Agrobacterium tumefaciens -mediated transformation of tobacco (Nicotiana tabaccum)

2017 ◽  
Vol 6 (4) ◽  
Author(s):  
Shanthala Mallikarjunaiah ◽  
Mahesh Pattabhiramaiah ◽  
Chethan Gowda
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Plasmid Dna ◽  
Restriction Enzymes ◽  
Kanamycin Resistance ◽  
Genetically Engineered ◽  
Nptii Gene ◽  
Helper Plasmid ◽  
Alkaline Lysis ◽  
Annexin Gene ◽  
Pcr Method

The present study involves the development of genetically engineered tobacco plants with annexin gene. The plasmid DNA of pUC 19 vector having the desired Annexin gene (3Kbp) and the plasmid DNA of the binary vector pGPTV (13 Kbp) were isolated from E.coli (DH5α strain) by alkaline lysis method. The purified pUC 19/Annexin and pGPTV plasmid were restriction digested using the restriction enzymes EcoRI and XbaI as a linearised band was eluted from the gel. The digested plasmid shown in the band pattern in the gel were cut by gel elution technique and purified from other reaction mixture. Then the dot spot test was done to calculate the concentration of pGPTV and Annexin gene. The recombinant PGPTV plasmid with the annexin gene in Agrobacterium tumefaciens MTCC 431 was mobilized and transferred to plant system through the mobilization helper plasmid pRK2013. The kanamycin resistance gene (NPT II) was used as a selective marker. The calli used for isolating the genomic DNA which was then amplified for confirmation of annexin gene. The nptII gene of 800 bp serves as a selectable marker system in plants and its amplification confirmed the presence of annexin gene in transgenic plants by PCR method.

Agrobacterium tumefaciens -mediated transformation of tomato (Solanum lycopersicum)

2017 ◽  
Vol 6 (6) ◽  
Author(s):  
Shanthala Mallikarjunaiah ◽  
Sowmya Moudgalya
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Plasmid Dna ◽  
Restriction Enzymes ◽  
Kanamycin Resistance ◽  
Genetically Engineered ◽  
Nptii Gene ◽  
Helper Plasmid ◽  
Alkaline Lysis ◽  
Annexin Gene ◽  
Pcr Method

The present investigation describes the development of genetically engineered tomato plants with annexin gene. The alkaline lysis method is used to isolate the plasmid DNA of pUC 19 vector having the desired Annexin gene (3Kbp) and the plasmid DNA of the binary vector pGPTV (13 Kbp) from E.coli (DH5α strain). The purified pUC 19/Annexin and pGPTV plasmid were restriction digested using the restriction enzymes EcoRI and XbaI as a linearised band was eluted from the gel. The digested plasmid shown in the band pattern in the gel were cut by gel elution technique and purified from other reaction mixture. Then the dot spot test was done to calculate the concentration of pGPTV and Annexin gene. The recombinant PGPTV plasmid with the annexin gene in Agrobacterium tumefaciens MTCC 431 was mobilized and transferred to plant system through the mobilization helper plasmid pRK2013. The kanamycin resistance gene (NPT II) was used as a selective marker. The calli used for isolating the genomic DNA which was then amplified for confirmation of annexin gene. The nptII gene of 800 bp serves as a selectable marker system in plants and its amplification confirmed the presence of annexin gene in transgenic plants by PCR method.

Evaluation of transgenic canola plants under field conditions

Genome ◽  
1992 ◽  
Vol 35 (1) ◽  
pp. 58-63 ◽  
Author(s):  
M. Arnoldo ◽  
C. L. Baszczynski ◽  
G. Bellemare ◽  
G. Brown ◽  
J. Carlson ◽  
...  
Keyword(s):  
Kanamycin Resistance ◽  
Genetically Engineered ◽  
Field Conditions ◽  
Enzyme Assays ◽  
Nptii Gene ◽  
Neomycin Phosphotransferase ◽  
Agrobacterium Mediated Transformation ◽  
Transgenic Canola ◽  
Progeny Analysis ◽  
Transformation Vectors

Eleven independent transgenic canola (Brassica napus ssp. oleifera L. cv. Westar and Regent) lines were evaluated in the field. The plants carried a neomycin phosphotransferase (NPTII) gene for kanamycin resistance that was introduced via Agrobacterium-mediated transformation. NPTII enzyme assays, Southern blot hybridizations and progeny analysis, confirmed the stable, heritable integration and expression of the introduced NPTII gene. A number of agronomic characteristics evaluated under field conditions, including maturity, yield, and oil and protein content, were all statistically comparable between the transformed and nontransformed plants. These results indicate that canola can be genetically engineered successfully, and that the Agrobacterium-based transformation system employed does not induce any adverse effects on the intrinsic agronomic and qualitative traits critical to the agricultural industry.Key words: transgenic field trial, canola, Agrobacterium-mediated transformation, vectors.

scholarly journals A Second T-Region of the Soybean-Supervirulent Chrysopine-Type Ti Plasmid pTiChry5, and Construction of a Fully Disarmed vir Helper Plasmid

