High-efficiency Transformation System Research Articles

Transient expression of the enhanced green fluorescence protein (egfp) gene in Tetraselmis subcordiformis (Chlorodendrales, Chlorophyta) with three exogenous promoters

:A stable transformation system for Tetraselmis subcordiformis had been established, but a low transformation rate limits its application to this alga. As a promoter is a key factor for transgenic research, a suitable promoter could increase the transformation rate. To improve a transformation system for T. subcordiformis, three commonly used promoters for algal transformation were selected. The CMV, CaMV35S and SV40 promoters were transferred to T. subcordiformis by glass-bead agitation and particle bombardment, with the enhanced green fluorescence protein (egfp) gene as the reporter gene. The transformation rate was calculated according to the GFP fluorescence in transformed algae. Results showed that the CaMV 35S promoter transformation efficiency was higher than that of CMV and SV40 promoters. This report will contribute to efforts to establish a high-efficiency transformation system and expands the fundamental research and applications of this alga.

Brachypodium distachyon.

The small grass Brachypodium distachyon has attributes that make it an excellent model for the development and improvement of cereal crops and bioenergy feedstocks. To realize the potential of this system, many tools have been developed (e.g., the complete genome sequence, a large collection of natural accessions, a high density genetic map, BAC libraries, EST sequences, microarrays, etc.). In this chapter, we describe a high-efficiency transformation system, an essential tool for a modern model system. Our method utilizes the natural ability of Agrobacterium tumefaciens to transfer a well-defined region of DNA from its tumor-inducing (Ti) plasmid DNA into the genome of a host plant cell. Immature embryos dissected out of developing B. distachyon seeds generate an embryogenic callus that serves as the source material for transformation and regeneration of transgenic plants. Embryogenic callus is cocultivated with A. tumefaciens carrying a recombinant plasmid containing the desired transformation sequence. Following cocultivation, callus is transferred to selective media to identify and amplify the transgenic tissue. After 2-5 weeks on selection media, transgenic callus is moved onto regeneration media for 2-4 weeks until plantlets emerge. Plantlets are grown in tissue culture until they develop roots and are transplanted into soil. Transgenic plants can be transferred to soil 6-10 weeks after cocultivation. Using this method with hygromycin selection, transformation efficiencies average 42 %, and it is routinely observed that 50-75 % of cocultivated calluses produce transgenic plants. The time from dissecting out embryos to having the first transgenic plants in soil is 14-18 weeks, and the time to harvesting transgenic seeds is 20-31 weeks.

Frontiers of torenia research: innovative ornamental traits and study of ecological interaction networks through genetic engineering

Advances in research in the past few years on the ornamental plant torenia (Torenia spps.) have made it notable as a model plant on the frontier of genetic engineering aimed at studying ornamental characteristics and pest control in horticultural ecosystems. The remarkable advantage of torenia over other ornamental plant species is the availability of an easy and high-efficiency transformation system for it. Unfortunately, most of the current torenia research is still not very widespread, because this species has not become prominent as an alternative to other successful model plants such as Arabidopsis, snapdragon and petunia. However, nowadays, a more global view using not only a few selected models but also several additional species are required for creating innovative ornamental traits and studying horticultural ecosystems. We therefore introduce and discuss recent research on torenia, the family Scrophulariaceae, for secondary metabolite bioengineering, in which global insights into horticulture, agriculture and ecology have been advanced. Floral traits, in torenia particularly floral color, have been extensively studied by manipulating the flavonoid biosynthetic pathways in flower organs. Plant aroma, including volatile terpenoids, has also been genetically modulated in order to understand the complicated nature of multi-trophic interactions that affect the behavior of predators and pollinators in the ecosystem. Torenia would accordingly be of great use for investigating both the variation in ornamental plants and the infochemical-mediated interactions with arthropods.

