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PubMed2025

Systematic Optimization Enables Near-Perfect In Vitro Transformation Efficiencies for Spirodela polyrhiza (Greater Duckweed).

The in vitro transformation of plants, or the delivery of foreign genetic material that is incorporated into their genomes, represents a powerful tool both for elucidating genotype-phenotype relationships and for generating plant cultivars which have desirable traits for agriculture and/or biotechnological applications. However, outside of a few model species, the processes involved in transformation are often inefficient and can take months to perform for many plant species, with several bottlenecks occurring at the different stages of calli induction, genetic transfection, and plant regeneration. While duckweeds - aquatic monocots whose species include some of the smallest and fastest-growing flowering plants on the planet - have distinguished themselves with several emerging biotechnological applications, they too are the subject of conflicting reports regarding their transformation potential and ability to be genetically manipulated. Here, we synthesized and optimized the protocols for in vitro transformation of duckweed Spirodela polyrhiza (Greater Duckweed) from start-to-finish: achieving >90% - 100% efficiencies for each of calli induction; transient and stable genetic transformation; visual marker-free selection of transformants; and regeneration of genetically modified plants with stable transgene expression for over 100 generations - and which in S. polyrhiza can be achieved over the course of weeks instead of months. The integrated, streamlined approaches for all stages of in vitro transformation overcome many bottlenecks and can help to pave the way for high-throughput functional genomics studies and synthetic biology applications in this biotechnologically-important species.

CRISPR/Cas9↗
PubMed1991

Genetic transformation of Arabidopsis thaliana zygotic embryos and identification of critical parameters influencing transformation efficiency.

An efficient procedure for Agrobacterium-mediated transformation of zygotic embryos derived from three different Arabidopsis thaliana ecotypes has been developed. This procedure yielded an average transformation rate of 76% for ecotype C24, and 15-20% for ecotypes Landsberg-erecta and Columbia. A critical step for optimal transformation was the preculture of embryos on a phytohormone-containing medium. Light and electron microscopical studies showed that, during preculture, procambium cells of embryos became highly susceptible to Agrobacterium infection. Transformed cells developed calli and regenerated shoots within 4-5 weeks of culture. A total of 1500 fertile transgenic plants were regenerated. In regenerated plants the presence of inserted DNA was verified by genomic Southern blot analysis, assays of enzymatic activities of reporter genes (neomycin phosphotransferase II and beta-glucuronidase) as well as by genetic segregation tests. R1 progenies of 45 randomly chosen transformed lines and 150 independent regenerants did not show any somaclonal variations as ascertained by both morphological and cytological criteria. Short duration (7-8 weeks), high efficiency, reproducibility and low frequency of somaclonal variation makes the zygotic embryo transformation particularly well-suited for T-DNA tagging mutagenesis.

Cells, Cultured↗
PubMedDate not supplied

Efficient transformation of mammalian cells using DNA interpolyelectrolyte complexes with carbon chain polycations.

A new method for mammalian cell transformation is proposed which is based on incorporation of plasmids into interpolyelectrolyte complexes (IPECs) with carbon chain polycations. The method is illustrated by examples of pRSV CAT and p beta-Gal plasmid IPECs with poly(N-ethyl-4-vinylpyridinium bromide) (C2PVP) and poly(N-ethyl-4-vinylpyridinium)-poly(N-cetyl-4-vinylpyridinium+ ++) bromides random copolymer (C16PVP). These IPECs are produced spontaneously due to formation of a cooperative system of interchain electrostatic bonds after mixing DNA and polycation solutions. The interaction of IPEC with normal mouse fibroblasts NIH 3T3, human T-lymphoma "Jurkat", and Mardin Darby canine kidney cells has been studied. The data obtained has revealed that plasmid incorporation into IPECs significantly enhances both DNA adsorption on the plasma membrane and DNA uptake into a cell. The in vitro transformation of NIH 3T3 cells was monitored by a standard cloramphenicol acetyltransferase (CAT) assay (pRSV CAT plasmid) and by detection of beta-galactosidase (beta-Gal) expression using 4-methylumbeliferril beta-D-galactopyranoside as a substrate (p beta-Gal plasmid). In both cases it has been proved that IPEC-incorporated plasmids possess an ability for efficient cell transformation. The transforming activity of IPECs depends on their composition and polycation chemical structure. Under optimal conditions the efficiency of cell transformation with IPECs is several fold higher than that observed during standard calcium phosphate precipitation. The mechanism of the phenomenon observed is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗
PubMed1968

Fractionated strands of bacterial deoxyribonucleic acid. 3. Transformation efficiencies and rates of phenotypic expression.

