Recovery of Primary Transformants of Valencia-type Peanut Using Agrobacterium tumefaciens1

1994 ◽  
Vol 21 (2) ◽  
pp. 84-88 ◽  
Author(s):  
Ming Cheng ◽  
David C. H. Hsi ◽  
Gregory C. Phillips
Keyword(s):  
Genetic Transformation ◽  
Marker Gene ◽  
Cell Types ◽  
Kanamycin Resistance ◽  
Competent Cell ◽  
Agrobacterium Strain ◽  
Protein Coding ◽  
Gus Reporter ◽  
Different Strains ◽  
Primary Transformants

Abstract Four regenerable seedling explants of peanut cv. New Mexico Valencia A, two different strains of Agrobacterium tumefaciens, and two transformation protocols were used in peanut genetic transformation experiments. The putative transformation-competent cell types were identified by transient expression of the β-glucuronidase (GUS) reporter gene, and were compared to the regeneration-competent cell types identified histologically in the four explant systems. One primary transformant plantlet and two primary transformant shoots were recovered from petiolule-with-blade-attached explants inoculated with Agrobacterium strain CKS (A208:pTi37ASE X pEMZ) following a long cocultivation time on the regeneration medium and using low selection pressure for kanamycin resistance. The leaves of the primary transformants expressed nopaline accumulation used as a marker gene, and the engineered 35S-15kD zein protein coding sequence as determined by western blot. The results from these experiments may be useful for developing reliable methods of genetic transformation for valencia-type peanut.

scholarly journals Efficiency of Genetic Transformation via Pollen-Tube Pathway of Jatropha (Jatropha curcas L.) Based on Histochemical and Molecular Analysis

2018 ◽  
Vol 45 (3) ◽  
pp. 316
Author(s):  
Agus Zainudin ◽  
Bambang Sapta Purwoko ◽  
Tri Joko Santoso ◽  
Sintho Wahyuning Ardie ◽  
And Trikoesoemaningtyas
Keyword(s):  
Pollen Tube ◽  
Genetic Transformation ◽  
Molecular Analysis ◽  
Plasmid Dna ◽  
Jatropha Curcas ◽  
Marker Gene ◽  
Pcr Analysis ◽  
Jatropha Curcas L ◽  
Gus Reporter ◽  
Combination Treatments

The genetic transformation via pollen-tube pathway is an alternative method to overcome the constraints imposed by genotype specificity in transformation and regeneration in jatropha (Jatropha curcas L.) tissue culture. Therefore, it is necessary to establish important parameters for efficient genetic transformation of jatropha via pollen-tube pathway. The objective of the research was to study the efficiency of direct transformation of jatropha via pollen-tube pathway based on histochemical and molecular analysis. Solution of purified pCAMBIA1301 DNA plasmid carrying a hptII marker gene and a gus reporter gene with concentration level of 0.05, 0.25, 0.50 µg µl-1 were applied to stigma of flowers at 1, 2, 4, 7, 10 h after pollination. Seedling of IP3A, IP3P and JcUMM18 jatropha’s genotypes derived from 15 combination treatments of plasmid DNA concentration and application time, also wild type was subjected to histochemical and molecular analyses. Based on those analyses, the efficiency of transformation via pollen-tube pathway of three jatropha genotypes ranged from 1.5-16.7%. PCR analysis showed that a number of positive plants were identified by using specific primers hptII and gus, i.e. 1-3 and 3-7 plants of the 15 combined treatments, respectively. It indicated that the transformation efficiency via the pollen-tube pathway varied in each jatropha genotype.<br /><br />Keywords: Jatropha curcas L., pCAMBIA1301, plasmid DNA, stigma-drip<br /><br />

scholarly journals Agrobacterium-mediated Genetic Transformation of Potato (Solanum tuberosum L.) var. Cardinal and Heera

2012 ◽  
Vol 10 (1) ◽  
pp. 81-86 ◽  
Author(s):  
A. Khatun ◽  
M. M. Hasan ◽  
M. A. A. Bachchu ◽  
M. Moniruzzaman ◽  
K. M. Nasiruddin
Keyword(s):  
Genetic Transformation ◽  
Selectable Marker ◽  
Binary Vector ◽  
Marker Gene ◽  
Kanamycin Resistance ◽  
Histochemical Analysis ◽  
Gene Encoding ◽  
Antisense Gene ◽  
Npt Ii ◽  
Transformation Experiment

