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PubMed2006

Genetic transformation of peanut (Arachis hypogaea L.) using cotyledonary node as explant and a promoterless gus::nptII fusion gene based vector.

We have generated putative promoter tagged transgenic lines in Arachis hypogaea cv JL-24 using cotyledonary node (CN) as an explant and a promoterless gus::nptII bifunctional fusion gene mediated by Agrobacterium transformation. MS medium fortified with 6-benzylaminopurine (BAP) at 4mg/l in combination with 0.1 mg/l alpha -napthaleneacetic acid (NAA) was the most effective out of the various BAP and NAA combinations tested in multiple shoot bud formation. Parameters enhancing genetic transformation viz. seedling age, Agrobacterium genetic background and co-cultivation periods were studied by using the binary vector p35SGUSINT. Genetic transformation with CN explants from 6-day-old seedlings co-cultivated with Agrobacterium GV2260 strain for 3 days resulted in high kanamycin resistant shoot induction percentage (45%); approximately 31% transformation frequency was achieved with p35S GUSINT in beta-glucuronidase (GUS) assays. Among the in vivo GUS fusions studied with promoterless gus::nptII construct, GUS-positive sectors occupied 38% of the total transient GUS percentage. We have generated over 141 putative T 0 plants by using the promoterless construct and transferred them to the field. Among these, 82 plants survived well in the green house and 5 plants corresponding to 3.54% showed stable integration of the fusion gene as evidenced by GUS, polymerase chain reaction (PCR) and Southern blot analyses. Twenty-four plants were positive for GUS showing either tissue-specific expression or blue spots in at least one plant part. The progeny of 15 T 0 plants indicated Mendelian inheritance pattern of segregation for single-copy integration. The tissue-specific GUS expression patterns were more or less similar in both T 0 and corresponding T 1 progeny plants. We present the differential patterns of GUS expression identified in the putative promoter-tagged transgenic lines in the present communication.

Arachis↗

PubMed1976

An approach to genetic transformation in the Xiphophorine fish.

The particular suitability of the Xiphophorine fish system for achieving genetic transformation is presented, and it was analyzed whether information carrying donor DNA might be available to the propigment cells of embryos of Xiphosphorus helleri, which are the target cells for the transformation. Heterologous 2H3H-labelled donor DNA from E. coli, which was taken for technical reasons instead of homologous fish DNA, undergoes degradation both after injection into the neural crest region and after injection into the yolk sac (molecular weight at O h: 50 X10(6); at 2 h: 1 X 10(6); at 5 h: less than 3 X 10(5); at 10 h: less than 1 X 10(4)). It is concluded therefore, that informative donor DNA is present for about 2 to 3 hours after injection. The DNA of the recipient embryo is labelled radioactively during that time at which informative DNA is present only, if the donor DNA is injected into the neural crest region. The probability that a foreign gene might become available to the propigment cells and might induce transformation is discussed.

Animals↗

PubMed1977

[Effect of calcium and magnesium ions on different stages of genetic transformation in Bacillus subtilis].

Optimal concentrations of magnesium and calcium ions under their combined effect on genetic transformation in Bacillus suttilis are 2 - 10(-2) M and 4 - 10(-2) M respectively. The same concentrations are optimal under the effect of each cation clone. Magnesium ions are efficient during irreversible DNA binding. In the presence of magnesium ions calcium ions stimulate more late stages of transformation. The greatest efficiency of transformation is shown in consecutive effect of magnesium ions at early stages of transformation and of calcium ions at late transformation stages. This suggests that magnesium and calcium ions stimulate the activity of nuclease, taking part at early and late transformation stages.

Bacillus subtilis↗

PubMed1983

The efficiency of genetic transformation of mammalian cells by transfection and microinjection depends on the transferred gene.

The efficiency of genetic transformation of mammalian cells was analysed with respect to the kind of the transferred gene and the selective system. Plasmids pAGO and pAG60 harboring the thymidine kinase gene of Herpes simplex virus type 1 and the bacterial neomycin resistance gene, respectively, were compared concerning their ability to transform mouse Ltk-aprt- cells. Using the calcium phosphate technique the neomycin resistance gene transformed at least ten times more efficiently than the thymidine kinase gene (3 X 10(-3) versus 2 X 10(-4] whereas the difference is even more impressive following microinjection of the plasmids into the nuclei (2 X 10(-1) versus 2.5 X 10(-3]. The neomycin system also proved to be more effective in secondary gene transfer experiments and, thus, seems to be the most convenient marker for cotransfer experiments.

