scholarly journals “Floral-dip” transformation of Amaranthus caudatus L. and hybrids A. caudatus × A. paniculatus L.

Biologija ◽  
2019 ◽  
Vol 64 (4) ◽  
Author(s):  
Olha Yaroshko ◽  
Maksym Vasylenko ◽  
Alena Gajdošová ◽  
Bogdan Morgun ◽  
Olesia Khrystan ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Lethal Dose ◽  
Pcr Analysis ◽  
Gus Gene ◽  
Gene Vector ◽  
Amaranthus Caudatus ◽  
Herbicide Resistant ◽  
Floral Dip ◽  
Transgenic Seeds ◽  
Positive Results

After “floral-dip” transformation of Amaranth plants with Agrobacterium tumefaciens strain GV3101 carrying pCBV19 gene vector that contained bar and gus genes, transgenic seeds were obtained. The functioning of the tran+sferred genes in Amaranthus tissues was confirmed with herbicide selection (PPT herbicide – phospinotricin) and gus gene activity. Positive results were obtained for cultivars “Karmin” and “Kremoviy rannii”. The percentage of GUS positive samples was 1% (for “Karmin”), 2.2% (for “Kremoviy rannii”) from the total initial quantity of plants that was prior to selection with the herbicide. The seeds of six amaranth cultivars were received after treatment with A. tumefaciens by the method “floral dip”. The lowest lethal dose of herbicide PPT was established – 40 mg/l. After spraying with herbicide, resistant plants were obtained for cultivars: “Kremoviy rannii” (21%) and “Karmin” (20%). After conduction of PCR analysis, positive results were obtained for four cultivars. The percentage of bar positive plants was 0.3% (“Helios”); 0.26% (“Sterkch”); 0.06% (“Kremoviy rannii”); 0.3% (“Rushnichok”) from total initial quantity of plants.

scholarly journals EXPRESSION OF REPORTER GENES IN TRANSFORMED INDICA RICE THROUGH Agrobacterium MEDIATED METHOD

2007 ◽  
Vol 20 (1) ◽  
pp. 01-08
Author(s):  
Shamsul H. Prodhan ◽  
A. Komamine
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Oryza Sativa L ◽  
Indica Rice ◽  
Kanamycin Resistance ◽  
Pcr Analysis ◽  
Gus Gene ◽  
Immature Inflorescence ◽  
Seed Fertility ◽  
Selection Medium ◽  
Mature Seeds

Genetic transformation of rice (Oryza sativa L.) mediated by Agrobacterium tumefaciens has been confirmed for japonica varieties and extended to include more recalcitrant indica varieties. Scutellum-derived calli from mature seeds of Kasalath and BR-5 were used. The Agrobacterium tumefaciens strain, EHA101, harboring the binary vector pIG121Hm/Km/GUS was used for transformation. The vector contains b-glucuronidase (GUS) gene as a reporter gene and hygromycin resistance (HPT) as well as kanamycin resistance gene (NPTII) as selection genes in the T-DNA region. After co-cultivation with the bacteria, calli were inoculated on selection medium in which hygromycin concentration was 50 mg/l for Kasalath and 20 mg/l for BR-5. Carbenicillin (500 mg/l) was used for removal of Agrobacterium after co-cultivation. Inclusion of acetosyringone 50–100 mM in the Agrobacterium suspension in co-culture medium increased the frequency of transformation. Frequency of transformed calli (hygromycin resistant cells) was 82% in Kasalath and 6% in BR-5. Regeneration efficiency from transformed calli in Kasalath was about 63% and in BR-5 was about 34%. Most of the transgenic plants were morphologically normal but seed fertility was lower than the control. In transformed calli, roots and immature inflorescence showed positive response in GUS assay. Presence of GUS, HPT and NPTII genes was confirmed by PCR analysis and PCR Southern blot analysis. Expression of GUS gene was 100% in T1 progeny of Kasalath, whereas that of HPT gene was 51%. BR-5 could not be tested because of low seed fertility of T0 plants. In T1 plants, seed fertility of transformed Kasalath was 79% which is lower than that of the respective non-transformants.DOI: http://dx.doi.org/10.3329/bjpbg.v20i1.10633

scholarly journals Agrobacterium-Mediated Stable Transformation of Medicinal Plant Andrographis paniculata Callus Expressing β-glucuronidase (GUS) Gene

2015 ◽  
Vol 18 (2) ◽  
pp. 92
Author(s):  
Erly Marwani ◽  
Agustina Tangapo ◽  
Fenny Martha Dwivany
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Gus Expression ◽  
Selective Medium ◽  
Stable Transformation ◽  
Andrographis Paniculata ◽  
Pcr Analysis ◽  
Induction Medium ◽  
Gus Gene ◽  
Glucuronidase Activity ◽  
Leaf Disks