Two potato varieties namely Cardinal and Heera were used in the Agrobacterium-mediated genetic transformation experiment to investigate the genetic transformation ability in the Biotechnology laboratory of the Department of Biotechnology, Bangladesh Agricultural University, Mymensingh, Bangladesh during 2006 to 2007. Agrobacterium tumefaciens strain LBA 4404 having a binary vector pB1121 of 14 KDa containing selectable marker gene npt II (neomycine phosphotransferase II) conferring kanamycin resistance, and the CIPK antisense gene encoding calcineurin B-like protein were used. Leaf and internodes were used as explants. Expression of the transgene (GUS) was confirmed by histochemical analysis. The variety Cardinal was found more suitable for expressing best GUS response (80% GUS positive) over Heera.DOI: http://dx.doi.org/10.3329/agric.v10i1.11068The Agriculturists 2012; 10(1): 81-86

scholarly journals Genetic transformation of Populus nigra X P. deltoides (black poplar, clone Gradizka)

2016 ◽  
Vol 14 (2) ◽  
pp. 187-191
Author(s):  
N. K. Kutsokon ◽  
V. A. Rudas ◽  
M. V. Shinkaruk ◽  
O. R. Lakhneko ◽  
B. V. Morgun ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Marker Gene ◽  
Kanamycin Resistance ◽  
Populus Nigra ◽  
Chain Reaction ◽  
Pcr Product ◽  
Gus Gene Expression ◽  
Commercially Important ◽  
Polymerase Chain ◽  
Gene Nptii

Aim. To carry out genetic transformation of poplar Populus nigra x P. deltoides clone Gradizka with the model gene construct pCB002 carrying selective gene of kanamycin resistance and marker gene of β-glucuronidase. Methods. Genetic transformation was performed with the using leaf, stem and petiole poplar explants. Transformants were selected on the medium with kanamycin, and transgene was identified by polymerase chain reaction (PCR) and histochemical GUS assay. Results. Successful transformants selected on kanamycin media were confirmed by the presence of PCR-product for the gene nptII with the length 700 bp, and gus gene expression was also observed. Conclusions. Protocol for genetic transformation of P. nigra x P. deltoides clone Gradizka established here will be used for poplar genetic modification to create new clones with commercially important traits. Keywords: genetic transformation, Populus sp., microclonal propagation.

Polyester Cloth–Based Hybridization Array System for Identification of Enterohemorrhagic Escherichia coli Serogroups O26, O45, O103, O111, O121, O145, and O157

2012 ◽  
Vol 75 (9) ◽  
pp. 1691-1697 ◽  
Author(s):  
BURTON W. BLAIS ◽  
MARTINE GAUTHIER ◽  
MYLÈNE DESCHÊNES ◽  
GEORGE HUSZCZYNSKI
Keyword(s):  
Escherichia Coli ◽  
Marker Gene ◽  
Enterohemorrhagic Escherichia Coli ◽  
Oligonucleotide Probes ◽  
E Coli ◽  
Polyester Cloth ◽  
Gene Profiles ◽  
Pcr Products ◽  
Different Strains ◽  
Immunoenzymatic Assay

A cloth-based hybridization array system (CHAS) was developed for the identification of foodborne colony isolates of seven priority enterohemorrhagic Escherichia coli (EHEC-7) serogroups targeted by U.S. food inspection programs. Gene sequences associated with intimin; Shiga-like toxins 1 and 2; and the antigenic markers O26, O45, O103, O111, O121, O145, and O157 were amplified in a multiplex PCR incorporating a digoxigenin label, and detected by hybridization of the PCR products with an array of specific oligonucleotide probes immobilized on a polyester cloth support, with subsequent immunoenzymatic assay of the captured amplicons. The EHEC-7 CHAS exhibited 100% inclusivity and 100% exclusivity characteristics with respect to detection of the various markers among 89 different E. coli strains, with various marker gene profiles and 15 different strains of non–E. coli bacteria.

scholarly journals Characteristics of the promoters derived from the single-stranded DNA components of Milk vetch dwarf virus in transgenic tobacco

2005 ◽  
Vol 86 (6) ◽  
pp. 1851-1860 ◽  
Author(s):  
Naomi Shirasawa-Seo ◽  
Yoshitaka Sano ◽  
Shigeo Nakamura ◽  
Taka Murakami ◽  
Shigemi Seo ◽  
...  
Keyword(s):  
Transgenic Tobacco ◽  
Cell Types ◽  
Gus Expression ◽  
Cauliflower Mosaic Virus ◽  
Dwarf Virus ◽  
Promoter Regions ◽  
Meristematic Tissue ◽  
Link Protein ◽  
Gus Reporter ◽  
Milk Vetch Dwarf Virus