Animals↗

PubMed2005

Reproducible genetic transformation system for two dermatophytes, Microsporum canis and Trichophyton mentagrophytes.

A reproducible genetic transformation system was developed for two major dermatophytes, Microsporum canis and Trichophyton mentagrophytes. Two circular transformation vectors carrying either the bacterial hygromycin B phosphotransferase gene (hph) or both the hph and green fluorescent protein (eGFP) genes under the control of a promoter sequence from Cochlibolus heterostrophus were introduced independently into the protoplasts by a polyethylene glycol (PEG)-mediated method. Polymerase chain reaction (PCR) showed that the hph gene was integrated randomly into the chromosomal DNA of the transformants through non-homologous recombination. Southern blotting analysis also demonstrated a single or multiple integration of the hph gene into the chromosomal DNA. Fluorescence due to eGFP gene expression was observed in the T. mentagrophytes transformants, and the transformants retained mitotic stability through subculture. This reproducible transformation system provides a method for the genetic manipulation of these pathogens, which will facilitate detailed molecular analysis of dermatophytosis.

Blotting, Southern↗

PubMed1995

Genetic transformation of the pathogenic fungus Wangiella dermatitidis.

Genetic transformation of Wangiella dermatitidis was studied using three plasmid vectors (pAN7-1, pWU44, and pKK5) and both electroporation and polyethyleneglycol-mediated methods. pAN7-1 contains the E. coli hygromycin B (HmB) phosphotransferase (hph) gene. Expression of the hph gene confers resistance to antibiotic HmB. Selection for resistance, indicative of transformation, resulted in 10 203 HmB-resistant colonies/micrograms pAN7-1 on medium containing 100 micrograms HmB/ml. Strains of W. dermatitidis used in this study have innate sensitivity to HmB at a critical inhibitory concentration of 20-40 micrograms/ml. Vectors pWU44 and pKK5 contain a URA5 gene from Podospora anserina. A ura5 auxotroph of W. dermatitidis was transformed to prototrophy with pWU44 or pKK5 by complementation. Transformation frequencies for these two plasmids were between 17-50 transformants/micrograms vector DNA. Southern blotting analysis and polymerase chain reaction detection of DNA from putative transformants confirmed transformation.

Base Sequence↗

PubMed1979

Genetic transformation in Streptococcus sanguis. Distribution of competence and competence factors in a collection of strains.

Forty-one strains of Streptococcus sanguis (37 of serogroup H and four of the newly-described serogroup W) were examined semiquantitatively for genetic transformation with streptomycin as marker. The material comprised eight reference laboratory strains and 33 recent isolates. Eighteen strains (16 of serogroup H and two of W) showed spontaneous competence in genetic transformation (without added competence factor, i.e. culture filtrate.). Individual culture filtrates from 19 spontaneously competent and ten incompetent strains were tested for competence-inducing effect on 23 spontaneously incompetent strains. Competence was induced in 16 of the strains, and 20 of the culture filtrates were active. There was considerable variation with respect to the number of recipient strains which were induced to competence by individual filtrates. Furthermore the recipients varied as regards the number of filtrates that were able to induce that particular strain. There was some relationship, but no complete association, between competence, competence-inducing ability and the occurrence of spreading zones around the colonies assumed to correspond generally to fimbriation. Thus, three incompetent strains had an active culture filtrate and one spontaneously competent strain had an inactive filtrate. Most, but not all, strains with spontaneous or inducible competence showed spreading, as did most of the strains from which broadly inducing filtrates could be produced.

Bacteriological Techniques↗

PubMedDate not supplied

[Genetic transformation of sugar beet: evolution of theoretical and experimental approaches].

The review is dedicated to several aspects of sugar beet (Beta vulgaris L.) biotechnology: in vitro cultivation, callus induction, plant regeneration and genetic transformation. Media composition, methods of plant regeneration via somatic embryogenesis and protoplast culture are analysed. The use of Agrobacterium tumefaciens and gold particle bombardment is the base for modern genetic transformation methods.

Beta vulgaris↗

PubMed2004

Improved genetic transformation of the thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1.

We improved genetic transformation of the thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1, by combining electroporation with a top agar method. Transformation was also improved when a disruptant of a putative type I restriction endonuclease (tll2230) was used as recipient cells. In particular, some constructs, with which wild type has never been transformed, were successfully integrated into the tll2230-disruptant. Single-crossover recombination was detected more frequently than the double-crossover recombination. In accordance with the presence of all the homologs of pil genes in Synechocystis sp. PCC 6803, we found that T. elongatus is naturally transformable with exogenous DNA.

