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 Receiving of genetic-modified wheat plants with heterolous ornitin-δ-aminotransferase gene

2018 ◽  
Vol 22 ◽  
pp. 222-227
Author(s):  
O. M. Honcharuk ◽  
O. V. Dubrovna
Keyword(s):  
Triticum Aestivum ◽  
Transgenic Plants ◽  
Bread Wheat ◽  
Binary Vector ◽  
Triticum Aestivum L ◽  
Callus Cultures ◽  
Pcr Analysis ◽  
Genetic Construct ◽  
Agrobacterium Mediated Transformation

Aim. Receiving of genetically modified plants of bread wheat with heterologous ornithine‑δ‑aminotransferase gene. Methods. Agrobacterium-mediated transformation of callus cultures in vitro, PCR-analysis. Results. By Agrobacterium-mediated transformation of the morphogenic calluses of bread wheat (Triticum aestivum L.) using the AGLO strain containing the binary vector pBi-OAT with the target ornithine-δ-aminotransferase (oat) and selective neomycinphosphotransferase II (nptII), transgenic plants-regenerators have been obtained. Conclusions. As a result of the genetic transformation of Zimoyarka variety, 12 wheat regenerants were obtained in the genome which revealed a complete integration of the genetic construct containing the oat and nptII transgenes. Keywords: Triticum aestivum L., Agrobacterium-mediated transformation, ornithine‑δ‑aminotransferase gene, PCR-analysis.

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 In vitro Regeneration and Agrobacterium-mediated Transformation of Potato (Solanum tuberosum L.) Cultivars Grown in Mexico

2013 ◽  
Vol 22 (2) ◽  
pp. 93-105 ◽  
Author(s):  
Rose Onamu ◽  
Juan P Legaria ◽  
Jaime C Sahagún ◽  
José L Rodríguez ◽  
Joel N Pérez
Keyword(s):  
Solanum Tuberosum ◽  
Solanum Tuberosum L ◽  
In Vitro Regeneration ◽  
Potato Cultivars ◽  
Pcr Analysis ◽  
Nptii Gene ◽  
Histochemical Assay ◽  
Gus Histochemical Assay ◽  
Binary Plasmid

Prior to Agrobacterium-mediated genetic transformation in vitro regeneration protocol was established for three potato cultivars (Alfa, Cambray Rosa Morelos and Atlantic) grown in Mexico using leaf, node and internodal explants. Regeneration protocol was developed with or without the intervention of callus. Two potato cultivars, namely, Cambray Rosa Morelos and Alpha were transformed using Agrobacterium tumefaciens strain LBA4404 harboring binary plasmid pBI121 containing the GUS and nptII genes. GUS histochemical assay and PCR analysis were conducted on rooted shoots grown in media without hormones but supplemented with antibiotics. Transformed shoots tested positive through GUS histochemical assay and integration of nptII gene was confirmed by PCR analysis DOI: http://dx.doi.org/10.3329/ptcb.v22i2.14193 Plant Tissue Cult. & Biotech. 22(2): 93-105, 2012 (December)

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.

Genetic Transformation for Pod Borer Resistance in Dolichos Bean [Lablab purpureus (L.) Sweet]

2021 ◽  
Author(s):  
J.K. Kshirsagar ◽  
S.V. Sawardekar ◽  
S.G. Bhave ◽  
N.B. Gokhale ◽  
A.L. Narangalkar ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Transformation Frequency ◽  
Vector System ◽  
Pcr Analysis ◽  
Nptii Gene ◽  
Lablab Purpureus ◽  
Cry Genes ◽  
Mendelian Segregation ◽  
Pcr Screening ◽  
Progeny Analysis

Background: Agrobacterium mediated genetic transformation experiments were carried out in Dolichos bean Cv. (Konkan Bhushan) showing better regenerability. Methods: Three cry genes viz. cry1Aabc, cry1Fa1 and cry2Aa were used for the transformation each of which were linked to CaMV35S promoter and nptII gene under control of nos promoter and terminator. A vector system consisting of the disarmed hyper virulent Agrobacterium tumefaciens strain EHA-105 harboring pBinAR or BinBt3 was used. Mature embryo axis with single cotyledon was used as explant. Kanamycin as well as PCR screening was carried out to assess the transformation frequency. Progeny analysis using PCR was also carried out to assess the transgene segregation and stable transformation.Result: Kanamycin concentration of 500 mg/l was found as optimum for selection of a transgenic turning leaf blades albino. Among five methods of colonization used, the method employing mild injury to explant with dipping in Agrobacterium culture for 20 minutes followed by co-cultivation for 48 hours, cefotaxime washing and sowing in soil resulted in maximum survival (74.80%) associated with maximum transformation frequency through PCR analysis (2.13%). Among three cry genes, the gene cry2Aa was found the most effective in transforming Dolichos bean. The progeny analysis of transformants has shown the 3:1 mendelian segregation ratio confirming stable transformation of transgene.

