scholarly journals Genetic Transformation of Peach Tissues by Particle Bombardment

1994 ◽  
Vol 119 (2) ◽  
pp. 367-373 ◽  
Author(s):  
Xiaojian Ye ◽  
Susan K. Brown ◽  
Ralph Scorza ◽  
John Cordts ◽  
John C. Sanford
Keyword(s):  
Fusion Gene ◽  
Gus Expression ◽  
Biolistic Transformation ◽  
Pcr Analysis ◽  
Nptii Gene ◽  
Embryonic Axes ◽  
Putative Transformants ◽  
Peach Genome ◽  
Callus Lines ◽  
Transgenic Callus

Physical and biological parameters affecting the efficiency of biolistic transformation of peach were optimized using ß-glucuronidase (GUS) as a reporter gene, such that efficiency of transient GUS expression in peach embryo-derived callus was increased markedly. Transient expression was also obtained in embryonic axes, immature embryos, cotyledons, shoot tips, and leaves of peach. Stable expression of a fusion gene combining neomycin phosphotransferase (NPTII) and ß-glucuronidase activities has been achieved in peach embryo calli. Sixty-five kanamycin-resistant callus lines were obtained from 114 pieces of bombarded calli after 4 months of selection. Nineteen of the 65 putative transformant lines produced shoot-like structures. Seven lines were examined to confirm stable transformation using the colorimetric GUS assay and PCR analysis. All seven lines showed GUS activity. PCR analysis confirmed that, in most of the putative transformants, the chimeric GUS/NPTII gene had been incorporated into the peach genome. The transgenic callus lines were very weakly morphogenic, presumably because the callus was 5 years old and no transgenic shoots developed from this callus. Results of this research demonstrate the feasibility of obtaining stable transgenic peach tissue by biolistic transformation.

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 Agrobacterium-Mediated Transformation of the RecalcitrantVandaKasem's Delight Orchid with Higher Efficiency

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Pavallekoodi Gnasekaran ◽  
Jessica Jeyanthi James Antony ◽  
Jasim Uddain ◽  
Sreeramanan Subramaniam
Keyword(s):  
Gene Expression ◽  
Genetic Transformation ◽  
Gus Expression ◽  
Pcr Analysis ◽  
Agrobacterium Mediated Transformation ◽  
Putative Transformants ◽  
Immersion Period ◽  
Cocultivation Medium

The presented study establishedAgrobacterium-mediated genetic transformation using protocorm-like bodies (PLBs) for the production of transgenicVandaKasem's Delight Tom Boykin (VKD) orchid. Several parameters such as PLB size, immersion period, level of wounding,Agrobacteriumdensity, cocultivation period, and concentration of acetosyringone were tested and quantified usinggusAgene expression to optimize the efficiency ofAgrobacterium-mediated genetic transformation of VKD's PLBs. Based on the results, 3-4 mm PLBs wounded by scalpel and immersed for 30 minutes inAgrobacteriumsuspension of 0.8 unit atA600nmproduced the highest GUS expression. Furthermore, cocultivating infected PLBs for 4 days in the dark on Vacin and Went cocultivation medium containing 200 𝜇M acetosyringone enhanced the GUS expression. PCR analysis of the putative transformants selected in the presence of 250 mg/L cefotaxime and 30 mg/L geneticin proved the presence ofwheatwin1,wheatwin2, andnptIIgenes.

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 Identification of Flower-Specific Promoters through Comparative Transcriptome Analysis in Brassica napus

2019 ◽  
Vol 20 (23) ◽  
pp. 5949 ◽  
Author(s):  
Yan Li ◽  
Caihua Dong ◽  
Ming Hu ◽  
Zetao Bai ◽  
Chaobo Tong ◽  
...  
Keyword(s):  
Brassica Napus ◽  
Candidate Genes ◽  
Oilseed Rape ◽  
Agronomic Traits ◽  
Expression System ◽  
Expression Patterns ◽  
Gus Expression ◽  
Pcr Analysis ◽  
Transcriptome Data ◽  
Sclerotinia Stem Rot

Brassica napus (oilseed rape) is an economically important oil crop worldwide. Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum is a threat to oilseed rape production. Because the flower petals play pivotal roles in the SSR disease cycle, it is useful to express the resistance-related genes specifically in flowers to hinder further infection with S. sclerotiorum. To screen flower-specific promoters, we first analyzed the transcriptome data from 12 different tissues of the B. napus line ZS11. In total, 249 flower-specific candidate genes with high expression in petals were identified, and the expression patterns of 30 candidate genes were verified by quantitative real-time transcription-PCR (qRT-PCR) analysis. Furthermore, two novel flower-specific promoters (FSP046 and FSP061 promoter) were identified, and the tissue specificity and continuous expression in petals were determined in transgenic Arabidopsis thaliana with fusing the promoters to β-glucuronidase (GUS)-reporter gene. GUS staining, transcript expression pattern, and GUS activity analysis indicated that FSP046 and FSP061 promoter were strictly flower-specific promoters, and FSP046 promoter had a stronger activity. The two promoters were further confirmed to be able to direct GUS expression in B. napus flowers using transient expression system. The transcriptome data and the flower-specific promoters screened in the present study will benefit fundamental research for improving the agronomic traits as well as disease and pest control in a tissue-specific manner.