Agar↗

PubMed2005

Enhancements and justice: problems in determining the requirements of justice in a genetically transformed society.

There is a concern that genetic engineering will exacerbate existing social divisions and inequalities, especially if only the wealthy can afford genetic enhancements. Accordingly, many argue that justice requires the imposition of constraints on genetic engineering. However, it would be unwise to decide at this time what limits should be imposed in the future. Decision makers currently lack both the theoretical tools and the factual foundation for making sound judgments about the requirements of justice in a genetically transformed society. Moreover, focusing on the uncertain inequities of the future may result in failure to give priority to more pressing inequities of the present. Especially in a country that recently has enacted tax legislation that will widen existing wealth disparities, concern about the distant threat of a genetic aristocracy appears misplaced.

Cognition Disorders↗

PubMed2006

[Genetic transformation of peach immature cotyledons with its antisenes ACO gene].

Genetic transformation of peach immature cotyledons with its ACO antisense gene was studied by using particle bombardment method through Agrobacterium tumefaciens. Kanr shoots and Kanr plantlet were obtained. The plantet with ACO antisenes gene through Agrobacterium tumefaciens was obtained by micrografting technique and survived for nearly one month. The results of the PCR, PCR-southern, genomes southern hybridization analysis and GUS color reaction of some Kanr materials showed in some degree that the peach ACO antisens gene was integrated into peach genomes.

Agrobacterium tumefaciens↗

PubMed1988

Identification of Moraxella bovis by qualitative genetic transformation and nutritional assays.

Strains of Moraxella bovis were identified definitively through the combined use of a qualitative genetic transformation assay and determination of the ability of the organism under examination to grow in a defined medium (medium MB). Except for weak transformation by DNA from strains of M. lacunata, M. nonliquefaciens, and M. (Branhamella) ovis, DNA samples from all other members of the genus Moraxella failed to transform either of the two M. bovis auxotrophs used in this study.

Culture Media↗

PubMed1982

Diagnosis of gonorrhea using a genetic transformation test on mailed clinical specimens.

The genetic transformation test (GTT), a technique used for the detection of gonococcal DNA in clinical specimens, was compared with culture testing for the diagnosis of gonorrhea. At the De Kalb County Venereal Disease Clinic, Decatur, Ga., 454 cervical and 160 rectal specimens from women and 191 urethral specimens from men were collected in duplicate. One of each of the two specimens from each anatomic site was immediately plated on Martin-Lewis medium and incubated; the other specimen was mailed to Philadelphia for a GTT. Using culture results as a standard, the GTT had a specificity of 98.1% although some "false-positive" GTT results were probably a reflection of false-negative culture results. The sensitivity of the GTT was greater than 96% except when specimens were collected with Culturettes (Marion Laboratories, Kansas City, Mo.). In situations where Gram staining is not appropriate or where on-site culture facilities are not available, GTT may be the method of choice for the diagnosis of gonorrhea.

Biological Assay↗

PubMed1980

Identification of pathogenic neisseriae by genetic transformation.

The detection of pathogenic neisseriae by genetic transformation of a naturally occurring proline auxotroph of Neisseria gonorrhoeae strain F62 is described. Of 169 clinical isolates of N. gonorrhoeae, approximately 90% gave a positive transformation assay. Twelve clinical isolates of N. meningitidis and stock cultures of the various meningococcal serogroups also gave a positive result. However, the sensitivity of the assay was found to be approximately 1000-fold lower with N. meningitidis as test organism. Eleven other members of the family Neisseriaceae failed to transform the recipient organism. Although proline requirement did not appear to limit the value of the assay greatly, it probably was the main reason for negative results. The sensitivity of the assay and its ability to detect non-viable gonococci suggests that this method merits further investigation as a possible aid to diagnosis of gonococcal infection in special circumstances.

Bacteriological Techniques↗

PubMed1986

Genetic transformation of rifampicin resistance in Lactobacillus acidophilus.

