and FeOCH3.

The conversion of methane to methanol (MTM) catalyzed by FeOCH3+ and FeCH3+ is investigated by means of multireference configuration interaction (MRCI), single-reference coupled clusters (CC), and density functional theory (DFT) approaches. Our dual purpose is the assessment of the applied methodologies and the performance of the proposed catalytic cycle, which involves both of the titled units. The investigated cycle aims to bypass the limitations of metal-oxide catalysts and offers an alternative promising method for efficient MTM transformation. From the technical viewpoint, we found that generally accurate electron correlation treatment is more important than accurately calculated geometries. The combination of optimal DFT geometries with MRCI and CC energetics provides a good compromise between accuracy and efficiency, although there are cases where multireference calculations must be used to obtain correct structures.

Indirect somatic embryogenesis and Agrobacterium-mediated transient transformation of ginger (Zingiber officinale Rosc.) using leaf sheath explants

ABSTRACTThis study has established an efficient and simple Agrobacterium-mediated transient transformation method for a valuable spice crop ‘ginger’ via indirect somatic embryogenesis. Leaf sheath explants were transformed using Agrobacterium tumefaciens strains EHA105 and LBA4404 harbouring vector pGFPGUSPlus containing β-glucuronidase (gus) reporter and hygromycin phosphotransferase (hptII) selection marker. Different parameters enhancing agro-infection were investigated to optimise transformation efficiency. High transformation frequency was achieved when explants were co-cultivated with bacterial cell density of 0.6 OD600 on medium containing 150 µM acetosyringone for 2 days, followed by selection regime of 40 mg/l hygromycin. Histochemical GUS analysis confirmed the transient expression of gus gene, which was further confirmed by PCR analysis of hptII and nptII genes specific primers. The origin and developmental stages of somatic embryos from leaf sheath explants were investigated with the help of scanning electron micrographs. This newly developed transient transformation system provides a basis for quick expression of marker genes in regenerated plantlets.Abbreviation: GUS - β-glucuronidase; PCR - Polymerase chain reaction; LB - Luria broth; BA - 6- Benzylaminopurine; 2,4-D - 2,4-Dichlorophenoxyacetic acid; TDZ - Thidiazuron; NAA - 1-Naphthaleneacetic acid

An efficient Agrobacterium-mediated soybean transformation method using green fluorescent protein as a selectable marker

ABSTRACTGenetic transformation plays a vital role in gene functional study and molecular breeding of soybean. Conventional soybean transformation methods using chemical selectable markers, such as antibiotic or herbicide resistance genes, rely on the identification of positive transgenic lines at advanced developmental stages, making selection procedure labor intensive and time consuming. Utilization of a visual maker to track the transgene would avoid the uncertainty and blindness in the transformation process. In this research, we used green fluorescent protein (GFP) as the selectable marker to detect transgenics at early stages of soybean development. Positive transformants were detected recurrently during each stage of the process based on visualization of the green fluorescence signal, which help us to discard the non-transgenic ones in each stage to reduce the unnecessary experimental cost and lab space. In addition, the positive transgenic seeds can be identified before planting for early detection of transgene and obtain homozygous lines in advance. The method established in this study is also a useful reference for other plant species.

Co-culturing on dry filter paper significantly increased the efficiency of Agrobacterium-mediated transformations of maize immature embryos.

