Genetic Transformation of Garlic (Allium sativum L.) by Particle Bombardment

Microprojectile bombardment was used to introduce DNA into embryogenic callus of garlic (Allium sativum L.) and produce stably transformed garlic plants. Embryogenic calluses, derived from garlic cultivar `GT96-1', were bombarded with plasmid DNA containing genes coding for hygromycin phosphotransferase and β-glucuronidase. Putatively transformed calluses were identified in the bombarded tissue after 4 months of selection on 20 mg·L-1 hygromycin B. The transgenic nature of the selected material was demonstrated by GUS histochemical assay and Southern blot hybridization analysis, and twenty transgenic plants were regenerated.

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Tandem arrangement of tubulin genes in the protozoan parasite Leishmania enriettii

Molecular and Cellular Biology ◽

10.1128/mcb.3.6.1070-1076.1983 ◽

1983 ◽

Vol 3 (6) ◽

pp. 1070-1076

Author(s):

S M Landfear ◽

D McMahon-Pratt ◽

D F Wirth

Keyword(s):

Tandem Repeat ◽

Blot Hybridization ◽

Protozoan Parasite ◽

Southern Blot Hybridization ◽

Beta Tubulin ◽

Developmentally Regulated ◽

Alpha Tubulin ◽

Tubulin Genes ◽

Southern Blot Hybridization Analysis ◽

Leishmania Enriettii

The arrangement of developmentally regulated alpha- and beta-tubulin genes has been studied in the parasitic protozoan Leishmania enriettii by using Southern blot hybridization analysis. The alpha-tubulin genes occur in a tandem repeat whose monomeric unit may be represented by a 2-kilobase PstI fragment. Similarly, the beta-tubulin genes probably occur in a separate tandem repeat consisting of approximately 4-kilobase units unlinked to the alpha-tubulin repeats.

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Tandem arrangement of tubulin genes in the protozoan parasite Leishmania enriettii.

Molecular and Cellular Biology ◽

10.1128/mcb.3.6.1070 ◽

1983 ◽

Vol 3 (6) ◽

pp. 1070-1076 ◽

Cited By ~ 69

Author(s):

S M Landfear ◽

D McMahon-Pratt ◽

D F Wirth

Keyword(s):

Tandem Repeat ◽

Blot Hybridization ◽

Protozoan Parasite ◽

Southern Blot Hybridization ◽

Beta Tubulin ◽

Developmentally Regulated ◽

Alpha Tubulin ◽

Tubulin Genes ◽

Southern Blot Hybridization Analysis ◽

Leishmania Enriettii

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Characterization of the pTFI91 -family replicon of Thiobacillus ferrooxidans plasmids

Canadian Journal of Microbiology ◽

10.1139/m95-048 ◽

1995 ◽

Vol 41 (4-5) ◽

pp. 354-365 ◽

Cited By ~ 6

Author(s):

Leena Chakravarty ◽

Thomas J. Zupancic ◽

Beth Baker ◽

Joseph D. Kittle ◽

Ilona J. Fry ◽

...

Keyword(s):

Dna Sequences ◽

Thiobacillus Ferrooxidans ◽

Blot Hybridization ◽

Marker Gene ◽

Southern Blot Hybridization ◽

Structural Features ◽

Broad Host Range ◽

Origin Of Replication ◽

E Coli ◽

Southern Blot Hybridization Analysis

Plasmids found in six strains of Thiobacillus ferrooxidans were mapped and compared in an effort to detect the origin of replication. Four strains yielded an identical 9.8-kb plasmid, pTFI91. Restriction mapping and Southern blot hybridization analysis were used to confirm this finding. Dissimilar plasmids found in two other strains contained a conserved 2.2-kb SacI region common to pTFI91. DNA sequence analysis of this region showed structural features common to bacterial plasmid replicons. A comparison of the pTFI91 origin with those of T. ferrooxidans pTF-FC2 and other broad host range vectors did not show significant homologous DNA sequences. To verify the replication function, a chloramphenicol acetyl transferase marker gene was ligated at the unique sites of pTFI91, and the plasmid was transformed into Escherichia coli DH5α cells but no transformants were identified. To test the replication of pTFI91 independent of DNA polymerase I in E. coli, different restriction fragments of pTFI91 were cloned into pHSG398 (Cmr, ColEI origin) and transformed into the polA1 mutant SF800, but chloramphenicol-resistant transformants were not detected. Electrotransformation of T. ferrooxidans TFI-70 and Pseudomonas putida ATCC 19151 also failed to yield transformants. The results suggested that the pTFI91 plasmid replicon does not function either in E. coli or in P. putida. Since pTFI91 contains the same origin of replication as other plasmids in several other T. ferrooxidans strains, this replicon may be commonly distributed in T. ferrooxidans.Key words: nucleotide sequence, origin of replication, plasmid DNA, replicon, Thiobacillus ferrooxidans.