This study was carried out to establish a stable genetic transformation in callus culture of Andrographispaniculata mediated by Agrobacterium tumefaciens. The leaf disks of A. paniculata were infected with A. tumefaciensLBA4404 carrying a binary vector pCAMBIA1304 that contain β-glucuronidase (GUS) and hygromycinphosphotransferase (hpt) genes. The infection was conducted by dipping method for one hour, followed byco-cultivation in the dark for three days. To examine transient GUS expression, the co-cultivated leaf disks wereassayed for β-glucuronidase activity and to obtain stable transformed callus, the co-cultivated leaf disks wereselected on the callus induction medium which contain 20 mg/l hygromycin for selection. The transformedcallus was periodically subcultured every three weeks into the fresh selection medium over the 15 weeksperiod. To test a stable transformation, the callus was subjected to PCR analysis for GUS gene detection. Theresults indicated that the co-cultivated leaf disks expressed GUS activity and proliferated to produce callus onthe selective medium. Analysis of PCR on the transformed callus indicated the presence 976 bp fragment thatconfi rmed the presence of β-glucuronidase gene. These fi ndings imply that the β-glucuronidase was stably integrated into A. paniculata callus culture.Keywords: Andrographis paniculata, Agrobacterium tumefaciens, andrographollide, transformed callus,β-glucuronidase gene.

scholarly journals Agrobacterium-mediated Genetic Transformation of Two Varieties of Jute (Corchorus capsularis L.)

1970 ◽  
Vol 18 (1) ◽  
pp. 7-16 ◽  
Author(s):  
Rakha Hari Sarker ◽  
G.M. Al-Amin ◽  
Fathi Hassan ◽  
M.I. Hoque
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Plant Tissue ◽  
Genomic Dna ◽  
Mature Embryo ◽  
Gus Expression ◽  
Pcr Analysis ◽  
Gus Gene ◽  
Corchorus Capsularis ◽  
Selection Of

Transformation experiments were carried out using different explants of two varieties of white jute (Corchorus capsularis L.), namely, CVL-1 and CVE-3 with Agrobacterium tumefaciens strain (LBA4404/pBI121) containing the GUS and nptII genes. Maximum transformation ability was obtained from petiole-attached cotyledons and mature embryo explants. Kanamycin at a concentration of 200 mg/l was found optimum for selection of transformed shoots developed frommature embryos. Histochemical assay revealed the stable expression of the GUS gene within the various tissues of transformed plantlets. Stable integration of GUS and nptII genes were confirmed by PCR analysis of genomic DNA isolated from these transformed shoots. Key words: Jute, Transformation, GUS expression, PCR analysis D.O.I. 10.3329/ptcb.v18i1.3245 Plant Tissue Cult. & Biotech. 18(1): 7-16, 2008 (June)

scholarly journals Agrobacterium tumefaciens-mediated Transformation of Embryogenic Callus and Somatic Embryos of the Banana cv “Ambon Lumut” (Musa acuminata)

2017 ◽  
Vol 2 (6) ◽  
pp. 599 ◽  
Author(s):  
Tifa R. Kusumastuti ◽  
Rizkita R. Esyantia ◽  
Fenny M. Dwivany
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Embryogenic Callus ◽  
Somatic Embryos ◽  
Crop Improvement ◽  
Pcr Analysis ◽  
Abiotic Factor ◽  
Gfp Gene ◽  
Culture Transformation ◽  
Biotechnological Approach

Banana is one of the major fruit crops, though its conventional breeding has limitations, such as sterility and high polyploidy  levels.  Biotechnological  approach  using genetic  transformation  crop for improvement  offers  an alternative  solution.  In  this  study  a  protocol  was developed  for  establishing genetic  transformation  from embryogenic callus and somatic embryos of the banana cv Ambon Lumut . Embryogenic callus was obtained in ID4 medium (MS-based medium) supplemented with 1 mg L-1 IAA, 4 mg L-1 2,4D, and 0.03 g L-1 active charcoal. Embryogenic callus was transferred into liquid mediu m to establish somatic embryos. Embryogenic callus and somatic embryos were used for Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain A GL1, containing pART-TEST7 p lasmid with gfp gene as a reporter and CaM V35S as a promoter, was used for transformations. The embryogenic callus and somatic embryos were transformed using heat-shock method followed by centrifugation  (2000 rpm) and co-cult ivation in liquid medium containing acetosyringone (100 M) for 3 days. Results of the GFP analysis showed transient expression from gfp gene reporter in transformed embryogenic callus and somatic embryos. Transformation efficiency in somatic embryos (85,9%) was higher than  that in embryogenic callus (32.09%). PCR analysis using CaMV primer showed bands that compatible with CaMV35S promoter at 507 bp. This is a report showing establisment of embryogenic callus and somatic embryo culture transformation by using A. tumefaciens-mediated transformation protocol of the local banana cv Ambon Lumut. This study proved  the huge potential for genetic transformation of banana cv Ambon Lumut for crop improvement, such as pest or disease  resistance and abiotic factor stress tolerance. Keywords: banana; embryogenic callus; somatic embryos.