scholarly journals Persistence of multipotent progenitors expressing AML1/ETO transcripts in long-term remission patients with t(8;21) acute myelogenous leukemia

Blood ◽  
1996 ◽  
Vol 87 (11) ◽  
pp. 4789-4796 ◽  
Author(s):  
T Miyamoto ◽  
K Nagafuji ◽  
K Akashi ◽  
M Harada ◽  
T Kyo ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Acute Myelogenous Leukemia ◽  
Fusion Gene ◽  
Phosphoglycerate Kinase ◽  
Myelogenous Leukemia ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Differentiation Potential ◽  
Acute Myelogenous

The leukemia-specific AML1/ETO fusion gene has been shown to be detected by reverse transcriptase polymerase chain reaction (RT-PCR) analysis in patients with t(8;21) acute myelogenous leukemia (AML) in long-term remission. In the present study, the AML1/ETO mRNA could be detected by RT-PCR in bone marrow (BM) and/or peripheral blood (PB) samples from all 18 patients who had been maintaining complete remission for 12 to 150 months (median, 45 months) following chemotherapy or PB stem cell transplantation (PBSCT), whereas it could not be detected in four patients who had been maintaining remission for more than 30 months following allogeneic BM transplantation (BMT). We surveyed the expression of AML1/ETO mRNA in clonogenic progenitors from BM in these cases. Notably, 51 of 2,469 colonies from clonogenic progenitors (2.1%) expressed the AML1/ETO mRNA in 18 cases who were RT- PCR+ in BM and/or PB samples. Expression was observed in various clonogenic progenitors, including granulocyte-macrophage colonies, mixed colonies, erythroid colonies, and megakaryocyte colonies. Furthermore, we analyzed the clonality of these progenitors by X- chromosome inactivation patterns of the phosphoglycerate kinase (PGK) gene in four female patients. The AML1/ETO mRNA+ progenitors showed the PGK allele identical to that detected in the leukemic blasts from the time of initial diagnosis. Normal constitutive hematopoiesis was sustained by polyclonal BM reconstitution in these patients. Accordingly, these committed progenitor cells that express AML1/ETO mRNA during remission likely have arisen from common t(8;21)+ pluripotent progenitor cells with at least trilineage differentiation potential. These data strongly suggest that the origin of the clonogenic leukemic progenitors of t(8;21) AML may be multipotent hematopoietic progenitors that acquired the t(8;21) chromosomal abnormality.

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)

Single-translocation and double-chimeric transcripts: detection of NUP98-HOXA9 in myeloid leukemias withHOXA11 or HOXA13 breaks of the chromosomal translocation t(7;11)(p15;p15)

Blood ◽  
2002 ◽  
Vol 99 (4) ◽  
pp. 1428-1433 ◽  
Author(s):  
Takashi Fujino ◽  
Akitaka Suzuki ◽  
Yoshikazu Ito ◽  
Kazuma Ohyashiki ◽  
Yoshiaki Hatano ◽  
...  
Keyword(s):  
Fusion Gene ◽  
Leukemia Cell ◽  
Chromosomal Translocation ◽  
Myelogenous Leukemia ◽  
Pcr Analysis ◽  
Chromosome 11 ◽  
Reverse Transcription Pcr ◽  
Class 1 ◽  
Chromosomal Breakpoints ◽  
Acute Myelogenous

It has been demonstrated that the chromosomal translocation t(7;11)(p15;p15) in patients with human acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) invariably involves fusion of the nucleoporin gene, NUP98, on chromosome 11 and the class 1 HOX gene, HOXA9, on chromosome 7, and that the fusion gene NUP98-HOXA9 is an important gene in myeloid leukemogenesis. Here are reported 2 novel chromosome 7p15 targets of the t(7;11)(p15;p15) chromosomal translocation in 2 patients with CML and myelodysplastic syndrome (MDS). Southern blot and polymerase chain reaction (PCR) analyses of leukemia cell DNA failed to show rearrangement of HOXA9,whereas NUP98 was found to be rearranged in both cases. Reverse transcription-PCR analysis using a NUP98 primer and a degenerate primer corresponding to the third helix of the homeodomain of HOXA demonstrated that NUP98 was fused in-frame to HOXA11 in the patient with CML and toHOXA13 in the patient with MDS. The chromosomal breakpoints on 7p15 were located within introns of HOXA11 orHOXA13 genes. In both patients chimericNUP98-HOXA9 transcripts were also observed. These findings suggest that AbdB-type HOXA genes are common targets of t(7;11)(p15;p15) chromosomal translocations and that a single translocation can produce more than oneNUP98-HOXA fusion gene, presumably because of altered splicing.