In the Agrobacterium tumefaciens-mediated transformations of maize immature embryos (IEs), the common co-culturing media used are MS or N6-based (MC). Here, we used a novel co-culturing method in which maize 'Qi319' IEs inoculated with Agrobacterium-harboring target vector were placed on dry filter paper (DC) in a petri dish. To compare the effects of the DC and MC co-culturing methods on transformation efficiency, we designed three experiments: (1) A. tumefaciens strain AGL1 independently carrying two plasmids, pXQD12 and pXQD70; (2) two A. tumefaciens strains, AGL1 and EHA105, carrying pXQD12; and (3) strains AGL1 and EHA105 each independently inoculated with pXQD12 and pXQD70 for different infiltration periods, 5, 10, 15, 20 and 25min. We used A. tumefaciens to inoculate IEs derived from maize ears 9-15d after pollination, and then IEs were placed in petri dishes for co-culturing. The DC treatment significantly increased the percentage of IEs expressing green fluorescence protein (%GFP), indicating positive transformants. DC-treated IEs had ~ 3 to 4 times the %GFP compared with MC-treated IEs at 8d after inoculation (3d co-culture and 5d restoration). The average regeneration frequency (%GFP positive regenerated calli of infected IEs) and stable transformation frequency (%GFP positive T0 plants of infected IEs) significantly increased with the DC treatment. Thus, the DC method may be used to develop a more efficient Agrobacterium-mediated transformation method for maize IEs.

Overexpression of SrDXS1 and SrKAH enhances steviol glycosides content in transgenic Stevia plants

BackgroundStevia rebaudiana produces sweet-tasting steviol glycosides (SGs) in its leaves which can be used as natural sweeteners. Metabolic engineering of Stevia would offer an alternative approach to conventional breeding for enhanced production of SGs. However, an effective protocol for Stevia transformation is lacking.ResultsHere, we present an efficient and reproducible method for Agrobacterium-mediated transformation of Stevia. In our attempts to produce transgenic Stevia plants, we found that prolonged dark incubation is critical for increasing shoot regeneration. Etiolated shoots regenerated in the dark also facilitated subsequent visual selection of transformants by green fluorescent protein during Stevia transformation. Using this newly established transformation method, we overexpressed the Stevia 1-deoxy-d-xylulose-5-phosphate synthase 1 (SrDXS1) and kaurenoic acid hydroxylase (SrKAH), both of which are required for SGs biosynthesis. Compared to control plants, the total SGs content in SrDXS1- and SrKAH-overexpressing transgenic lines were enhanced by up to 42–54% and 67–88%, respectively, showing a positive correlation with the expression levels of SrDXS1 and SrKAH. Furthermore, their overexpression did not stunt the growth and development of the transgenic Stevia plants.ConclusionThis study represents a successful case of genetic manipulation of SGs biosynthetic pathway in Stevia and also demonstrates the potential of metabolic engineering towards producing Stevia with improved SGs yield.

Construction and Transient Expression of Chimeric Cassettes Containing CaMV 35S or OsAER1 Promoter and GUS Gene Fusion in Tobacco

<p>Reporter gene assays are commonly used to study the expression pattern of a gene and the promoter activity. The purpose of this study was to assemble the chimeric gene constructs consisting of CaMV 35S promoter orOsAER1 gene promoter connected to the β-glucuronidase (GUS) reporter gene encoding the β-glucuronidase enzyme and to obtain an efficient method for Agrobacterium tumefaciens-mediated transient transformation of tobacco sprouts. The CaMV 35S promoter fragment reamplified from pCAMBIA1301 binary vector and the OsAER1 gene promoter fragment amplified from rice cv. Awan Kuning were ligated into pCAMBIA1300int::gus::tNOS to produce binary vectors pCAMBIA1300int::p35S::gus::tNOS and pCAMBIA1300int::prOsAER1::gus::tNOS. The vectors were used for transient transformation of 5–day old tobacco seedlings. The transformation was carried out using two bacterial cultures with densities of OD600 = 0.5 or OD600 = 1.0 combined with a vacuum for 15 or 30 minutes. Tobacco seedlings transformed with pCAMBIA1300int::p35S::gus::tNOS showed higher transformation efficiency as compared tothe ones transformed with pCAMBIA1300int::prOsAER1::gus::tNOS. A higher efficiency was obtained from transformation using bacterial culture with density of OD600 = 0.5 in combination with a vacuum for 30 minutes. Expression of GUS gene in the tobacco sprouts transformed with CaMV 35S promoter construct was observed through out the sprouts area (root, hypocotyl, cotyledon, and leaf), where as expression of GUS gene was observed in root, hypocotyl, and cotyledon, but not in leaf on tobacco sprouts transformed with OsAER1 promoter construct. These results indicate that the transient transformation is a quick and simple method for testing a chimeric gene construct.</p>