Transformation of Cucumber Flowering Integrator (<i>CFL</i> Gene) into <i>Cyclamen persicum</i> Mediated by <i>Agrobacterium</i>

By employing cyclamen young leaves, petioles and callus from Cyclamen persicum as explants, transformation of cucumber flowering integrator ( CFL gene) into the genome of Cyclamen persicum mediated by Agrobacterium tumefaciens was conducted to compare the transformation efficiencies of different explants and to optimize the sonication-assisted transformation procedure mediated by Agrobacterium . The results demonstrated that the CFL gene was integrated into the genome of regenerated Cyclamen plants, which were validated by PCR and RT-PCR and by GUS histochemical assay. The results also showed that young leaves were the best receptor for transformation among employed explants, which the GUS-positive rates of regenerated plants can reach 27.25%. Furthermore, GUS positive rate can be increased four times by using the sonication-assisted transformation procedure. In this study, we successfully introduced the CFL gene into the genome of Cyclamen persicum by using modified high-efficiency genetic transformation system, which might be an alternative way for developing early flowering Cyclamen variety.

Optimization of electroporation conditions for Arthrobacter with plasmid PART2

A prerequisite for genetic studies of Arthrobacter is a high efficiency transformation system that allows for DNA transfer, transposon mutagenesis, and expression of specific genes. In this study, we develop a detailed electroporation method through a systematic examination of the factors involved in the entire electroporation process. Key features of this procedure, including the addition of penicillin to cells during the early log phase of growth and the presence of 0.5 M sorbitol in the electroporation and recovery media, produced the greatest increases in transformation efficiency and consistency of results. The transformation rate also varied depending on the electrical parameters, DNA concentration, and recovery time period. Using optimum conditions, we generally achieved an efficiency of 6.8 × 10 7 transformants per microgram of PART2 for Arthrobacter sp. A3. This protocol was also successfully applied to other Arthrobacter species. Therefore, we conclude that the proposed method is rapid, simple and convenient, which allows a transformation trial to be accomplished in minutes.

Establishment of high efficiency genetic transformation system of maize mediated by <I>Agrobacterium tumefaciens</I>

In order to establish high-frequency regeneration and high-efficiency genetic transformation system in maize, the significance of the 11 factors influencing maize embryonic callus induction and 9 factors affecting embryonic callus differentiation was researched by orthogonal experiment. The results showed that genotype had highly significant impact on induction of embryonic callus. The concentration of 6-BA, AgNO3, 2,4-D, ABA, and medium are the significant factors. The Multi-comparison showed that ABA 2 mg/L has a significant influence. Among the callus differentiation factors, the genotype and 6-BA concentration showed a strong main effect, the concentrations of NAA, medium, KT and 2,4-D had significant impacts on callus differentiation. Southern blotting analysis demonstrated that the resistant callus rate under the selection pressure of 25 mg/L hygromycin was a reliable indicator for system optimization in resistance screening. The concentration of acetosyringone (AS) showed sensitive differences among genotypes. The highest transformation rate was found with the optimized combination of 24-25 degrees C for co-culture temperature, 0.7 ODx15 min for Agrobacterium tumefa-ciens concentration and incubation-time, and pH 5.5-6.2. By this optimized combination, the survival rate of resistant calli as an index for the stable transformation rates of inbred lines Huangzao 4 and Zong 31 by introducing GUS gene into maize inbred lines was as high as 48.6% and 46.2%, respectively.

Research note: High efficiency transformation of the diatom Phaeodactylum tricornutum with a promoter from the diatom Cylindrotheca fusiformis

SUMMARYA high efficiency transformation system was established for the pennate diatom Phaeodactylum tricornutum Bohlin using a plasmid containing fucoxanthin chlorophyll a/c binding protein (fcp) promoter/terminator and nitrate reductase (NR) promoter/terminator that are derived from the pennate diatom Cylindrotheca fusiformis. The plasmid that contains the zeocin resistance gene (ble) with the fcp promoter and enhanced green fluorescent protein gene (egfp) with the NR promoter was introduced into P. tricornutum using microparticle bombardment. Transformants (650 ± 58 per 108 cells) were obtained. The yield of transformants was between 1.5 and 130 times higher than previously reported P. tricornutum transformation systems. Four to seven copies of the ble gene were integrated into genomic DNA of the transformants. This high efficiency transformation system of P. tricornutum is expected to provide a powerful tool for high‐throughput analysis of gene function using homologous recombination or RNAi.