GUS Gene Transformation in Rice (Oryza sativa L.) Variety BRRI Dhan-30 Mediated by Agrobacterium tumefaciens

2008 ◽  
Vol 7 (3) ◽  
pp. 530-536 ◽  
Author(s):  
M. Asaduzzama ◽  
M.A. Bari ◽  
M. Rahman ◽  
M. Minami ◽  
K. Matsushima ◽  
...  
Keyword(s):  
Oryza Sativa ◽  
Agrobacterium Tumefaciens ◽  
Oryza Sativa L ◽  
Gene Transformation ◽  
Gus Gene

scholarly journals Piquiá Shells (Caryocar villosum): A Fruit by-Product with Antioxidant and Antiaging Properties in Caenorhabditis elegans

2020 ◽  
Vol 2020 ◽  
pp. 1-19 ◽  
Author(s):  
Mariana Roxo ◽  
Herbenya Peixoto ◽  
Pille Wetterauer ◽  
Emerson Lima ◽  
Michael Wink
Keyword(s):  
Caenorhabditis Elegans ◽  
Lethal Dose ◽  
Pcr Analysis ◽  
Transcriptional Level ◽  
Dependent Manner ◽  
Wild Type ◽  
Fluorescent Reporter ◽  
Triterpenoid Saponins ◽  
Promising Source

In a context of rising demand for sustainable antiaging interventions, fruit processing by-products are a promising source of bioactive compounds for the production of antiaging dietary supplements. Piquiá (Caryocar villosum) is a native Amazonian fruit consisting of 65% nonedible shells. In the present study, the phytochemical profile of a hydroalcoholic extract of piquiá shells (CV) was characterized by LC-MS/MS analysis. Its antioxidant and antiaging activities were investigated using the nematode Caenorhabditis elegans as an in vivo model. CV is mainly composed by hydrolysable tannins and triterpenoid saponins. The extract enhanced stress resistance of wild-type and mutant worms by reducing the intracellular levels of reactive oxygen species (ROS) and by increasing their survival against a lethal dose of the prooxidant juglone. These effects involved the upregulation of sod-3 and downregulation of gst-4 and hsp-16.2, studied through the GFP fluorescent reporter intensity and at the transcriptional level by qRT-PCR analysis. CV extended the lifespan of wild-type worms in a DAF-16/FoxO- and SKN-1/Nrf-dependent manner. Taken together, our findings indicate piquiá shells as potential candidates for nutraceutical applications. Further studies are needed to validate the relevance of our findings to antiaging interventions in humans.

scholarly journals Identifying Optimum Herbicide Mixtures to Manage and Avoid Fenoxaprop-p-Ethyl Resistant Phalaris minor in Wheat

2016 ◽  
Vol 34 (4) ◽  
pp. 787-794 ◽  
Author(s):  
T. ABBAS ◽  
M.A. NADEEM ◽  
A. TANVEER ◽  
R. AHMAD
Keyword(s):  
Grain Yield ◽  
Lethal Dose ◽  
Wheat Plant ◽  
Mixture Component ◽  
Herbicide Resistant ◽  
Phytotoxic Effects ◽  
Herbicide Mixtures ◽  
Susceptible Populations ◽  
Repeated Experiment ◽  
Different Doses

ABSTRACT Use of herbicide mixtures has been advocated as most effective strategy for avoidance and management of herbicide resistant weeds. Effect of twelve selected treatments of four herbicides (clodinafop-propargil, metribuzin, pinoxaden and sulfosulfuron) two-way mixtures at different doses was investigated against fenoxaprop-p-ethyl resistant and susceptible populations of P. minor grown along the wheat plants. In repeated experiment, herbicides mixtures were applied at 3 to 4 leaf stage of P. minor under greenhouse conditions. All the herbicide mixtures were effective to control resistant as well as susceptible P. minor. Mixtures having 75% lethal dose of each mixture component provided best control against P. minor. Mixtures with 50% lethal dose of each herbicide also provided more than 80% control of P. minor. Surviving P. minor plants after exposure to herbicide mixtures showed reduced growth and seed production potential. No mixture combination produced phytotoxic effects on wheat plant up to 75% of lethal dose of each mixture component. Mixtures including clodinafop-propargil + metribuzin, pinoxaden + sulfosulfuron and pinoxaden + metribuzin at 100% dose of each mixture component produced minor phytotoxic effects on wheat plants and caused no reduction in terms of ultimate growth and grain yield. However, mixture of sulfosulfuron + clodinafop-propargil at 100% dose of each component was phytotoxic to wheat and caused significant reduction in term of growth and grain yield. So, farmers can use these mixtures even at 75% of recommended dose of mixture component to control susceptible and resistant P. minor in wheat.