Preferential binding of guanine-rich ribopolymers to one of the complementary strands of denatured deoxyribonucleic acid (DNA) of Diplococcus pneumoniae permitted the fractionation of the complements in CsCl density gradients. Phenotypic expression of the newly acquired genes for four drug resistances was more rapid in cells transformed by the heavy fractions than in those transformed either by light fractions or by unfractionated DNA. The efficiencies of transformation with the two complements were nearly equal for the four markers tested. Both efficiency and expression results were the same whether we assayed the residual activity or the activity obtained by annealing the fractions with excess recipient DNA.

DNA, Bacterial↗
PubMed1996

High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens.

Transformants of maize inbred A188 were efficiently produced from immature embryos cocultivated with Agrobacterium tumefaciens that carried "super-binary" vectors. Frequencies of transformation (independent transgenic plants/embryos) were between 5% and 30%. Almost all transformants were normal in morphology, and more than 70% were fertile. Stable integration, expression, and inheritance of the transgenes were confirmed by molecular and genetic analysis. Between one and three copies of the transgenes were integrated with little rearrangement, and the boundaries of T-DNA were similar to those in transgenic dicotyledons and rice. F1 hybrids between A188 and five other inbreds were transformed at low frequencies.

Agrobacterium tumefaciens↗
PubMed2004

Efficient transformation of Cellulomonas flavigena by electroporation and conjugation with Bacillus thuringiensis.

The conjugative self-transmissible plasmid pHT73, harbored in Bacillus thuringiensis var. kurstaki, was demonstrated to be transferred to Cellulomonas flavigena, a cellulolytic bacterium. Both conjugation and transformation procedures yielded resistant colonies; however, chromosomal integration was observed only when bacterial conjugation occurred. The efficiency of conjugation was 10% of recipient strain, which is considered a very efficient process. When the plasmid pHT73 was introduced by transformation, erythromycin-resistant cells contained the plasmid as an episome with no arrangements, as assayed by Southern blot analysis. In contrast, conjugated-resistant cells harbor the plasmid integrated into the chromosome. These data suggest a common mechanism of cell communication between nonrelated bacterial species with similar ecological habitats, and also that both electroporation and conjugation can be used to transform C. flavigena efficiently.

Bacillus thuringiensis↗
PubMed1992

High efficiency transformation of Fusarium solani f. sp. cucurbitae race 2 (mating population V).

A cosmid vector, suitable for library construction and DNA transformation in filamentous fungi, has been constructed and a reliable and highly efficient PEG-mediated DNA transformation system for F. solani f. sp. cucurbitae, based on resistance to hygromycin B, has been developed for use with this vector. This transformation system yielded 10(4) transformants per micrograms of DNA when using 10(7) protoplasts. Factors important in achieving high efficiency included: the maintenance of an osmoticum in all transformation steps, PEG 4000 concentration, and the ratio of transforming vector DNA to protoplasts. Approximately 60% of transformants stably integrated vector DNA. Molecular analysis revealed multiple copies of the plasmid integrated into the genome at one or more sites. The frequency of transformation achieved will facilitate the isolation of genes from this fungus by complementation.

Base Sequence↗
PubMed1994

Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.

A large number of morphologically normal, fertile, transgenic rice plants were obtained by co-cultivation of rice tissues with Agrobacterium tumefaciens. The efficiency of transformation was similar to that obtained by the methods used routinely for transformation of dicotyledons with the bacterium. Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the R0, R1 and R2 generations. Sequence analysis revealed that the boundaries of the T-DNA in transgenic rice plants were essentially identical to those in transgenic dicotyledons. Calli induced from scutella were very good starting materials. A strain of A. tumefaciens that carried a so-called 'super-binary' vector gave especially high frequencies of transformation of various cultivars of japonica rice that included Koshihikari, which normally shows poor responses in tissue culture.

Agrobacterium tumefaciens↗
PubMed2005

Efficient transformation of Legionella pneumophila by high-voltage electroporation.

A simple and reproducible method has been developed to transform Legionella pneumophila by electroporation. Effects of different conditions, including electric field strength, pulse length, DNA quality and cell density, were evaluated. Using our method, an efficiency of up to 6 x 10(7) transformants/microg DNA was obtained. This optimized transformation procedure should efficiently facilitate gene manipulations in L. pneumophila, such as plasmid transfer, transposon mutagenesis, library transformation for complementation cloning, etc.