Predicted promoter regions of Milk vetch dwarf virus (MDV) components (C1–C11) were isolated and fused with a β-glucuronidase (GUS) reporter gene and the characteristics of the promoters were examined. In transgenic tobacco calli, promoters of MDV C4 (encoding a cell-cycle link protein), C5 and C7 (both encoding unknown proteins), C6 (encoding a nuclear-shuttle protein) and C8 (encoding a movement protein) generated a stronger level of GUS expression than the Cauliflower mosaic virus 35S RNA promoter (P35S). In leaves of transgenic tobacco plants, the promoters of C5 and C8 conferred a level of GUS activity comparable to that of P35S. Histochemical GUS analysis showed that the promoters of C4–C9, the latter encoding a capsid protein, were active in phloem and meristematic tissue. The promoter of C8 was also active in mesophyll and cortex cell types. A low level of activity was found for the promoters of C11, which encodes a master replication-initiator protein (Rep), and C1, C2, C3 and C10, which encode additional Reps, in both transgenic tobacco calli and plants.

scholarly journals Detection of Oil Palm Root Penetration by Agrobacterium-Mediated Transformed Ganoderma boninense, Expressing Green Fluorescent Protein

2017 ◽  
Vol 107 (4) ◽  
pp. 483-490 ◽  
Author(s):  
Nisha Govender ◽  
Mui-Yun Wong
Keyword(s):  
Oil Palm ◽  
Fluorescent Protein ◽  
Fusion Gene ◽  
Early Stage ◽  
Marker Gene ◽  
Induction Medium ◽  
Root Penetration ◽  
Ganoderma Boninense ◽  
Agrobacterium Strain ◽  
Green Fluorescent

A highly efficient and reproducible Agrobacterium-mediated transformation protocol for Ganoderma boninense was developed to facilitate observation of the early stage infection of basal stem rot (BSR). The method was proven amenable to different explants (basidiospore, protoplast, and mycelium) of G. boninense. The transformation efficiency was highest (62%) under a treatment combination of protoplast explant and Agrobacterium strain LBA4404, with successful expression of an hyg marker gene and gus-gfp fusion gene under the control of heterologous p416 glyceraldehyde 3-phosphate dehydrogenase promoter. Optimal transformation conditions included a 1:100 Agrobacterium/explant ratio, induction of Agrobacterium virulence genes in the presence of 250 μm acetosyringone, co-cultivation at 22°C for 2 days on nitrocellulose membrane overlaid on an induction medium, and regeneration of transformants on potato glucose agar prepared with 0.6 M sucrose and 20 mM phosphate buffer. Evaluated transformants were able to infect root tissues of oil palm plantlets with needle-like microhyphae during the penetration event. The availability of this model pathogen system for BSR may lead to a better understanding of the pathogenicity factors associated with G. boninense penetration into oil palm roots.

scholarly journals Identifying inaccuracies in gene expression estimates from unstranded RNA-seq data

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Mikhail Pomaznoy ◽  
Ashu Sethi ◽  
Jason Greenbaum ◽  
Bjoern Peters
Keyword(s):  
Gene Expression ◽  
Differential Expression Analysis ◽  
Cell Types ◽  
Library Preparation ◽  
Rna Seq ◽  
Protein Coding ◽  
Protein Coding Genes ◽  
Machine Learning Model ◽  
Specific Manner ◽  
Library Preparation Protocol

Abstract RNA-seq methods are widely utilized for transcriptomic profiling of biological samples. However, there are known caveats of this technology which can skew the gene expression estimates. Specifically, if the library preparation protocol does not retain RNA strand information then some genes can be erroneously quantitated. Although strand-specific protocols have been established, a significant portion of RNA-seq data is generated in non-strand-specific manner. We used a comprehensive stranded RNA-seq dataset of 15 blood cell types to identify genes for which expression would be erroneously estimated if strand information was not available. We found that about 10% of all genes and 2.5% of protein coding genes have a two-fold or higher difference in estimated expression when strand information of the reads was ignored. We used parameters of read alignments of these genes to construct a machine learning model that can identify which genes in an unstranded dataset might have incorrect expression estimates and which ones do not. We also show that differential expression analysis of genes with biased expression estimates in unstranded read data can be recovered by limiting the reads considered to those which span exonic boundaries. The resulting approach is implemented as a package available at https://github.com/mikpom/uslcount.

scholarly journals DualSeqDB: the host–pathogen dual RNA sequencing database for infection processes

2020 ◽  
Vol 49 (D1) ◽  
pp. D687-D693
Author(s):  
Javier Macho Rendón ◽  
Benjamin Lang ◽  
Marc Ramos Llorens ◽  
Gian Gaetano Tartaglia ◽  
Marc Torrent Burgas
Keyword(s):  
Pathogenic Bacteria ◽  
High Throughput Sequencing ◽  
Homo Sapiens ◽  
Cell Types ◽  
Natural Hosts ◽  
Host Infection ◽  
Infection Processes ◽  
Different Strains