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Leptospira interrogans serovar sejroe infection in a group of laboratory dogs

Laboratory Animals ◽

10.1258/002367795781088261 ◽

1995 ◽

Vol 29 (3) ◽

pp. 300-306 ◽

Cited By ~ 14

Author(s):

E. Scanziani ◽

L. Crippa ◽

Anna M. Giusti ◽

M. Luini ◽

Maria L. Pacciarini ◽

...

Keyword(s):

Interstitial Nephritis ◽

Urine Analysis ◽

Blot Hybridization ◽

Southern Blot Hybridization ◽

Leptospira Interrogans ◽

Normal Ranges ◽

Southern Blot Hybridization Analysis ◽

Antibody Titres ◽

Renal Infection ◽

Endonuclease Digestion

Interstitial nephritis was seen histologically in 19 (59%) out of 32 pure-breed beagle dogs (16 males and 16 females) subjected to standard safety tests. In these animals no clinical abnormalities were observed and all the tested parameters (haematology, biochemistry and urine analysis) were within the normal ranges. Leptospiral antibody titres ranging from 1:100 to 1:6400, against a serovar ( hardio) belonging to the Sejroe serogroup, were detected by the microscopic agglutination test (MAT) in the serum of the 19 dogs with interstitial nephritis. All animals without renal lesions were seronegative. Leptospiral antigen was detected immunohistochemically in the kidneys of 4 dogs; leptospires were detected in Warthin-Starry stained sections of one dog. Leptospires were isolated from the kidneys of 3 of the 4 dogs examined by bacterial culture. The isolated strains were typed as serovar sejroe by restriction endonuclease digestion and Southern blot hybridization analysis of their DNA. It was concluded that Leptospira interrogans serovar sejroe, was responsible for an asymptomatic chronic renal infection which was widespread in this group of laboratory dogs.

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088 Microprojectile Bombardment-mediated Transformation of Rhododendron `Catawbiense Album' L.

HortScience ◽

10.21273/hortsci.34.3.456d ◽

1999 ◽

Vol 34 (3) ◽

pp. 456D-456

Author(s):

Jane E. Knapp ◽

Mark H. Brand

Keyword(s):

Microprojectile Bombardment ◽

Blot Hybridization ◽

Shoot Proliferation ◽

Southern Blot Hybridization ◽

Pcr Analysis ◽

Marker Genes ◽

Selective Agent ◽

Putative Transformants ◽

High Level

Horticultural improvements in Rhododendron require long periods of time to produce flowering plants by traditional breeding methods. In addition, new trait development by conventional genetics is limited to existing germplasm. Genetic engineering approaches to horticultural improvement offer the possibility for introduction of new traits using foreign DNA from any source. To this end, we have developed a system for the genetic transformation of Rhododendron based on microprojectile bombardment. Leaves from in vitro-grown plantlets of R. `Catawbiense Album' L. were bombarded with the marker genes uidA (GUS) in combination with nptII or hph. Two days post-bombardment, explants were transferred to shoot iniation medium containing either 50 mg/L kanamycin or 2.5 mg/L hygromycin. After 4 weeks, proliferating tissues were transferred to media containing increased levels of selective agent (100 mg/L kanamycin or 5 mg/L hygromycin, respectively). Shoots that regenerated were then excised from necrotic tissues and transferred to shoot proliferation medium containing the high level of selective agent. PCR analysis of putative transformants revealed the presence of the transgenes. Southern blot hybridization confirmed stable transgene integration. Histochemical GUS assays of transformed tissues indicated uniform expression throughout the transgenic plant. With the development of an efficient transformation system, the introduction of genes to confer useful horticultural traits becomes feasible.

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Southern blot-hybridization analysis of HBV DNA in patients with HBsAg seropositive chronic liver diseases.

Kanzo ◽

10.2957/kanzo.26.565 ◽

1985 ◽

Vol 26 (5) ◽

pp. 565-571 ◽

Cited By ~ 1

Author(s):

Osamu YOKOSUKA ◽

Masao OMATA ◽

Fumio IMAZEKI ◽

Yoshimi ITO ◽

Junko MORI ◽

...