High efficiency electrotransformation of Lactococcus lactis spp. lactis cells pretreated with lithium acetate and dithiothreitol.

BackgroundA goal for the food industry has always been to improve strains of Lactococcus lactis and stabilize beneficial traits. Genetic engineering is used extensively for manipulating this lactic acid bacterium, while electropolation is the most widely used technique for introducing foreign DNA into cells. The efficiency of electrotransformation depends on the level of electropermealization and pretreatment with chemicals which alter cell wall permeability, resulting in improved transformation efficiencies is rather common practice in bacteria as in yeasts and fungi. In the present study, treatment with lithium acetate (LiAc) and dithiothreitol (DTT) in various combinations was applied to L. lactis spp. lactis cells of the early-log phase prior to electroporation with plasmid pTRKH3 (a 7.8 kb shuttle vector, suitable for cloning into L. lactis). Two strains of L. lactis spp. lactis were used, L. lactis spp. lactis LM0230 and ATCC 11454. To the best of our knowledge these agents have never been used before with L. lactis or other bacteria.ResultsElectrotransformation efficiencies of up to 105 transformants per μg DNA have been reported in the literature for L. lactis spp.lactis LM0230. We report here that treatment with LiAc and DDT before electroporation increased transformation efficiency to 225 ± 52.5 × 107 transformants per μg DNA, while with untreated cells or treated with LiAc alone transformation efficiency approximated 1.2 ± 0.5 × 105 transformants per μg DNA. Results of the same trend were obtained with L. lactis ATCC 11454, although transformation efficiency of this strain was significantly lower. No difference was found in the survival rate of pretreated cells after electroporation. Transformation efficiency was found to vary directly with cell density and that of 1010 cells/ml resulted in the highest efficiencies. Following electrotransformation of pretreated cells with LiAc and DDT, pTRKH3 stability was examined. Both host-vector systems proved to be reproducible and highly efficient.ConclusionThis investigation sought to improve still further transformation efficiencies and to provide a reliable high efficiency transformation system for L. lactis spp. lactis. The applied methodology, tested in two well-known strains, allows the production of large numbers of transformants and the construction of large recombinant libraries.

From genomes to function: haloarchaea as model organisms

Haloarchaea are adapted to high-salt environments and accumulate equally high salt concentrations in the cytoplasm. The genomes of representatives of six haloarchaeal genera have been fully or partially sequenced, allowing the analysis of haloarchaeal properties in silico. Transcriptome and proteome analyses have been established for Halobacterium salinarum and Haloferax volcanii. Genetic systems are available including methods that allow the fast in-frame deletion or modification of chromosomal genes. The high-efficiency transformation system of Hf. volcanii allows the isolation of genes essential for a biological process by complementation of loss-of-function mutants. For the analysis of haloarchaeal biology many molecular genetic, biochemical, structural and cell biological methods have been adapted to application at high salt concentrations. Recently it has become clear that several different mechanisms allow the adaptation of proteins to the high salt concentration of the cytoplasm. Taken together, the wealth of techniques available make haloarchaea excellent archaeal model species.

Identification of an ARS element and development of a high efficiency transformation system for Pichia guilliermondii.

An Autonomously Replicating Sequence element adjacent to the RIB1 gene encoding GTP cyclohydrolase II of the yeast Pichia guilliermondii was identified by transformation experiments. Detailed sequence analysis unveiled two potential ARS elements located 5' and 3' of the RIB1 open reading frame. The chromosomal fragment containing the ARS-like sequence 3' to the RIB1 structural gene, called PgARS, conferred high transformation frequencies of 10(4)-10(5) transformants/microg of DNA to a pUC19-derived plasmid in P. guilliermondii. The PgARS element also conferred autonomous replication to hybrid plasmids in this host. Based on this element a series of Escherichia coli shuttle vectors for efficient transformation of the flavinogenic yeast P. guilliermondii was developed.

