scholarly journals Genetic transformation of bean callus via Agrobacterium- mediated DNA transfer

2003 ◽  
Vol 9 (3-4) ◽  
Author(s):  
E. Eissa Ahmed
Keyword(s):  
Southern Blotting ◽  
Southern Hybridization ◽  
Marker Gene ◽  
Transient Gene Expression ◽  
Callus Cultures ◽  
Hybridization Technique ◽  
Coding Region ◽  
Herbicide Resistant ◽  
Gusa Gene ◽  
Media Data

Callus cultures were induced from hypocotyl of young bean seedlings. Callus developed and maintenaned on B5 medium supplemented with 2mg/1 2,4-D and 1 mg/1 kinetin. The results demonstrate that A. tumefacins-mediated transformation is a convenient method to obtain transient gene expression in callus of bean. The results have shown that the bean callus co-cultivated with A. tumefaciens can be transformed to get heibicide Finale (glufosinate-ammonium) resistant GUS positive tissues. Southern blot analysis of transformed calli showed integration of gusA marker gene carried by a binary vector. Transformed calli were selected on herbicide containing media. Data of molecular analysis (Southern blotting) confirmed the insertion of gusA gene in the genome of herbicide resistant calli with bar gene. There are three evidences that calli are stable transformants: (1) herbicide resistance, (2) GUS activity which is indicative since the coding region containing an intron, (3) the results of Southern hybridization technique.

scholarly journals Production of transgenic bean callus via genetic transformation by DNA-coated tungsten particles

2003 ◽  
Vol 9 (3-4) ◽  
Author(s):  
E. Eissa Ahmed
Keyword(s):  
Gene Expression ◽  
Genetic Transformation ◽  
Transient Gene Expression ◽  
Callus Cultures ◽  
Ms Medium ◽  
Mannitol Dehydrogenase ◽  
Dehydrogenase Gene ◽  
Half Strength ◽  
Media Data ◽  
Callus Lines

Callus cultures were induced from hypocotyl of young bean seedlings. The B5 medium completed with 1 mg/1 KIN and 2mg/1 2,4-D proved the best. Callus developed and maintenaned on B5 medium supplemented with 1mg/1 kinetin and 2mg/I 2,4-D. The B5 medium supplemented with 1mg/1 KIN and 2mg/1 2,4-D induced much more callus than half strength MS medium supplemented with 0.5 or 0.75mg/1 BA and 0.1 mg/1 NAA. The results demonstrate that GeneboosterTM is convenient method to obtain transient gene expression in callus of bean. The results have shown that the bean callus shot by GeneboosterTM can be transformed to get (kanamycin-resistant and stress mannitol­tolerant) calli. The presence of mannitol-dehydrogenase gene (mt/) was verified by PCR, showing the integration of mt/ gene carried by two plasmids. Co-transformed calli were selected after bombardment on kanamycin, mannitol and (kanamycin+mannitop-containing media. Data of molecular analysis (PCR) confirmed the insertion of mtl gene in the genome of mannitol-tolerant callus lines.

scholarly journals High-velocity microprojectile mediated DNA delivery into Phaseolus vulgaris callus cells

2000 ◽  
Vol 6 (4) ◽  
Author(s):  
E. Eissa Ahmed ◽  
Gy. Bisztray ◽  
I. Velich
Keyword(s):  
Phaseolus Vulgaris ◽  
High Velocity ◽  
Callus Tissue ◽  
Marker Gene ◽  
Transient Gene Expression ◽  
Dna Delivery ◽  
Callus Cultures ◽  
Physical Factors ◽  
Foreign Dna ◽  
High Level

We report the method for the establishment of rapidly growing callus cultures of Phaseolus vulgaris and the conditions required for efficient transformation using high velocity microprojectiles and high level of transient gene expression. Using hypocotyl explant and vertical culture on B5 medium with lmg/1 kinetin and 2 mg/1 2,4-D, we can recommend to get a rapidly growing callus from bean which is a good starting material to introduce foreign DNA into bean cells. The GeneBooster particle delivery system was used for the bombardment of bean callus and the Hgm resistance gene (Hgmr) was used as a selectable marker gene. 25mg/I hygromycin (Hgm) concentration was sufficient to kill the control callus. We used the standard physical factors, the appropriate pressure of N2 gas for the bombardment of the callus tissue, the shooting distance and the size of tungsten particles used as microprojectiles. Selective and nonselective tests were made by transferring the healthy green and white calluses, subcultured for 4 months on selective and nonselective medium. Several Hgm resistant calli had been obtained. Selective pressure was maintained over a period of 10 months.