Keyword(s):

Southern Blot ◽

Liver Diseases ◽

Blot Hybridization ◽

Southern Blot Hybridization ◽

Hbv Dna ◽

Chronic Liver ◽

Hybridization Analysis ◽

Chronic Liver Diseases ◽

Southern Blot Hybridization Analysis

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Protein C deficiency Hong Kong 1 and 2: hereditary protein C deficiency caused by two mutant alleles, a 5-nucleotide deletion and a missense mutation

Blood ◽

10.1182/blood.v80.1.126.126 ◽

1992 ◽

Vol 80 (1) ◽

pp. 126-133 ◽

Cited By ~ 11

Author(s):

Y Sugahara ◽

O Miura ◽

P Yuen ◽

N Aoki

Keyword(s):

Amino Acid ◽

Protein C ◽

Catalytic Domain ◽

Stop Codon ◽

Blot Hybridization ◽

Southern Blot Hybridization ◽

Expression Vectors ◽

Protein C Deficiency ◽

Mutant Proteins ◽

Southern Blot Hybridization Analysis

Abstract We characterized a mutant protein C gene from an individual with no detectable protein C antigen in blood plasma. Southern blot hybridization analysis with human protein C cDNA demonstrated neither gross deletion nor rearrangement of the gene. Sequencing all the exons and exon-intron boundaries of the gene except the 3′ noncoding region showed two mutant alleles. The one, derived from the mother, represents a deletion of 5 nucleotides (nt) (CCCGC) in the end of exon VI (mutation I), predicted to result in the generation of a new stop codon due to a reading frameshift and the premature termination of translation. The other, derived from the father, represents a point mutation (G to A) in exon IX (mutation II), resulting in an amino acid substitution, Gly-376(GGC) to Asp(GAC), in the catalytic domain of the protein. Allele-specific oligonucleotide probe hybridization confirmed the presence of the two mutations. Mutation I would result in a truncated polypeptide of 169 amino acid residues that lacks the heavy chain. Mutation II gives rise to an alteration of a highly conserved amino acid, Gly-376. These data indicate that this patient is a compound heterozygote of the two mutant alleles, each one inherited from each parent. Transient expression assays using COS-7 cells transfected with mutated protein C expression vectors suggested that each of the two mutations leads to the protein C deficiency by causing an impairment of secretion of the respective mutant proteins.

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A DNAβ Associated with Tomato Yellow Leaf Curl China Virus Is Required for Symptom Induction

Journal of Virology ◽

10.1128/jvi.78.24.13966-13974.2004 ◽

2004 ◽

Vol 78 (24) ◽

pp. 13966-13974 ◽

Cited By ~ 207

Author(s):

Xiaofeng Cui ◽

Xiaorong Tao ◽

Yan Xie ◽

Claude M. Fauquet ◽

Xueping Zhou

Keyword(s):

Transgenic Plants ◽

Stop Codon ◽

Blot Hybridization ◽

Southern Blot Hybridization ◽

Tomato Yellow Leaf ◽

Cauliflower Mosaic Virus ◽

Start Codon ◽

35S Promoter ◽

Yellow Leaf ◽

Leaf Curl

ABSTRACT We report here that all 25 isolates of Tomato yellow leaf curl China virus (TYLCCNV) collected from tobacco, tomato, or Siegesbeckia orientalis plants in different regions of Yunnan Province, China, were associated with DNAβ molecules. To investigate the biological role of DNAβ, full-length infectious clones of viral DNA and DNAβ of TYLCCNV isolate Y10 (TYLCCNV-Y10) were agroinoculated into Nicotiana benthamiana, Nicotiana glutinosa, Nicotiana. tabacum Samsun (NN or nn), tomato, and petunia plants. We found that TYLCCNV-Y10 alone could systemically infect these plants, but no symptoms were induced. TYLCCNV-Y10 DNAβ was required, in addition to TYLCCNV-Y10, for induction of leaf curl disease in these hosts. Similar to TYLCCNV-Y10, DNAβ of TYLCCNV isolate Y64 was also found to be required for induction of typical leaf curl diseases in the hosts tested. When the βC1 gene of TYLCCNV-Y10 DNAβ was mutated, the mutants failed to induce leaf curl symptoms in N. benthamiana when coinoculated with TYLCCNV-Y10. However, Southern blot hybridization analyses showed that the mutated DNAβ molecules were replicated. When N. benthamiana and N. tabacum plants were transformed with a construct containing the βC1 gene under the control of the Cauliflower mosaic virus 35S promoter, many transgenic plants developed leaf curl symptoms similar to those caused by a virus, the severity of which paralleled the level of βC1 transcripts, while transgenic plants transformed with the βC1 gene containing a stop codon after the start codon remained symptomless. Thus, expression of a βC1 gene is adequate for induction of symptoms of viral infection in the absence of virus.