High-efficiency transformation of Pichia stipitis based on its URA3 gene and a homologous autonomous replication sequence, ARS2

This paper describes the first high-efficiency transformation system for the xylose-fermenting yeast Pichia stipitis. The system includes integrating and autonomously replicating plasmids based on the gene for orotidine-5'-phosphate decarboxylase (URA3) and an autonomous replicating sequence (ARS) element (ARS2) isolated from P. stipitis CBS 6054. Ura- auxotrophs were obtained by selecting for resistance to 5-fluoroorotic acid and were identified as ura3 mutants by transformation with P. stipitis URA3. P. stipitis URA3 was cloned by its homology to Saccharomyces cerevisiae URA3, with which it is 69% identical in the coding region. P. stipitis ARS elements were cloned functionally through plasmid rescue. These sequences confer autonomous replication when cloned into vectors bearing the P. stipitis URA3 gene. P. stipitis ARS2 has features similar to those of the consensus ARS of S. cerevisiae and other ARS elements. Circular plasmids bearing the P. stipitis URA3 gene with various amounts of flanking sequences produced 600 to 8,600 Ura+ transformants per micrograms of DNA by electroporation. Most transformants obtained with circular vectors arose without integration of vector sequences. One vector yielded 5,200 to 12,500 Ura+ transformants per micrograms of DNA after it was linearized at various restriction enzyme sites within the P. stipitis URA3 insert. Transformants arising from linearized vectors produced stable integrants, and integration events were site specific for the genomic ura3 in 20% of the transformants examined. Plasmids bearing the P. stipitis URA3 gene and ARS2 element produced more than 30,000 transformants per micrograms of plasmid DNA. Autonomously replicating plasmids were stable for at least 50 generations in selection medium and were present at an average of 10 copies per nucleus.

Enhanced stable expression of aVibrio luciferase under the control of the ?-translational enhancer in transgenic plants

A fusion gene usingluxA andluxB genes ofVibrio species has been designed to express light autonomously in plants.LuxA:luxB was introduced into plants by a high-efficiency transformation system consisting of a high-copy virulence helper plasmid pUCD2614 and T-vector pUCD2715 containingluxA:luxB. The expression ofluxA:luxB fusion gene was optimized by adjusting the spacing between the genes and by placing the translational efficiency of its mRNA under the control of the Ω-3 translational enhancer. The resulting transgenic plants synthesized luciferase at levels greater than 1% of the total leaf protein. These plants produced light autonomously and light intensity was enhanced by the addition of aldehyde. That theluxA:luxB fusion has been optimized enables its use as a reporter for gene activity in plants during development and under various stress-inducing conditions. These results show that a specific protein from an introduced foreign gene can be produced with high efficiency in cultivated plants and such a system is therefore amenable for production of desired proteins through conventional farming methods.

A high efficiency transformation system for the basidiomycete Ustilago violacea employing hygromycin resistance and lithium-acetate treatment

A basidiomycete phytopathogenic fungus, Ustilago violacea, was transformed with pUCH1, a bacterial plasmid containing the hygromycin (Hyg)-resistance (R) hygB gene fused to a promoter from the ascomycete Cochliobolus heterostrophus. After lithium acetate/polyethylene glycol treatment of whole sporidial cells, U. violacea transformants appeared on Hyg-agar at a frequency of 60–80 per μg pUCH1 DNA. The HygR phenotype was 100% stable in these transformants for at least 30 generations of mitotic growth under non-selective conditions. Southern DNA-DNA hybridization revealed multiple integrations of the pUCHI plasmid into the U. violacea nuclear DNA. In addition, Escherichia coli transformants appeared at a frequency of 12 per Jiμg nuclear fraction DNA from HygR U. violacea transformants; these E. coli consistently contained a deleted pUCHI plasmid. This latter result suggested the low-frequency production of circular molecules by recombination within the integrated sequences.