scholarly journals Distribution of the Intermedilysin Gene among the Anginosus Group Streptococci and Correlation between Intermedilysin Production and Deep-Seated Infection with Streptococcus intermedius

2000 ◽  
Vol 38 (1) ◽  
pp. 220-226
Author(s):  
Hideaki Nagamune ◽  
Robert A. Whiley ◽  
Takatsugu Goto ◽  
Yasuko Inai ◽  
Takuya Maeda ◽  
...  
Keyword(s):  
Southern Hybridization ◽  
Marker Gene ◽  
Toxin Production ◽  
Clear Correlation ◽  
Toxin Gene ◽  
Hemolysis Assay ◽  
Streptococcus Intermedius ◽  
Triggering Factor ◽  
Peripheral Infection ◽  
Human Specific

ABSTRACT The distribution of intermedilysin, a human-specific cytolysin, among the anginosus group streptococci and the correlation of toxin production and infection by Streptococcus intermedius were investigated. PCR and Southern hybridization specific for the intermedilysin gene revealed that the toxin gene exists only in S. intermedius and no homologue to the toxin gene is distributed in S. anginosus and S. constellatus . Thus, the intermedilysin gene is useful as a marker gene of S. intermedius . Moreover, a human-specific hemolysis assay and Western blotting with intermedilysin-specific antibodies clearly demonstrated that the intermedilysin production level in isolates from deep-seated infections, such as brain and liver abscesses, is higher (6.2- to 10.2-fold, respectively) than in strains from normal habitats, such as dental plaque, or from peripheral infection sites. However, other candidate virulence factors of S. intermedius , such as chondroitin sulfate depolymerase, hyaluronidase, and sialidase activities, did not show such a clear correlation between enzymatic activity and isolation sites or disease severity. From these results, intermedilysin is likely to be the pathogenic or triggering factor of significance in inducing deep-seated infections with S. intermedius .

Use of functional genes to quantify denitrifiers in the environment

2006 ◽  
Vol 34 (1) ◽  
pp. 101-103 ◽  
Author(s):  
L. Philippot
Keyword(s):  
Real Time Pcr ◽  
Southern Hybridization ◽  
Most Probable Number ◽  
Functional Genes ◽  
Hybridization Technique ◽  
Dna Arrays ◽  
Competitive Pcr ◽  
Probable Number ◽  
Composition And Structure ◽  
Micro Organisms

During the last decade, application of molecular methods using cultivation-independent approaches has provided new insights into the composition and structure of denitrifying communities in various environments. However, little is known about their abundance, and quantification is still performed using cultivation-based approaches, which are not only biased by the inability to cultivate of many micro-organisms but also fastidious and time-consuming. Two types of cultivation-independent approaches have recently been developed to quantify denitrifiers. The first type, which is based on the hybridization technique, comprises the use of Southern hybridization and DNA arrays. The second type, based on PCR, comprises the use of MPN (most probable number)-PCR, competitive PCR or real-time PCR. In this review, these different approaches will be presented with examples of their application in environmental studies.

Genetic transformation of Stella De Oro daylily by particle bombardment

2003 ◽  
Vol 83 (4) ◽  
pp. 873-876 ◽  
Author(s):  
A. N. Aziz ◽  
R. J. Sauvé ◽  
S. Zhou
Keyword(s):  
Southern Blotting ◽  
Basal Medium ◽  
Callus Cultures ◽  
Naphthaleneacetic Acid ◽  
Ms Medium ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Semi Solid ◽  
Independent Transformation

Daylily (Hemerocallis sp. ‘Stella de Oro’) callus cultures initiated from ovules were bombarded with gold particles coated with plasmid harboring Basta® resistance gene. Resulting putative transgenic calli were selected after 3 wk on semi-solid Murashige and Skoog’s (MS) basal medium supplemented with 10 mg L-1 1-naphthaleneacetic acid, 2 mg L-1 6-benzylaminopurine and 3 mg L-1 phosphinothricin (PPT). Surviving calli regenerated shoots after 2 mo on semi-solid MS medium supplemented with 2 mg L-1 thiadiazuron and 1 mg L-1 PPT. Polymerase chain reaction and Southern blotting were used to confirm independent transformation events. Key words: Basta® resistance, in vitro, Hemerocallis

Toward Understanding the Function of Amelogenin Using Transgenic Mice

1996 ◽  
Vol 10 (2) ◽  
pp. 208-214 ◽  
Author(s):  
K. Ibaraki-O'Connor ◽  
K. Nakata ◽  
M.F. Young
Keyword(s):  
Southern Blotting ◽  
Signal Sequence ◽  
Transgenic Animals ◽  
Full Length ◽  
Pcr Analysis ◽  
Coding Region ◽  
Chain Reaction ◽  
Mammalian Expression ◽  
Ectopic Production ◽  
Polymerase Chain