Lactobacillus acidophilus strain 100-33, originally isolated from swine faeces, was transformed to rifampicin resistance with DNA from spontaneous rifampicin-resistant mutants derived from it. Cells of the recipient strain were treated with lysozyme and mutanolysin, mixed with donor DNA and polyethylene glycol and grown on a regeneration medium overnight. After 48 h incubation, the numbers of rifampicin-resistant cells in the populations of regenerated cells were estimated from numbers of colonies. Efficiency of the lysozyme/mutanolysin treatment (the ratio of the number of osmotically fragile cells after the enzyme treatment to the initial cell number) was about 99%. The regeneration frequency of the enzyme-treated cells varied from 5 to 67%. The transformation frequency varied from about 0.2 X 10(-8) to 8.0 X 10(-8) transformants per regenerated cell per microgram DNA. To our knowledge, this method for genetic transformation is the first to be reported for a Lactobacillus strain.

DNA, Bacterial↗

PubMed2026

Magnetic nanoparticle-mediated genetic transformation and gene editing system in loquat (Eriobotrya japonica).

Loquat (Eriobotrya japonica Lindl.) is a valuable subtropical fruit tree whose genetic improvement has been significantly constrained by the absence of an efficient genetic transformation system. Although Agrobacterium-mediated transformation is the most widely used method, it proves ineffective in loquat due to the species' recalcitrance to in vitro regeneration. Pollen-based transformation offers a promising alternative by bypassing the need for tissue culture. However, the pollen wall poses a major physical barrier to the uptake of exogenous DNA. In this study, we investigated magnetic nanoparticle (MNP)-mediated transformation as a novel strategy for loquat. We confirmed that loquat pollen contains tricolporate apertures with diameters ranging from 3.0 to 5.0 μm, which are structurally suitable for the entry of MNPs-DNA. Based on this finding, we developed and optimized a transformation protocol using polyethyleneimine-coated Fe3O4 nanoparticles to deliver genetic material into loquat pollen grains. Using this approach, we successfully generated stable transgenic loquat lines, including both overexpression and gene-edited mutants. To our knowledge, this is the first report of successful MNP-mediated pollen transformation in a woody plant species. This work establishes a robust and efficient genetic transformation platform for loquat, providing a valuable tool for functional genomics and molecular breeding, as well as a potentially applicable strategy for other recalcitrant woody plants.

Eriobotrya↗

PubMedDate not supplied

[Genetic transformation of Nicotiana africana Merxm. with plasmids containing lox recombination].

An efficient genetic transformation method for african tobacco Nicotiana africana Merxm. has been established. African tobacco is a valuable source for cytoplasmic male sterility (CMS) and nuclear encoded resistance to potato virus Y (PVY). N. africana transgenic plants have been obtained using both Agrobacterium-mediated and direct transformation of leaf explants with gold particle bombardment using particle inflow gun. Plasmid vectors containing phosphinothricin resistance gene (bar gene) coding region without promoter and independent 35S promoter between lox sites (lox-bar-35S-lox) and nptII gene were used. Transgenic plants were selected according to growth capacity on the selective medium containing 50 mg/l kanamycin. PCR analyses of kanamycin-resistant plants confirmed the presence of nptII and bar genes in their genome. Agrobacterium-mediated transformation of root explants has proved to be the most efficient transformation method for N. africana.

Agrobacterium tumefaciens↗

PubMed1996

Separation of abnormal cell wall composition from penicillin resistance through genetic transformation of Streptococcus pneumoniae.

Compared with most penicillin-susceptible isolates of Streptococcus pneumoniae, penicillin-resistant clinical isolate Hun 663 contains mosaic penicillin-binding protein (PBP) genes encoding PBPs with reduced penicillin affinities, anomalous molecular sizes, and also cell walls of unusual chemical composition. Chromosomal DNA prepared from Hun 663 was used to transform susceptible recipient cells to donor level penicillin resistance, and a resistant transformant was used next as the source of DNA in the construction of a second round of penicillin-resistant transformants. The greatly reduced penicillin affinity of the high-molecular-weight PBPs was retained in all transformants through both genetic crosses. On the other hand, PBP pattern and abnormal cell wall composition, both of which are stable, clone-specific properties of strain Hun 663, were changed: individual transformants showed a variety of new, abnormal PBP patterns. Furthermore, while the composition of cell walls resembled that of the DNA donor in the first-round transformants, it became virtually identical to that of susceptible pneumococci in the second-round transformants. The findings indicate that genetic elements encoding the low affinity of PBPs and the penicillin resistance of the bacteria are separable from determinants that are responsible for the abnormal cell wall composition that often accompanies penicillin resistance in clinical strains of pneumococci.

Amino Acid Sequence↗