scholarly journals Application of sonication-assisted Agrobacterium-mediated transformation in Chenopodium rubrum L

2011 ◽  
Vol 49 (No. 6) ◽  
pp. 255-260 ◽  
Author(s):  
J.I. Flores Solís ◽  
P. Mlejnek ◽  
K. Studená ◽  
S. Procházka
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Transient Expression ◽  
Chenopodium Rubrum ◽  
Limiting Factors ◽  
Gus Gene ◽  
Efficient Tool ◽  
Factors Affecting ◽  
Agrobacterium Mediated Transformation ◽  
Glucuronidase Activity ◽  
Gene Coding

Chenopodium rubrum belongs to the plant species in which standard Agrobacterium-mediated transformation procedures remain inefficient. We demonstrate that the employment of sonication-assisted Agrobacterium-mediated transformation (SAAT) effectively enhanced transient expression of GUS gene coding for b-glucuronidase in Chenopodium rubrum. Further the results indicated that the age of seedlings is one of the limiting factors affecting the potency of Agrobacterium tumefaciens infection. Histochemical detection of b-glucuronidase activity revealed that two-days-old seedlings were much more susceptible to infection than ten-days-old ones. According to our results SAAT technology could provide an efficient tool for obtaining stable transformants when applied to two-days-old seedlings of Chenopodium rubrum.

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

scholarly journals Novel psbA1 Gene from a Naturally Occurring Atrazine-Resistant Cyanobacterial Isolate

2002 ◽  
Vol 68 (3) ◽  
pp. 1358-1366 ◽  
Author(s):  
Kannika Sajjaphan ◽  
Nir Shapir ◽  
Adam K. Judd ◽  
Lawrence P. Wackett ◽  
Michael J. Sadowsky
Keyword(s):  
Amino Acid ◽  
Oxygen Evolution ◽  
Selection Pressure ◽  
Pcr Analysis ◽  
Sequence Analyses ◽  
Conserved Residues ◽  
Herbicide Resistant ◽  
16S Ribosomal Dna ◽  
Naturally Occurring ◽  
Dimethyl Urea

ABSTRACT A naturally occurring atrazine-resistant cyanobacterial isolate, strain SG2, was isolated from an atrazine-containing wastewater treatment system at the Syngenta atrazine production facility in St. Gabriel, La. Strain SG2 was resistant to 1,000 μg of atrazine per ml but showed relatively low resistance to diuron [3-(3,4-dichlorophenyl)-1,1-dimethyl urea]. Analyses of 16S ribosomal DNA indicated that strain SG2 falls into the Synechocystis/Pleurocapsa/Microcystis group. Photosynthetically driven oxygen evolution in strain SG2 was only slightly inhibited (about 10%) by 2,000 μg of atrazine per ml, whereas in the control strain Synechocystis 6803, oxygen evolution was inhibited 90% by 1,000 μg of atrazine per ml. No atrazine accretion, mineralization, or metabolites were detected when strain SG2 was grown with [14C]atrazine. Strain SG2 contained three copies of the psbA gene, which encodes the D1 protein of the photosystem II reaction center. Nucleotide sequence analyses indicated that the psbA2 and psbA3 genes encoded predicted proteins with the same amino acid sequence. However, the psbA1 gene product contained five extra amino acids, which were not found in PsbA proteins from five other cyanobacteria. Moreover, the PsbA1 protein from strain SG2 had an additional 13 amino acid changes compared to the PsbA2/PsbA3 proteins and contained 10 amino acid alterations compared to conserved residues found in other cyanobacteria. Reverse transcriptase PCR analysis indicated that the psbA1 gene and the psbA2/psbA3 gene(s) were expressed in photosynthetically grown cells in the presence of atrazine. These results suggest that strong selection pressure conferred by the continual input of atrazine has contributed to the evolution of a herbicide-resistant, yet photosynthetically efficient, psbA gene in a cyanobacterium.

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