Virus-induced Phytoene Desaturase (PDS) Gene Silencing Using Tobacco Rattle Virus in Lilium × formolongi

Gene function analysis is challenging for Lilium due to the lack of an efficient method for stable genetic transformation. Virus-induced gene silencing (VIGS) is an attractive tool for determining gene function in plants. This study reported that Tobacco rattle virus (TRV)-based VIGS of a PHYTOENE DESATURASE (PDS) ortholog (LhPDS) can be achieved in Lilium × formolongi seedlings, with a survival rate of 92%, using the inoculation method of rubbing plus injection. Compared with untreated and mock-treated seedlings, the photobleached leaf phenotype of silenced plants significantly correlates with down-regulation of endogenous LhPDS (P ≤ 0.05). In addition, the silencing phenomenon can be observed in the growing points of plants, indicating that systemic viral infection was achieved using the protocol. The results indicate that TRV-based VIGS can be used to characterize gene function in Lilium × formolongi. This work lays the foundation for gene function analysis and molecular breeding in Lilium spp.

Agrobacterium-mediated transformation of the ascomycete mushroom Morchella importuna using polyubiquitin and glyceraldehyde-3-phosphate dehydrogenase promoter-based binary vectors.

Morchella importuna is a worldwide distributed edible mushroom with high ecological and economic values, but the molecular and genetic research about this mushroom has been hindered due to lack of an efficient transformation method. Here, we report for the first time the successful transformation of M. importuna by using a hypervirulent Agrobacterium tumefaciens strain bearing the constructed binary plasmid p1391-U-GUS. The selectable markers used were the genes for hygromycin resistance under the control of the polyubquitin promoter from M. importuna. The reporter genes were those for enhanced green fluorescent protein (EGFP) and the β-Glucuronidase (GUS) under the control of glyceraldehyde-3-phosphate dehydrogenase promoter and polyubquitin promoter respectively. The presence of the reporter gene EGFP in the transformants was confirmed by the fluorescence and confocal microscope and molecular analysis and that of the reporter gene GUS was verified by enzyme activity and molecular analysis. The analysis results of both reporter genes indicated that Agrobacterium-mediated transformation was successfully performed in M. importuna.

An efficient method for Agrobacterium-mediated genetic transformation of chilli pepper (Capsicum annuum L.)

In this study, we have describe a highly efficient transformation protocol for two elite Indian cultivars of chilli pepper (Capsicum annuum L.), Pusa Sadabahar and Pusa Jwala. We used hypocotyls and cotyledons derived from young seedlings as explants, standardized regeneration medium for enhanced regeneration, and optimized parameters for promoting efficient transformation in these chilli cultivars. The optimized regeneration medium consisting of MS medium augmented with 1 mg l−1 indole-3-acetic acid and 5 mg l−1 6-benzyl amino purine induced profuse shoot regeneration from the hypocotyl and cotyledon explants, with a regeneration frequency of 81% in Pusa Sadabahar and 78% in Pusa Jwala. The transformation protocol was standardized using Agrobacterium tumefaciens strain LBA4404 harbouring pCAMBIA2301 construct with GUS reporter and NPT-II marker genes. The results demonstrated that the hypocotyl explants are more amenable to transformation than cotyledonary explants in both the cultivars studied. Further, co-cultivation of the hypocotyl explants with agrobacterial cells (OD600 = 0.2–0.5) for the duration of 72 h was found to be optimum for obtaining high transformation efficiency of about 30% in both the cultivars. The transgenic status of chilli transformants recovered in the selection medium having 30 mg l−1 kanamycin was confirmed by GUS activity, PCR, Southern and RT-PCR analyses. About 85–90% of rooted transgenic plants survived hardening and acclimatization processes. Using this protocol we produced several independent transgenic lines of each chilli cultivar.