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CYTOKININ EFFECTS ON TOMATO SEED QUALITY, FRUIT YIELD, AND RIPENING IN TRANSGENIC PLANTS CARRYING THE ipt GENE

HortScience ◽

10.21273/hortsci.27.6.661c ◽

1992 ◽

Vol 27 (6) ◽

pp. 661c-661

Author(s):

Ann C. Smigocki ◽

Iris J. Honeczy

Keyword(s):

Transgenic Plants ◽

Southern Blot ◽

Seed Quality ◽

Blot Hybridization ◽

Southern Blot Hybridization ◽

Isopentenyl Transferase ◽

Ipt Gene ◽

Tomato Seed ◽

Selective Media ◽

Antisense Orientation

Lycopersicon esculentum cv. UC82b cotyledons were co-cultivated with A. tumefaciens carrying vectors with modified isopentenyl transferase (ipt) genes. The ipt gene was placed under the control of the RUBISCO promoter in both the sense and antisense orientation. Over 50 transformants were recovered on kanamycin-containing media. Seeds from RO plants were germinated on selective media and R1 plants transformed with the ipt gene identified by PCR and Southern blot hybridization. Phenotypes of the R1 plants, whether transformed with the ipt gene in the sense or antisense orientation, were comparable to the control plants transformed with an inactive cytokinin gene. Fruit weights from both were similar to those from control plants, however, yields were reduced and ripening delayed. Most fruit had no seeds or very few small seeds. Cytokinin levels are being determined in order to correlate them to the observed phenotypes.

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Optimization of regeneration and Agrobacterium-mediated transformation of Stevia (Stevia rebaudiana Bertoni): a commercially important natural sweetener plant

Scientific Reports ◽

10.1038/s41598-020-72751-8 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Pooja Taak ◽

Siddharth Tiwari ◽

Bhupendra Koul

Keyword(s):

Transgenic Plants ◽

Blot Hybridization ◽

Stevia Rebaudiana ◽

Southern Blot Hybridization ◽

Nodal Explants ◽

Naphthalene Acetic Acid ◽

Steviol Glycosides ◽

Root Induction ◽

Commercially Important

Abstract Stevia rebaudiana Bertoni is a commercially important zero calorie natural-sweetener herb which produce sweet compounds known as steviol glycosides. Rising demands of steviol glycosides by food and beverage industries has led to an increase in its cultivation in various countries. Unfortunately, stevia cultivation faces 2–25% yield penalty due to weeds which further adds to its cultivation cost. To resolve this major challenge, Agrobacterium-mediated genetic transformation of in vitro derived stevia-nodal explants using herbicide resistance gene (bar) has been optimized, for the production of stable transgenic stevia plants. Several parameters including explant type, pre-incubation duration, acetosyringone (As) concentration, Agrobacterium cell density, Agro-inoculation duration, co-cultivation duration, selection regime and plant growth regulators (PGRs) combination and concentration, have been successfully optimized. Among the two types of explants used, nodal explants showed a higher regeneration response of 82.85%, with an average of 25 shoots/explant. The best PGRs combination and concentration for shoot-induction, shoot-elongation and root-induction was found to be 6-benzyladenine (1.0 mg l−1) + naphthalene acetic acid (0.5 mg l−1), gibberellic acid (1.0 mg l−1), and half-strength MS medium, respectively. The two-step selection (phosphinothricin) regime resulted in an average transformation efficiency of 40.48% with nodal explants. Molecular characterization of putative transformants through PCR, RT-PCR, qRT-PCR and Southern-blot hybridization confirmed the presence, stability, expression as well as copy number of bar gene respectively. Compared to the non-transgenic plants, the T0 transgenic plants successfully tolerated 8 mg l−1 glufosinate ammonium sprays. Thus, the optimized protocol can be useful for the introduction of other genes (inter-kingdom transfer) into stevia genome.

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