scholarly journals Glyphosate selection of maize transgenic callus lines among genotypes of Ukrainian plant breeding

1970 ◽  
pp. 237-242
Author(s):  
I. O. Nitovska ◽  
I. K. Komarnytskyi ◽  
B. V. Morgun
Keyword(s):  
Plant Breeding ◽  
Transgenic Maize ◽  
Pcr Analysis ◽  
Agrobacterium Mediated Transformation ◽  
Maize Germplasm ◽  
Maize Transformation ◽  
Callus Lines ◽  
Selection Of ◽  
Maize Callus

Aim. Glyphosate selection has a number of advantages over other commonly used selectable markers for maize. There is some natural variability within maize germplasm for degree of sensitivity to glyphosate. We investigated the selective effect of glyphosate for production transgenic maize callus after Agrobacterium-mediated transformation among geno-types of Ukrainian plant breeding. Methods. Agrobacterium-mediated transformation, glyphosate selection in vitro, and PCR analysis were used to obtain transgenic maize callus and to confirm its status. Results. An efficient selectable marker system for production transgenic maize callus lines tolerant to herbicide glyphosate was proposed. Calluses of four maze genotypes of Ukrainian plant breeding and pCB135 vector containing CP4epsps gene were used in Agrobacterium-mediated transformation experiments. Three callus maize lines of DK267×PLS61 genotype containing CP4epsps gene were obtained. Conclusions. The use of glyphosate as a selective agent after Agrobacterium-mediated transformation proved to be effective for transgenic maize callus lines production containing the gene CP4epsps. The success of Agrobacterium-mediated transformation of maize callus strongly depended on the genotype of source ma-terial. Keywords: Agrobacterium-mediated maize transformation, CP4epsps gene, glyphosate selection, PCR analysis.

Adenosine Signaling in Priapism and Novel Therapies

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1006-1006
Author(s):  
Chen Ning ◽  
Jiaming Wen ◽  
Yujin Zhang ◽  
Harinder S. Juneja ◽  
Lin Qi ◽  
...  
Keyword(s):  
Animal Models ◽  
Conflicts Of Interest ◽  
Sexual Interest ◽  
Mrna Level ◽  
Marker Gene ◽  
Pcr Analysis ◽  
Enzyme Therapy ◽  
General Utility ◽  
Enos Mrna

Abstract Abstract 1006 Objective: Priapism is abnormal prolonged penile erection occurring without sexual interest. The condition is prevalent among men with sickle cell disease (SCD). Priapism is a urological emergency which needs early intervention to avoid the risks of penile fibrosis and eventual erectile dysfunction. Due to poorly understood pathogenesis of priapism no effective approaches to manage the disorder. Recent studies have revealed excess adenosine (Ado) in priapism via Ado A2B receptor (ADORA2B), suggesting novel therapeutic possibilities. Here, we aim to conduct preclinical studies to assess the efficacy and safety of Ado-based therapy in priapism. Materials and Methods: ADA-deficient mice (ADA−/−) and SCD Berkeley mice are two independent priapism animal models. We treated both mice with polyethylene glycol-modified ADA (PEG-ADA) to lower penile adenosine level or PSB1115, a selective ADORA2BR antagonist. The erectile function are measured by the changes of intracavernosal pressure (ICP) induced by cavernous nerve stimulation (CNS). Penile fibrosis is evaluated by histological studies and RT-PCR analysis of fibrotic marker gene. In vitro human microvascular endothelial cells (HMECs) were used to determine mechanism underlying ADORA2B-induced priapism. Results: Both ADA−/− and SCD mice with elevated Ado levels in penile tissue displayed priapic feature defined by prolonged and heightened erectile in response to CNS. Chronic reduction of accumulation of penile Ado levels by PEG-ADA enzyme therapy or PSB1115 corrected the priapic feature in both priapic animal models and further prevented progression of penile fibrosis. Significantly, both HIF-1α and eNOS mRNA level were elevated but reversed by PSB1115 treatment in penile tissues of ADA−/− mice and SCD mice. Finally, we provide in vitro direct evidence that PSB1115 treatment or siRNA knockdown HIF-1α in HMECs significantly reduced Ado-induced eNOS mRNA, implicating that ADORA2B-mediated HIF-1α induction contributes to elevated eNOS mRNA and underlies Ado-mediated priapism. Conclusions: PEG-ADA and PSB1115 are effective and safe to treat priapism and exacerbation of the disease by decreasing penile Ado levels or interfering its signaling. This study provides direct preclinical evidence for the novel and general utility of PEG-ADA enzyme therapy or ADORA2B antagonists for priapism and sets up a solid foundation for future clinical trials to assess the usefulness of Adobased therapeutics to treat priapism. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Deciphering the role of fungal calmodulin gene using Host-mediated gene silencing approach in apple