Edetic Acid↗
PubMed1985

[Genetic transformation of somatic cells. I. Clone of Chinese hamster cells defective in thymidine kinase and characterized by high transformation efficiency].

A characteristics is given of clone A238 of the Chinese hamster cells deficient in thymidine kinase (TK). The isolation procedure is described. Upon transformation with the aid of DNA of plasmids, containing thymidine kinase gene (tk-gene) of Herpes simplex virus type 1 (HSV1) clone A238 cells show frequency (7.10(-5) and efficiency (130 TK+ colonies per 1 microgram of plasmid DNA) compatible with those of mouse line LMtk- cells. Modified transformation and selection conditions of clone A238 cells expressing TK-gene of HSV1 are demonstrated. A simple method is described for discriminating somatic cells, expressing either their proper or a virus TK-gene according to the cloning efficiency of cells on the HAT medium containing thymidine in concentration 100 micrograms/ml. It is shown that at the fixed total DNA concentrations a complete replacement of the eukaryotic carrier DNA for the plasmid DNA, containing no tg gene of HSV1, decreases but only insignificantly the frequency and efficiency of transformation.

Animals↗
PubMed1992

Efficient transformation of Claviceps purpurea using pyrimidine auxotrophic mutants: cloning of the OMP decarboxylase gene.

A homologous transformation system was developed for the phytopathogenic fungus Claviceps purpurea. Orotidine-5'-monophosphate decarboxylase (OMPD)-deficient mutants were obtained by UV mutagenesis and selection for resistance against 5-fluoroorotate. These mutants could be complemented well by the corresponding genes of Aspergillus niger (pyrA) and Neurospora crassa (pyr4), yielding significantly higher transformation rates (and lower copy numbers per transformant) than the phleomycin resistance system. The homologous OMPD gene was isolated from a lambda genomic library by heterologous hybridization with the pyr4 gene of N. crassa, identified by complementation of Aspergillus and Claviceps mutants, and used to confirm homologous integration in Claviceps. The pyr transformation system also proved to be very efficient in cotransformation experiments using the bacterial beta-glucuronidase gene (uidA) as a reporter gene, which was also efficiently expressed during the parasitic cycle: honeydew produced by plants infected with pyr/uidA cotransformants was shown to contain significant levels of beta-glucuronidase activity.

Blotting, Southern↗
PubMed1993

Low temperature protocol for efficient transformation of Mycobacterium smegmatis spheroplasts.

Spheroplasts of Mycobacterium smegmatis LM15, strain 607, were prepared by a combined treatment with glycine and lysozyme. The spheroplasts were tested for ability to take up and express purified mycobacteriophage DNA. Exposure of 1.0 x 10(8) to 1.0 x 10(9) spheroplasts to saturating DNA (1 microgram) for 15 min at 5 degrees C resulted in a transfection efficiency of approximately 0.009% . The transfer of the beta-lactamase marker with DNA purified from strain LM15 to spheroplasts of a beta-lactamase-negative mutant, strain LM144, was achieved. The DNA-treated cultures, after reversion to the bacillary form, contained 20-fold more penicillin-resistant cells than the nontreated control culture. Approximately 80% of the penicillin-resistant colonies from the DNA-treated cells were positive for beta-lactamase Cell-free extracts of penicillin-resistant transformants contained beta-lactamase activity that ranged from 0.046 to 0.134 micromol of benzylpenicillin hydrolyzed/min per mg protein. This low temperature procedure is recommended for high efficiency transformation of M. smegmatis.

Mycobacteriophages↗
PubMed2001

Efficient transformation of the yellow fever mosquito Aedes aegypti using the piggyBac transposable element vector pBac[3xP3-EGFP afm].