The purpose of this study was to establish transgenic mouse lines as a tool to investigate the function of amelogenin during mineralization by causing ectopic production of amelogenin and studying its effect. The mouse amelogenin (mAme) was cloned from a 16-day-old whole mouse embryo cDNA library and was determined to be "full-length" mouse amelogenin (with a complete coding region) by comparison with the mouse amelogenin reported previously by Snead et al. (1985) and Lau et al. (1992). The overexpression construct contained: (1) the rat osteocalcin (OC) promoter (1.8 kb); (2) the adenovirus splicing casettes, including introgenic (Int) sequence (0.3 kb); (3) the full-length mAme cDNA (0.8 kb); and (4) the polyadenylation signal sequence from the pSG5 mammalian expression vector. Both Southern blotting and polymerase chain-reaction (PCR) analyses were performed, by means of a specific probe and a pair of oligodeoxynucleotides to OclntmAme(A)+, respectively. The animals which showed transgene-positive in both analyses were further used to establish F1 animals. Heterozygocity was confirmed with F1 animals by PCR analysis of DNA from the F0 x FVB/N pups. Three independent transgenic F1 heterozygous lines (640t, 706t, and 708t) have now been established. The generation of F2 homozygous lines is under way. The heterozygous transgenic animals are currently being analyzed for alterations in the morphology and structure of various bone tissues.

scholarly journals Structure and expression of B-myc, a new member of the myc gene family

1988 ◽  
Vol 8 (8) ◽  
pp. 3168-3174
Author(s):  
S Ingvarsson ◽  
C Asker ◽  
H Axelson ◽  
G Klein ◽  
J Sümegi
Keyword(s):  
Southern Blotting ◽  
Cdna Libraries ◽  
Rat Tissues ◽  
Coding Region ◽  
Specific Expression ◽  
Protein Coding ◽  
Myc Gene ◽  
The Brain ◽  
Mouse Somatic Cell ◽  
Specific Mrna

The myc family of genes contains five functional members. We describe the cloning of a new member of the myc family from rat genomic and cDNA libraries, designated B-myc. A fragment of cloned B-myc was used to map the corresponding rat locus by Southern blotting of DNA prepared from rat X mouse somatic cell hybrids. B-myc mapped to rat chromosome 3. We have previously mapped the c-myc to rat chromosome 7 (J. Sümegi, J. Spira, H. Bazin, J. Szpirer, G. Levan, and G. Klein, Nature [London] 306:497-498, 1983) and N-myc and L-myc to rat chromosomes 6 and 5, respectively (S. Ingvarsson, C. Asker, Z. Wirschubsky, J. Szpirer, G. Levan, G. Klein, and J. Sümegi, Somat. Cell Mol. Genet. 13:335-339, 1987). A partial sequence of B-myc had extensive sequence homology to the c-myc protein-coding region, and the detection of intron homology further indicated that these two genes are closely related. The DNA regions conserved among the myc family members, designated myc boxes, were highly conserved between c-myc and B-myc. A lower degree of homology was detected in other parts of the coding region in c-myc and B-myc not present in N-myc and L-myc. A 1.3-kilobase B-myc-specific mRNA was detected in most rat tissues, with the highest expression in the brain. This resembled the expression pattern of c-myc, although at different relative levels, and was in contrast to the more tissue-specific expression of N-myc and L-myc. B-myc was expressed at uniformly high levels in all fetal tissues and during subsequent postnatal development, in contrast to the stage-specific expression of c-myc.

scholarly journals Herbicide-tolerant tobacco mutants selected in situ and recovered via regeneration from cell culture

1978 ◽  
Vol 32 (1) ◽  
pp. 85-89 ◽  
Author(s):  
David N. Radin ◽  
Peter S. Carlson
Keyword(s):  
Cell Culture ◽  
Resistant Cell ◽  
Callus Cultures ◽  
Wild Type ◽  
Tobacco Plants ◽  
Herbicide Resistant ◽  
Cell Clones ◽  
Herbicide Tolerant ◽  
Sexual Crosses

SUMMARYThe herbicides Bentazone and Phenmedipharm kill the leaves of intact tobacco plants but do not affect callus cultures. Tolerant mutants were isolated by treating leaves of previously γ-irradiated haploid plants with herbicide then excising and culturing the green herbicide-resistant cell clones on the otherwise yellowed leaves. Among plants subsequently regenerated were a total of ten stable independently isolated mutants. Sexual crosses show these ten represent four Bentazone and two Phenmedipharm loci; all mutants were recessive to wild type.

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