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.

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.

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.

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.

scholarly journals AGROBACTERIUM-MEDIATED TRANSFORMATION OF TWO TOMATO CULTIVARS (LYCOPERSICON ESCULENTUM MILL.) CV. SANDRA AND ROCKY

2021 ◽  
Vol 52 (3) ◽  
pp. 745-755
Author(s):  
G. H. Danial ◽  
D. A. Ibrahim ◽  
G. Q. Song
Keyword(s):  
Marker Gene ◽  
Kanamycin Resistance ◽  
Transformation Frequency ◽  
Pcr Analysis ◽  
Nptii Gene ◽  
Agrobacterium Mediated Transformation ◽  
Nos Terminator ◽  
Transgenic Tomato Plants ◽  
Tomato Cultivars

An efficient protocol for Agrobacterium-mediated transformation of tomato cultivars Sandra and Rocky was conducted to examine the possibility of producing transgenic tomato plants cultivars harbouring the nptII gene, conferring kanamycin resistance. To achieve this aim, tomato cotyledon explants were transformed using EHA105 Agrobacterium tumefaciens strain harboring the binary vectors pBI121 which contains Gus gene, and neomycin phosphotransferase II (nptII) as selectable marker gene under the control of a CaMV35S promoter and nopaline synthase (nos) Terminator. Transformant detection was carried out in three distinct ways. First antibiotic selection, Kanamycin at a concentration of100 mgl-1 found to be efficient for this purpose. Second histochemical GUS assay revealed the presence of blue colored zones in a number of shoots and leaves for both in vitro and the greenhouse-grown transgenic plants. Third PCR analysis indicated positive result by showing the fragment for nptII gene in tested transformants, while was absent in non-transgenic control (wild type). On the other hand, the results showed that Sandra cultivar was more efficient for regeneration and subsequently transformation frequency than Rocky cultivar, which record 26.66% of transformation frequency compared with 11.57% in Rocky cultivar.

scholarly journals Obtaining of transgenic alfalfa (Medicago sativa L.) and peanut (Arachis hypogaea L.) plants resistant to the herbicide Pursuit by Agrobacterium-mediated transformation

1970 ◽  
pp. 243-246
Author(s):  
S. M. Nifantova ◽  
I. K. Komarnytskyi ◽  
M. V. Kuchuk
Keyword(s):  
Gene Selection ◽  
Molecular Size ◽  
Pcr Analysis ◽  
Nptii Gene ◽  
Transformation Protocol ◽  
Genetic Construct ◽  
Agrobacterium Mediated Transformation ◽  
Transgenic Alfalfa ◽  
Arachis Hypogaea L ◽  
Selective Agents

Aim. The production of alfalfa and peanut cultivars with new properties is necessary. The purpose of this work was to develop Agrobacterium-mediated transformation protocol and to construct transgenic alfalfa and peanut plants resistant to herbicide Pursuit Methods. Genetic transformation was carried out using cocultivation of peanut and alfalfa explants with Agrobacterium tumefaciens strain GV3101 carrying genetic construct pCB004 containing mutant ahas/als gene and nptII gene. Selection was held on the solidified callus inducing medium with 50 mkg/l Pursuit. The selected callus clones were put on the regeneration medium with the same selective agents. Obtained regeneration lines were analysed using PCR-analysis. Results. 17 peanut and 14 alfalfa regeneration lines had positive signals after PCR analysis with DNA fragments of required molecular size for ahas/als and nptII genes. Conclusions. Transgenic alfalfa and peanut plants resistant to the herbicide Pursuit were obtained.Keywords: alfalfa, peanut, ahas/als gene, transformation.

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

Progeny Analysis of Glyphosate Selected Transgenic Soybeans Derived from Agrobacterium -Mediated Transformation

Crop Science ◽  
2000 ◽  
Vol 40 (3) ◽  
pp. 797-803 ◽  
Author(s):  
Thomas E. Clemente ◽  
Bradley J. LaVallee ◽  
Arlene R. Howe ◽  
Dannette Conner-Ward ◽  
Renee J. Rozman ◽  
...  
Keyword(s):  
Agrobacterium Mediated Transformation ◽  
Progeny Analysis

scholarly journals Sugar Beet Improvement using Agrobacterium-mediated Transformation technology

2018 ◽  
pp. 1-5
Author(s):  
Abo-Bakr A. Youssef ◽  
Wessam M. Rslan
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Sugar Beet ◽  
Tumor Development ◽  
Technical Information ◽  
Gus Expression ◽  
Kanamycin Resistance ◽  
Gus Activity ◽  
Dot Blot ◽  
Glyphosate Tolerance ◽  
Agrobacterium Mediated Transformation

Since discovering Agrobacterium tumefaciens distinctive capacity to incorporate a specified part of their transfer-DNA (T-DNA) into eukaryotic cells, the bacteria were commonly used for crop transformation originally of dicotyledonous crops and subsequently of nearly all organisms. To achieve this, the tumor-inducing (Ti) plasmid was changed to extract phytohormone and opine biosynthetic proteins (cytokinin and auxin) so as not to interfere with ordinary morphological growth. Overall, the conversion mediated by Agrobacterium was easier, more effective and less costly relative to other technologies. It also results in insertions with small copy count. Tumor development in crops has also proved the susceptibility of explants from field-grown sugar beet crops to Agrobacterium tumefaciens. Early efforts by Agrobacterium tumefaciens to transform sugar beet were unsuccessful, primarily owing to inability to regenerate crops from stably modified callus or suspended cells. A genotype-independent method was defined under which cotyledonary explants of various sugar beet genotypes are inoculated with Agrobacterium tumefaciens comprising whether kanamycin tolerance and GUS activity or kanamycin resistance, GUS activity and glyphosate tolerance. GUS expression, NPT dot blot as well as EPSPS assays verified the presence of transgenes; progeny showed Mendelian genetically modified inheritance and glyphosate tolerance at deadly concentrations to control plants. Unfortunately, there was no publication of technical information of the technique. Here we reviewed the concept Agrobacterium-mediated transformation and how to be applicable

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