We report efficient germ-line transformation in the yellow fever mosquito Aedes aegypti accomplished using the piggyBac transposable element vector pBac[3xP3-EGFP afm]. Two transgenic lines were established and characterized; each contained the Vg-Defensin A transgene with strong eye-specific expression of the enhanced green fluorescent protein (EGFP) marker gene regulated by the artificial 3xP3 promoter. Southern blot hybridization and inverse PCR analyses of genomic DNA demonstrated a precise piggyBac-mediated, single copy insertion of the pBac[3xP3-EGFP afm,Vg-DefA] transposon in each transgenic line. For each line, genetic analysis confirmed stability and integrity of the entire transposon construct in the mosquito genome through the G2-G6 generations. Successful establishment of homozygous transgenic lines indicated that in both cases a non-lethal integration of the transposon into the mosquito genome had occurred. The 3xP3-EGFP marker was tested in mosquitoes with different genetic backgrounds. In white-eyed transgenic mosquitoes, the strong eye-specific expression of GFP was observed throughout all stages of development, starting from newly hatched first instar larvae to adults. A similar level and pattern of fluorescence was observed in red-eyed mosquitoes that were generated by crossing the 3xP3-EGFP transformants with the kh(w) white-eye mosquitoes transformed with the Drosophila cinnabar gene. Importantly, the utility of the 3xP3-EGFP, as marker gene for transformation of wild type mosquitoes, was demonstrated by strong eye-specific GFP expression in larval and pupal stages of black-eyed hybrids of the 3xP3-EGFP white-eye transformants and the wild type Rockefeller/UGAL strain. Finally, analysis of the Vg-DefA transgene expression in transformants from two established lines demonstrated strong blood-meal activation and fat-body-specific expression regulated by the Vg 1.8-kb 5' upstream region.

Aedes↗
PubMed1989

Heat shock at an elevated temperature improves transformation efficiency of protoplasts from Podospora anserina.

We have developed an improved transformation procedure for the filamentous fungus Podospora anserina. This procedure is based on the observation that a heat shock at an elevated temperature (48 degrees C) improves the competence of P. anserina protoplasts for transformation 5- to 10-fold. This is observable only if the heat shock is applied before the addition of transforming DNA. An increase in competence is observed immediately after the heat shock, and heat-shocked cells are still competent after 20-30 min. The mechanism by which heat shock improves competence remains unclear. The modified transformation procedure gives as many as 200-500 stable transformants per microgram of plasmid DNA containing the P. anserina ura5 gene. This should allow direct cloning of P. anserina genes from a cosmid library.

Ascomycota↗
PubMed1997

High efficiency transformation of Bacillus brevis by electroporation.

Various conditions in the Bacillus brevis transformation by electroporation were investigated to increase the efficiency. Competent cell volume and presence of polyethylene glycol 6000 and single stranded DNA on pulsing were critical for the efficiency. As a result, we established the improved method by which 10(7)-10(8) transformants/microgram plasmid DNA could be obtained.

Bacillus↗
PubMed1990

Optimum conditions for efficient transformation of Streptomyces venezuelae protoplasts.

Optimum conditions for protoplast regeneration and transformation of Streptomyces venezuelae ETH14630 have been established. Protoplasts from mycelium grown to the stationary phase and treated with lysozyme in P medium under mild conditions gave the best regeneration frequency. Transformation of protoplasts with naked DNA was very efficient using either polyethylene glycol of mol. wt. 4000 or 6000, at concentrations of 28.5% or 36% (w/v) respectively. About 10(7) transformants/micrograms DNA could be isolated using protoplasts derived from cells cultivated to the early exponential growth phase in LB medium containing 0.2%-0.6% glycine and subsequently treated at 30 degrees -32 degrees C with 20 mg lysozyme/ml in P medium for 30 min. Selection of the transformants occurred on MRYE plates containing less than 10(5) regenerating protoplasts per plate. Higher protoplasts densities considerably decreased the regeneration frequency of the transformants.

Biotechnology↗
PubMed1995

A stable and efficient transformation system for Butyrivibrio fibrisolvens OB156.

A 9.5-kb shuttle vector capable of replication and selection in both Escherichia coli and Butyrivibrio fibrisolvens was constructed. Plasmid pUC118 provided replication functions and ampicillin resistance selection in E. coli. In B. fibrisolvens, replication was controlled by the native plasmid pRJF1 from strain OB156, and selectability was provided by a 3.5-kb fragment of plasmid pAM beta 1 containing the erythromycin resistance gene. Optimum conditions for transformation were 15 kV/cm, 2 h recovery, and plating in an agar overlay on medium containing 10 micrograms erythromycin/ml. Maximum efficiency was 1.1 x 10(5) transformants per micrograms plasmid DNA (average 3 x 10(4)), and restriction mechanisms reduced efficiency by a factor of 2 x 10(2). Nonselective growth for 200 generations gave no measurable loss of plasmid.

Escherichia coli↗