Genotype-independent Agrobacterium rhizogenes-mediated root transformation of chickpea: a rapid and efficient method for reverse genetics studies

Background Chickpea (Cicer arietinum L.), an important legume crop is one of the major source of dietary protein. Developing an efficient and reproducible transformation method is imperative to expedite functional genomics studies in this crop. Here, we present an optimized and detailed procedure for Agrobacterium rhizogenes-mediated root transformation of chickpea.ResultsTransformation positive roots were obtained on selection medium after two weeks of A. rhizogenes inoculation. Expression of green fluorescent protein further confirmed the success of transformation. We demonstrate that our method adequately transforms chickpea roots at early developmental stage with high efficiency. In addition, root transformation was found to be genotype-independent and the efficacy of our protocol was highest in two (Annigiri and JG-62) of the seven tested chickpea genotypes. Next, we present the functional analysis of chickpea hairy roots by expressing Arabidopsis TRANSPARENT TESTA 2 (AtTT2) gene involved in proanthocyanidins biosynthesis. Overexpression of AtTT2 enhanced the level of proanthocyanidins in hairy roots that led to the decreased colonization of fungal pathogen, Fusarium oxysporum. Furthermore, the induction of transgenic roots does not affect functional studies involving infection of roots by fungal pathogen.ConclusionsTransgenic roots expressing genes of interest will be useful in downstream functional characterization using reverse genetics studies. It requires 1 day to perform the root transformation protocol described in this study and the roots expressing transgene can be maintained for 3–4 weeks, providing sufficient time for further functional studies. Overall, the current methodology will greatly facilitate the functional genomics analyses of candidate genes in root-rhizosphere interaction in this recalcitrant but economically important legume crop.

A transient transformation system for gene characterization in upland cotton (Gossypium hirsutum)

BackgroundGenetically modified cotton accounts for 64% of the world’s cotton growing area (22.3 million hectares). The genome sequencing of the diploid cotton progenitors Gossypium raimondii and Gossypium arboreum as well as the cultivated Gossypium hirsutum has provided a wealth of genetic information that could be exploited for crop improvement. Unfortunately, gene functional characterization in cotton is lagging behind other economically important crops due to the low efficiency, lengthiness and technical complexity of the available stable transformation methods. We present here a simple, fast and efficient method for the transient transformation of G. hirsutum that can be used for gene characterization studies.ResultsWe developed a transient transformation system for gene characterization in upland cotton. Using β-glucuronidase as a reporter for Agrobacterium-mediated transformation assays, we evaluated multiple transformation parameters such as Agrobacterium strain, bacterial density, length of co-cultivation, chemicals and surfactants, which can affect transformation efficiency. After the initial characterization, the Agrobacterium EHA105 strain was selected and a number of binary constructs used to perform gene characterization studies. 7-days-old cotton seedlings were co-cultivated with Agrobacterium and transient gene expression was observed 5 days after infection of the plants. Transcript levels of two different transgenes under the control of the cauliflower mosaic virus (CaMV) 35S promoter were quantified by real-time reverse transcription PCR (qRT-PCR) showing a 3–10 times increase over the levels observed in non-infected controls. The expression patterns driven by the promoters of two G. hirsutum genes as well as the subcellular localization of their corresponding proteins were studied using the new transient expression system and our observations were consistent with previously published results using Arabidopsis as a heterologous system.ConclusionsThe Agrobacterium-mediated transient transformation method is a fast and easy transient expression system enabling high transient expression and transformation efficiency in upland cotton seedlings. Our method can be used for gene functional studies such as promoter characterization and protein subcellular localization in cotton, obviating the need to perform such studies in a heterologous system such as Arabidopsis.