2000 ◽  
Vol 13 (10) ◽  
pp. 1081-1091 ◽  
Author(s):  
Karuppaiah Palanichelvam ◽  
Philippe Oger ◽  
Steven J. Clough ◽  
Chung Cha ◽  
Andrew F. Bent ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Ti Plasmid ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
Nptii Gene ◽  
Helper Plasmid ◽  
Selection For ◽  
Border Repeats ◽  
The Right ◽  
Binary Plasmid

Agrobacterium tumefaciens Chry5, which is particularly virulent on soybeans, induces tumors that produce a family of Amadori-type opines that includes deoxyfructosyl glutamine (Dfg) and its lactone, chrysopine (Chy). Cosmid clones mapping to the right of the known oncogenic T-region of pTiChry5 conferred Amadori opine production on tumors induced by the nopaline strain C58. Sequence analysis of DNA held in common among these cosmids identified two 25-bp, direct repeats flanking an 8.5-kb segment of pTiChry5. These probable border sequences are closely related to those of other known T-regions and define a second T-region of pTiChry5, called T-right (TR), that confers production of the Amadori opines. The oncogenic T-left region (TL) was located precisely by identifying and sequencing the likely border repeats defining this segment. The two T-regions are separated by approximately 15 kb of plasmid DNA. Based on these results, we predicted that pKYRT1, a vir helper plasmid derived from pTiChry5, still contains all of TR and the leftmost 9 kb of TL. Consistent with this hypothesis, transgenic Arabidopsis thaliana plants selected for with a marker encoded by a binary plasmid following transformation with KYRT1 coinherited production of the Amadori opines at high frequency. All opine-positive transgenic plants also contained TR-DNA, while those plants that lacked TR-DNA failed to produce the opines. Moreover, A. thaliana infected with KYRT1 in which an nptII gene driven by the 35S promoter of Cauliflower mosaic virus was inserted directly into the vir helper plasmid yielded kanamycin-resistant transformants at a low but detectable frequency. These results demonstrate that pKYRT1 is not disarmed, and can transfer Ti plasmid DNA to plants. A new vir helper plasmid was constructed from pTiChry5 by two rounds of sacB-mediated selection for deletion events. This plasmid, called pKPSF2, lacks both of the known T-regions and their borders. pKPSF2 failed to transfer Ti plasmid DNA to plants, but mobilized the T-region of a binary plasmid at an efficiency indistinguishable from those of pKYRT1 and the nopaline-type vir helper plasmid pMP90.

scholarly journals Nonspecific transmission of the NPTII gene from E. coli helper plasmid to a plasmid of Agrobacterium tumefaciens.

1994 ◽  
Vol 41 (2) ◽  
pp. 129-132
Author(s):  
K Leśniewicz ◽  
W Grygorowicz ◽  
S Bartkowiak ◽  
H Augustyniak
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Nptii Gene ◽  
Helper Plasmid ◽  
E Coli

scholarly journals Multiple Displacement Amplification as a Solution for Low Copy Number Plasmid Sequencing

2021 ◽  
Vol 12 ◽  
Author(s):  
Kuan Yao ◽  
Narjol González-Escalona ◽  
Maria Hoffmann
Keyword(s):  
Antibiotic Resistance ◽  
Single Molecule ◽  
Plasmid Dna ◽  
Copy Number ◽  
Sequence Data ◽  
Multiple Displacement Amplification ◽  
Fast Method ◽  
Alkaline Lysis ◽  
Low Copy Number ◽  
Long Read

Plasmids play a major role in bacterial adaptation to environmental stress and often contribute to antibiotic resistance and disease virulence. Although the complete sequence of each plasmid is essential for studying plasmid biology, most antibiotic resistance and virulence plasmids in Salmonella are present only in a low copy number, making extraction and sequencing difficult. Long read sequencing technologies require higher concentrations of DNA to provide optimal results. To resolve this problem, we assessed the sufficiency of multiple displacement amplification (MDA) for replicating Salmonella plasmid DNA to a satisfactory concentration for accurate sequencing and multiplexing. Nine Salmonella enterica isolates, representing nine different serovars carrying plasmids for which sequence data are already available at NCBI, were cultured and their plasmids isolated using an alkaline lysis extraction protocol. We then used the Phi29 polymerase to perform MDA, thereby obtaining enough plasmid DNA for long read sequencing. These amplified plasmids were multiplexed and sequenced on one single molecule, real-time (SMRT) cell with the Pacific Biosciences (Pacbio) Sequel sequencer. We were able to close all Salmonella plasmids (sizes ranged from 38 to 166 Kb) with sequencing coverage from 24 to 2,582X. This protocol, consisting of plasmid isolation, MDA, and multiplex sequencing, is an effective and fast method for closing high-molecular weight and low-copy-number plasmids. This high throughput protocol reduces the time and cost of plasmid closure.