PML/RARα Mediates Resistance to Apoptosis and Down-Regulation of the NF-κB Pathway in Acute Promyelocytic Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4283-4283
Author(s):  
Zaida Garcia-Casado ◽  
Jose Cervera ◽  
Juan C. Pajuelo ◽  
Ana Valencia ◽  
Carlos Garcia ◽  
...  
Keyword(s):  
Acute Promyelocytic Leukemia ◽  
Fusion Gene ◽  
Chimeric Protein ◽  
Promyelocytic Leukemia ◽  
Pcr Analysis ◽  
Down Regulation ◽  
Bcl2 Family ◽  
Real Time Quantitative Pcr ◽  
Apoptotic Pathways ◽  
Wbc Count

Abstract Acute promyelocytic leukemia (APL) is genetically characterized by the translocation t(15;17) that results in the fusion gene PML/RARα. The chimeric protein renders hematopoietic progenitor cells resistant to FAS-, TNF- and IFN-induced apoptosis and caspase-3 activation, supporting a role for apoptosis impairment in the pathogenesis of APL. To better investigate apoptosis deregulation in APL, we analyzed the expression profile of 53 newly diagnosed APL patients (27M/26F; median age: 41 yr. (range: 9–80); median WBC count x 109/L: 2.67 (range: 0.5–128); median platelet count x 109/L: 19 (range: 4.4–135); FAB subtype: 39 M3/14 M3v) using the OncoChip®, a cDNA microarray especially designed for analyzing genes involved in cancer which contains 6,386 genes. After array processing, a total of 371 and 249 known genes were found to be ≥2 fold down- or up-regulated, respectively. To verify the results of microarray analysis, six differentially expressed genes (JAG1, JUN, CDKN1C, FAS, TRAIL, TRAF6 and MMP9) were tested by real-time quantitative PCR analysis (Q-RT-PCR). As initially hypothesized, numerous genes involved in apoptotic pathways were deregulated. In particular, we found a significant down-regulation of genes involved in the activation of NF-κB and of genes related to TNF-mediated apoptosis (FAS, TRAIL, TNFSF13B), with 46 and 24 genes deregulated, respectively. Expression changes in other genes implicated in apoptosis were also identified, being of special interest those affecting to the BCL2 family. Thus, anti-apoptotic genes BCL2 and BCL11A were up-regulated while pro-apoptotic members of the family were down-regulated in the analyzed series. Genes involved in the regulation of p53-dependent apoptosis (such as APAF1, p53DINP1, ATM), as well as numerous genes involved in diverse mechanisms of DNA repair, were also inhibited. Finally, the protein kinase C-δ (PKCδ) and several interferon- and interleukin-related genes were also infraexpressed in APL. These findings suggest that inactivation of apoptotic pathways is a common event in the pathogenesis of APL and may have important implications in the design of therapeutic protocols. It was of particular interest the unexpected inhibition of the NF-κB pathway. It could be in accordance with the hypothesis that NF-κB is an inductor of apoptosis under some circumstances. The role of NF-κB in promoting or repressing cell death in APL should be further investigated.

scholarly journals INCREASED SHOOT PROLIFERATION OF APPLES CO-CULTIVATED WITH AGROBACTERIUM TUMEFACIENS

HortScience ◽  
1992 ◽  
Vol 27 (6) ◽  
pp. 661a-661
Author(s):  
F.A. Hammerschlag ◽  
R.H. Zimmerman ◽  
A.C. Smigocki
Keyword(s):  
Shoot Proliferation ◽  
Shoot Formation ◽  
Pcr Analysis ◽  
Axillary Shoot ◽  
Putative Transformants ◽  
Proliferation Medium ◽  
Shoot Production ◽  
Dna Fragment ◽  
Different Levels

`McIntosh' apple shoots were inoculated in vitro with Agrobacterium tumefaciens strain tms328::Tn5 (tms) carrying a functional cytokinin gene. Callus tissue, removed from the infected stems, produced shoots on shoot proliferation medium. After three subcultures, axillary shoot production from a tms-infected putative transformant was eight times that of controls. Subsequent shoot production on three different levels of BA (3, 6 and 10 uM) was significantly greater than from controls on all levels of BA. PCR analysis of putative transformants revealed an expected 503 bp DNA fragment corresponding to the amplified portion of the cytokinin gene. After 6 months of in vitro propagation, proliferation rates of shoots obtained from the original transformants were similar to the controls and the expected PCR fragment of 503 bp could only be detected by Southern analysis. Even though the T-DNA appears to be lost from the apple genome, the data suggest that the tms strain may be useful in co-infection experiments to induce shoot formation, thus avoiding difficult regeneration procedures.

Sign in / Sign up

Export Citation Format

Share Document