Establishment of an efficient genetic transformation method in Dunaliella tertiolecta mediated by Agrobacterium tumefaciens

Microalgae are photosynthetic microorganisms widely used for the production of highly valued compounds, and recently they have been shown to be promising as a system for the heterologous expression of proteins. Several transformation methods have been successfully developed, from which the Agrobacterium tumefaciens-mediated method remains the most promising. However, microalgae transformation efficiency by A. tumefaciens is shown to vary depending on several transformation conditions.The present study aimed to establish an efficient genetic transformation system in the green microalgae Dunaliella tertiolecta using the A. tumefaciens method. The parameters assessed were the infection medium, the concentration of the A. tumefaciens and co-culture time. As a preliminary screening, the expression of the gusA gene and the viability of transformed cells were evaluated and used to calculate a novel parameter called Transformation Efficiency Index (TEI). The statistical analysis of TEI values showed five treatments with the highest gusA gene expression. To ensure stable transformation, transformed colonies were cultured on selective medium using hygromycin B and the DNA of resistant colonies were extracted after five subcultures and molecularly analyzed by PCR. Results revealed that treatments which use solid infection medium, A. tumefaciens OD600 = 0.5 and co-culture times of 72 h exhibited the highest percentage of stable gusA expression. Overall, this study established an efficient, optimized A. tumefaciens-mediated genetic transformation of D. tertiolecta, which represents a relatively easy procedure with no expensive equipment required. This simple and efficient protocol opens the possibility for further genetic manipulation of this commercially-important microalgae for biotechnological applications.

Optimasi Sistem Transformasi Gen Xiloglukanase Pada Eucalyptus pellita F. Muell Menggunakan Agrobacterium tumefaciens

Eucalyptus pellita is a type of woody plant that is widely used as raw materials of pulp and paper so that the need for wood from this type of plant is increasing. Improvements in wood quality such as cellulose deposition and increased growth rates are needed to support the supply of raw materials for the pulp and paper industry. One technology to change the composition of wood is the modification of plant cell walls through the transformation of xylolukanase gene which in other plants such as Populus alba and Acacia mangium have been shown to increase cellulose deposition and spur growth. The purpose of this study was to obtain an efficient xyloglucanase transformation method in E. pellita using Agrobacterium tumefaciens. Sprouts E. pellita 006 and 06A with different ages of 8 and 15 days are used as plant material for transformation. The sonication treatment of the sprouts prior to the transformation was also applied to determine the effect on transformation efficiency. Transformation is done by soaking the seeds that have been through the treatment of sonication and without sonication on the suspension of Agrobacterium carrying plasmid pAa XEG300 and subsequently grown on the selection medium. Sprouts E. pellita 006 aged 15 days without sonication treatment showed the highest percentage of regeneration in the selection media that is equal to 72%. Gene integration testing through DNA amplification with specific primers showed a ribbon of xyloglucanase with a size of 709 bp.

An efficient Agrobacterium-mediated transformation method for aflatoxin generation fungus Aspergillus flavus.

Aspergillus flavus often invade many important corps and produce harmful aflatoxins both in preharvest and during storage stages. The regulation mechanism of aflatoxin biosynthesis in this fungus has not been well explored mainly due to the lack of an efficient transformation method for constructing a genome-wide gene mutant library. This challenge was resolved in this study, where a reliable and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for A. flavus NRRL 3357 was established. The results showed that removal of multinucleate conidia, to collect a homogenous sample of uninucleate conidia for use as the transformation material, is the key step in this procedure. A. tumefaciens strain AGL-1 harboring the ble gene for zeocin resistance under the control of the gpdA promoter from A. nidulans is suitable for genetic transformation of this fungus. We successfully generated A. flavus transformants with an efficiency of ∼ 60 positive transformants per 106 conidia using our protocol. A small-scale insertional mutant library (∼ 1,000 mutants) was constructed using this method and the resulting several mutants lacked both production of conidia and aflatoxin biosynthesis capacity. Southern blotting analysis demonstrated that the majority of the transformants contained a single T-DNA insert on the genome. To the best of our knowledge, this is the first report of genetic transformation of A. flavus via ATMT and our protocol provides an effective tool for construction of genome-wide gene mutant libraries for functional analysis of important genes in A. flavus.