Preparation of Plasmid DNA by Alkaline Lysis with SDS: Midipreparation

2006 ◽  
Vol 2006 (1) ◽  
pp. pdb.prot3901 ◽  
Author(s):  
Joseph Sambrook ◽  
David W. Russell
Keyword(s):  
Plasmid Dna ◽  
Alkaline Lysis

scholarly journals Successful genetic transformation in date palm (Phoenix dactylifera)

2014 ◽  
Vol 11 (2) ◽  
pp. 171-176 ◽  
Author(s):  
L Hassan
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Agrobacterium Rhizogenes ◽  
Reporter Gene ◽  
Hairy Roots ◽  
Date Palm ◽  
Binary Vector ◽  
Nptii Gene ◽  
Neomycin Phosphotransferase ◽  
Gus Gene ◽  
Nos Terminator

The introduction of foreign genes into most of the Phoenix spp using recombinant DNA technology is not a straight forward task. In Phoenix spp application of this technology towards successful transformation proved to be a more difficult one – so far no report on the successful regeneration of transgenic date palm plants has been published. We developed an efficient and reproducible variety-independent method for producing transgenic date palm (Phoenix spp) via Agrobacterium-mediated transformation. Agrobacterium rhizogenes strains LBA 9402 were used and for cotransformation experiments the strain LBA 9402 with the binary vector pBIN19 with the p35S GUS INT gene was used. Off-shoot segments from different Phoenix spp cultivars were infected with Agrobacterium rhizogenes. The development of ‘hairy roots’ at a high frequency only on infected tissue pieces showed that transformation is possible. Various parameters like, effect of different genotypes on root initiation, root number and root length have been studied. Regeneration of transformed root cultures to plantlets was also attempted. Histochemical GUS assay and polymerase chain reaction analysis of hairy roots confirmed the presence of GUS gene. Agrobacterium tumifaciensmediated transformation was also performed using the leaves of off-shoot explants. Agrobacterium tumefaciens strains: I) GV3101 with the vir plasmid pMP90 the strain C58C1 ATHV with the vir-plasmid pTiBo542 (=pEHA101; Hood et al. 1986) was used. The nptII gene (neomycin phosphotransferase) was used as a selectable marker gene. The ?-Glucuronidase-gene (GUS-Gene: Jefferson et al. 1987) under control of the Ubi- and 35S-Promotors, with an Intron (Vancanneyt et al. 1990), was used as the reporter gene. We also used the genetically engineered Agrobacterium tumefaciens strain LBA4404 as a vector for infection in the transformation experiment, which contains plasmid pBI121 of 14 KDa (binary vector). This binary vector contains following genes within the right border (RB) and left border (LB) region of the construct: The udiA gene (Jefferson, 1986) predetermining GUS (?-glucuronidase), driven by CaMV promoter and NOS terminator. This reporter gene can be used to assess the efficiency of transformation. The nptII gene (Herrera-Estrella et al., 1983) encoding neomycin phosphotransferase II (nptII) conferring kanamycin resistance, driven by NOS promoter and NOS terminator. The bacterium also contains plasmid pAL4404 which is a disarmed Ti plasmid (132 KDa) containing the virulence genes. For the confirmation of transgenes, calli were taken from the growing callus mass for DNA isolation. PCR- and Southern analysis was performed to determine the integration and the copy number of the transgene. The GUS-test was performed to demonstrate ß-glucuronidase expression. The transgenic plantlets were kept in a hardening room for four weeks and they will be transferred to a growth chamber with controlled environment for further establishment. DOI: http://dx.doi.org/10.3329/jbau.v11i2.19841 J. Bangladesh Agril. Univ. 11(2): 171-176, 2013

A rapid immunoprecipitation assay for neomycin phosphotransferase II expression in transformed bacteria and plant tissues

1990 ◽  
Vol 68 (6) ◽  
pp. 983-987
Author(s):  
Chris L. Baszczynski
Keyword(s):  
Brassica Napus ◽  
Bovine Serum Albumin ◽  
Kanamycin Resistance ◽  
Plant Tissues ◽  
Nptii Gene ◽  
Neomycin Phosphotransferase ◽  
Kanamycin Resistance Gene ◽  
Dot Blot ◽  
Immunoprecipitation Assay ◽  
Neomycin Phosphotransferase Ii

Anti-kanamycin antibodies produced in rabbits, following coupling of the antibiotic to bovine serum albumin, were used to immunoprecipitate radioactively labelled phosphorylated kanamycin from transformed bacterial or plant extracts in a novel assay system, for the detection of neomycin phosphotransferase II (NPTII) activity. Radioactive counts in the immunoprecipitated pellet give a semiquantitative measure of the kanamycin phosphorylation and hence the amount of NPTII activity. This assay is sensitive, uses very small amounts of radioactivity, and is very rapid, allowing many samples to be processed within a few hours. Immunoprecipitated counts from reactions with bacteria carrying a kanamycin resistance gene or from tobacco and Brassica napus plants transformed with NPTII gene-containing vectors were consistently higher than counts from nontransformed controls. Results obtained with this assay correlate well with those from the previously described gel overlay and dot-blot assays, but can be obtained in an appreciably shorter time frame.Key words: anti-kanamycin antibodies, immunoprecipitation, neomycin phosphotransferase II assay, transformation.

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