2021 ◽  
Author(s):  
Shalini Verma ◽  
Manju Modgil ◽  
Arjun Chauhan
Keyword(s):  
Gene Silencing ◽  
Transgene Expression ◽  
Selective Medium ◽  
Pcr Analysis ◽  
Nptii Gene ◽  
Calmodulin Gene ◽  
Putative Transformants ◽  
Marssonina Coronaria

Abstract Premature leaf fall caused by Marssonina coronaria is one of the most destructive diseases of apple in India. In this study, host induced gene silencing approach was exploited to develop resistance to this disease in an apple cultivar ‘Red Chief’. Calmodulin gene (CaM) having its role in fungal differentiation, development and pathogenicity was selected as target. hpRNAi construct was prepared from the conserved off target free partial gene sequence of CaM and used for transformation trials. Upto 6% kanamycin resistant shoots were obtained on selective medium having 5–6 mg/l kan after 7 weeks of coculture. In PCR analysis of 13 RNAi putative transformants, 10 lines were found positive with CaM and nptII gene specific primers and six lines showed hybridization signal. Semi qRT-PCR revealed variable levels of transgene expression among RNAi lines which seems to be related to copy number of integrated gene. In vitro detached leaf assay revealed lesion development and disease progression in wild type after 5 dpi but not visible in five CaM RNAi lines. Microscopic examination of infected control leaves showed fully developed, septate mycelium, and conidia along with necrosis of whole tissue while three transformants showed reduced growth and differentiation of fungus and in rest three, hyphal development and necrosis were strongly restricted. We conclude that trafficking of dsRNA/ siRNA from apple plants to pathogen might have triggered the down regulation of fungal CaM gene which confirms that deciphering the role of CaM through HIGS lead to resistance to Marssonina blotch in apple.

scholarly journals Micropropagation Cultures for Genetic Transformation of Grapevine

HortScience ◽  
2006 ◽  
Vol 41 (4) ◽  
pp. 972C-972
Author(s):  
Manjul Dutt ◽  
Dennis J. Gray ◽  
Zhijian T. Li ◽  
Sadanand Dhekney ◽  
Marilyn M. Van Aman
Keyword(s):  
Genetic Transformation ◽  
Fluorescent Protein ◽  
Marker Gene ◽  
In Vitro Cultures ◽  
35S Promoter ◽  
Nptii Gene ◽  
Embryogenic Cultures ◽  
Wide Range ◽  
Over Time

A major drawback to the use of embryogenic cultures for transformation of grapevine is that their ability to undergo genetic transformation is cultivar-dependent. Also, depending on cultivar, embryogenic cultures are difficult to impossible to maintain over time, reducing their utility for use in genetic transformation. An alternative to the use of embryogenic cultures for transformation of grapevine is the use of micropropagation cultures, which are easier to initiate from a wide range of grapevine cultivars and can be maintained over time without loss of function. Vitis vinifera `Thompson Seedless' was used as a model for genetic transformation using micropropagation cultures. In vitro cultures were initiated from apical meristems of actively growing vines and maintained in C2D medium containing 4 μM of 6-benzylaminopurine (C2D4B). Shoot tips and nodes were collected from proliferating in vitro cultures for transformation studies. A variety of wounding techniques, including nicking, sonication, and fragmenting of meristematic tissues was employed in order to enable Agrobacterium infection. We used a construct containing a bidirectional 35S promoter complex with a marker gene composed of a bifunctional fusion between an enhanced green fluorescent protein (EGFP) gene and a neomycin phosphotransferase (NPTII) gene in one direction and a hybrid lytic peptide gene in the other. Transgenic shoots growing in C2D4B medium containing 200 mg·L-1 each of carbenicillin and cefotaxime and 20 mg·L-1 of kanamycin were selected based on GFP fluorescence. Transgenic shoots were rooted and transferred to a greenhouse. To date, 18 transgenic lines have been generated. Details on the transformation procedure will be discussed.

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