Overexpression of VpPR10.1 by an efficient transformation method enhances downy mildew resistance in V. vinifera.

Putrescine and spermidine increase the transformation efficiency of Vitis vinifera L. cv. Thompson seedless. Accumulation of VpPR10.1 in transgenic V. vinifera Thompson seedless, likely increases its resistance to downy mildew. A more efficient method is described for facilitating Agrobacterium-mediated transformation of Vitis vinifera L. cv. Thompson Seedless somatic embryogenesis using polyamines (PAs). The efficacies of putrescine, spermidine and spermine are identified at a range of concentrations (10µM, 100µM and 1mM) added to the culture medium during somatic embryo growth. Putrescine (PUT) and spermidine (SPD) promote the recovery of proembryonic masses (PEM) and the development of somatic embryos (SE) after co-cultivation. Judging from the importance of the time-frame in genetic transformation, PAs added at the co-cultivation stage have a stronger effect than delayed selection treatments, which are superior to antibiotic treatments in the selection stage. Best embryogenic responses are with 1mM PUT and 100µM SPD added to the co-culture medium. Using the above method, a pathogenesis-related gene (VpPR10.1) from Chinese wild Vitis pseudoreticulata was transferred into Thompson Seedless for functional evaluation. The transgenic line, confirmed by western blot analysis, was inoculated with Plasmopara viticola to test for downy mildew resistance. Based on observed restrictions of hyphal growth and increases in H2O2 accumulation in the transgenic plants, the accumulation of VpPR10.1 likely enhanced the transgenic plants resistance to downy mildew.

Agrobacterium tumefaciens-Mediated Transformation of the King Tuber Medicinal Mushroom Lentinus tuber-regium (Agaricomycetes).

Lentinus tuber-regium is a sclerotium-forming basidiomycetous mushroom. It has increasingly aroused people's attention for its medicinal effects. In this study, we investigated the applicability of the Agrobacterium tume-faciens-mediated transformation (ATMT) method in L. tuber-regium. A. tumefaciens strain GV 3101 harboring the vector pPEH was used to transform the mycelium of L. tuber-regium strain ACCC50657. The genes for hygromycin B phosphotransferase (hph) and enhanced green fluorescent protein (egfp) under the control of the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene promoter of Pleurotus ostreatus were employed as the selection marker and reporter gene, respectively. The optimal cocultivation temperature and time for transformation were 3 days and 4 days at 25°C and 20°C, respectively. Southern blot analysis showed the variation in the copy number and position of hph, which indicated random integration of hph. Polymerase chain reaction and fluorescence microscopy indicated that the P. ostreatus gpd promoter can drive the fused hph-egfp gene expression in L. tuber-regium. This is the first report that the ATMT method was successfully applied to L. tuber-regium. This reliable and efficient transformation method could be a powerful tool for strain genetic improvement and gene function study in L. tuber-regium.

Research Article Efficient method for Agrobacterium-mediated genetic transformation of tobacco nodal segments

Research Article Efficient method for Agrobacterium-mediated genetic transformation of tobacco nodal segments

Ag(i)-Catalyzed solvent-free CO2 capture with homopropargylic amines: an efficient access to 1,3-oxazinan-2-ones

An efficient solvent-free CO2 capture and transformation method was developed through using homopropargylic amines in the presence of AgOAc and DBU, and a series of 1,3-oxazinan-2-ones was obtained in excellent yields.

グルタチオンと対数期初期細胞を用いる酵母Saccharomyces cerevisiaeの簡便,迅速,高効率形質転換法

グルタチオンと対数期初期細胞を用いる酵母Saccharomyces cerevisiaeの簡便,迅速,高効率形質転換法