Abstract Despite antibiotic resistance being a matter of growing concern worldwide, the bacterial mechanisms of pathogenesis remain underexplored, restraining our ability to develop new antimicrobials. The rise of high-throughput sequencing technology has made available a massive amount of transcriptomic data that could help elucidate the mechanisms underlying bacterial infection. Here, we introduce the DualSeqDB database, a resource that helps the identification of gene transcriptional changes in both pathogenic bacteria and their natural hosts upon infection. DualSeqDB comprises nearly 300 000 entries from eight different studies, with information on bacterial and host differential gene expression under in vivo and in vitro conditions. Expression data values were calculated entirely from raw data and analyzed through a standardized pipeline to ensure consistency between different studies. It includes information on seven different strains of pathogenic bacteria and a variety of cell types and tissues in Homo sapiens, Mus musculus and Macaca fascicularis at different time points. We envisage that DualSeqDB can help the research community in the systematic characterization of genes involved in host infection and help the development and tailoring of new molecules against infectious diseases. DualSeqDB is freely available at http://www.tartaglialab.com/dualseq.

scholarly journals Agrobacterium-mediated genetic transformation of two varieties of Brassica: optimization of protocol

1970 ◽  
Vol 34 (2) ◽  
pp. 287-301 ◽  
Author(s):  
MMA Khan ◽  
ABMAHK Robin ◽  
MAN Nazim-Ud-Dowla ◽  
SK Talukder ◽  
L Hassan
Keyword(s):  
Brassica Napus ◽  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Brassica Rapa ◽  
Binary Vector ◽  
Gus Assay ◽  
Key Words Transformation ◽  
Gus Reporter ◽  
Best Response ◽  
Transformation Experiment

 Two rapeseed varieties, namely Tori-7 and BARI Sarisha-8, respectively, from Brassica rapa and Brassica napus were selected to observe the transformation ability. Petioles were inoculated in Agrobacterium tumefaciens strain LBA 4404 carrying a binary vector pBl2l with GUS (reporter) and nptII (kanamycin resistant) gene. The transformation experiment was performed by optimizing two important factors: preculture time and co-cultivation time and also selected out the best variety. Infection was most effective when explants were pre-cultured for 72 hours (80% GUS positive). and co-cultivated for 72 hours (72% GUS positive). The variety Tori-7 showed the best response to GUS assay (65% GUS positive). Callus induction was the highest in Tori-7, which were 6% with 72 hours of preculture period and 9% in 48 hours of co-cultivation. Number of putative transformed plantlets were highest in Tori-7 (7 plants) followed by BARI Sarisha-8 (3 plants). Key words: Transformation; Brassica; GUS; Agrobacterium. DOI: 10.3329/bjar.v34i2.5802Bangladesh J. Agril. Res. 34(2): 287-301, June 2009

scholarly journals Cell-type-specific analysis of alternative polyadenylation using single-cell transcriptomics data

2019 ◽  
Vol 47 (19) ◽  
pp. 10027-10039 ◽  
Author(s):  
Eldad David Shulman ◽  
Ran Elkon
Keyword(s):  
Gene Regulation ◽  
Single Cell ◽  
Alternative Polyadenylation ◽  
Cell Types ◽  
Protein Coding ◽  
Multiple Datasets ◽  
Transcriptomics Data ◽  
Cell Type Specific ◽  
Transcriptional Gene Regulation ◽  
Different Cell Types

AbstractAlternative polyadenylation (APA) is emerging as an important layer of gene regulation because the majority of mammalian protein-coding genes contain multiple polyadenylation (pA) sites in their 3′ UTR. By alteration of 3′ UTR length, APA can considerably affect post-transcriptional gene regulation. Yet, our understanding of APA remains rudimentary. Novel single-cell RNA sequencing (scRNA-seq) techniques allow molecular characterization of different cell types to an unprecedented degree. Notably, the most popular scRNA-seq protocols specifically sequence the 3′ end of transcripts. Building on this property, we implemented a method for analysing patterns of APA regulation from such data. Analyzing multiple datasets from diverse tissues, we identified widespread modulation of APA in different cell types resulting in global 3′ UTR shortening/lengthening and enhanced cleavage at intronic pA sites. Our results provide a proof-of-concept demonstration that the huge volume of scRNA-seq data that accumulates in the public domain offers a unique resource for the exploration of APA based on a very broad collection of cell types and biological conditions.

Sign in / Sign up

Export Citation Format

Share Document