scholarly journals Agrobacterium tumefaciens mediated transformation of the aquatic carnivorous plant Utricularia gibba

2020 ◽  
Author(s):  
Araceli Oropeza-Aburto ◽  
Sergio Alan Cervantes-Perez ◽  
Victor A Albert ◽  
Luis Rafael Herrera-Estrella
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
In Vitro Selection ◽  
Carnivorous Plant ◽  
Carnivorous Plants ◽  
35S Promoter ◽  
Transformation Protocol ◽  
Utricularia Gibba

Abstract Background The genus Utricularia belongs to Lentibulariaceae, the largest family of carnivorous plants, which includes terrestrial, epiphytic and aquatic species. The development of specialized structures that evolved for carnivory is a feature of this genus that has been of great interest to biologists since Darwin‘s early studies. Utricularia gibba is itself an aquatic plant with sophisticated bladder traps having one of the most complex suction mechanisms for trapping prey. However, the molecular characterization of the mechanisms that regulate trap development and the biophysical processes involved in prey trapping are still largely unknown due to the lack of a simple and reproducible gene transfer system. Results Here, we report the establishment of a simple, fast and reproducible protocol for genetic transformation of U. gibba based on the T-DNA of Agrobacterium tumefaciens . An in vitro selection system using Phosphinotricin as a selective agent was established for U. gibba . Plant transformation was confirmed by histochemical GUS assays and PCR and qRT-PCR analyses. We report on the expression pattern of the 35S promoter and of the promoter of a trap-specific ribonuclease gene in transgenic U. gibba plants. Conclusions The genetic transformation protocol reported here is an effective method for studying developmental biology and functional genomics of this genus of carnivorous plants and advances the utility of U. gibba as a model system to study developmental processes involved in trap formation.

scholarly journals Agrobacterium tumefaciens mediated transformation of the aquatic carnivorous plant Utricularia gibba

2020 ◽  
Author(s):  
Araceli Oropeza-Aburto ◽  
Sergio Alan Cervantes-Perez ◽  
Victor A Albert ◽  
Luis Rafael Herrera-Estrella
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
In Vitro Selection ◽  
Carnivorous Plant ◽  
Carnivorous Plants ◽  
35S Promoter ◽  
Transformation Protocol ◽  
Utricularia Gibba

Abstract Background The genus Utricularia belongs to Lentibulariaceae, the largest family of carnivorous plants, which includes terrestrial, epiphytic and aquatic species. The development of specialized structures that evolved for carnivory is a feature of this genus that has been of great interest to biologists since Darwin‘s early studies. Utricularia gibba is itself an aquatic plant with sophisticated bladder traps having one of the most complex suction mechanisms for trapping prey. However, the molecular characterization of the mechanisms that regulate trap development and the biophysical processes involved in prey trapping are still largely unknown due to the lack of a simple and reproducible gene transfer system. Results Here, we report the establishment of a simple, fast and reproducible protocol for genetic transformation of U. gibba based on the T-DNA of Agrobacterium tumefaciens . An in vitro selection system using Phosphinotricin as a selective agent was established for U. gibba . Plant transformation was confirmed by histochemical GUS assays and PCR and qRT-PCR analyses. We report on the expression pattern of the 35S promoter and of the promoter of a trap-specific ribonuclease gene in transgenic U. gibba plants. Conclusions The genetic transformation protocol reported here is an effective method for studying developmental biology and functional genomics of this genus of carnivorous plants and advances the utility of U. gibba as a model system to study developmental processes involved in trap formation.

scholarly journals Agrobacterium tumefaciens mediated transformation of the aquatic carnivorous plant Utricularia gibba

2020 ◽  
Author(s):  
Araceli Oropeza-Aburto ◽  
Sergio Alan Cervantes-Perez ◽  
Luis Rafael Herrera-Estrella
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
In Vitro Selection ◽  
Carnivorous Plant ◽  
35S Promoter ◽  
Specific Expression ◽  
Transformation Protocol ◽  
Utricularia Gibba

Abstract Background Utricularia genus belongs to Lentibulariaceae family and has the highest number of species including terrestrials, epiphytes and aquatics plants. The development of specialized structures that evolved for carnivory is a feature of this genus that has been of great interest to biologist since the early studies of Darwin. Utricularia gibba is an aquatic carnivorous plant with sophisticated bladders traps that have one of the most complex suction mechanisms for trapping a prey. However, the molecular characterization of trap developmental and the biophysical processes involved in prey trapping are still largely unknown due to the lack of simple and reproducible gene transfer system for carnivorous plants.Results Here, we report the establishment of a simple, fast and effective protocol for the genetic transformation of U. gibba based on the T-DNA of Agrobacterium tumefaciens . An in vitro selection system using Phosphinotricin as selective agent was established for U. gibba. We report the tissue specific expression of the 35S promoter and the promoter of a trap specific ribonuclease gene. Plant transformation was confirmed by PCR and Real Time PCR in U. gibba plants.Conclusions We conclude that the genetic transformation protocol we developed is an effective method to study developmental biology and functional genomics of carnivory and propose U. gibba as model to study the developmental processes involved in trap formation.

scholarly journals In vitro plant regeneration and Agrobacterium-mediated genetic transformation of a carnivorous plant, Nepenthes mirabilis

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Sissi Miguel ◽  
Cindy Michel ◽  
Flore Biteau ◽  
Alain Hehn ◽  
Frédéric Bourgaud
Keyword(s):  
Genetic Transformation ◽  
Shoot Regeneration ◽  
Carnivorous Plant ◽  
Feeding Strategies ◽  
In Vitro Plant Regeneration ◽  
Rt Pcr ◽  
Transformation Protocol ◽  
Positive Method ◽  
Stem Segments

Abstract In nutrient-poor habitats, carnivorous plants have developed novel feeding strategies based on the capture and digestion of prey and the assimilation of prey-derived nutrients by specialized traps. The Nepenthes genus, comprising nearly 160 species, presents a remarkable pitcher-shaped trap, leading to great interest among biologists, but the species of this genus are listed as threatened. In this work, we developed a protocol for reproducing Nepenthes mirabilis through shoot regeneration from calli. The cultivation of stem segments of N. mirabilis on MS medium containing thidiazuron induced organogenic calli after 10 weeks. Subcultured calli exposed to 6-benzylaminopurine showed shoot regeneration in 3 weeks with considerable yields (143 shoots/g of calli). Excised shoots transferred to medium with indole-3-butyric acid allowed rooting in 4 weeks, and rooted plantlets had a 100% survival rate. Based on this method, we also developed an Agrobacterium-mediated genetic transformation protocol using calli as explants and ipt as a positive method of selection. Twelve weeks post infection, regenerated shoots were observed at the surface of calli. Their transgenic status was confirmed by PCR and RT-PCR. In conclusion, this study provides an efficient method for regenerating Nepenthes and the first protocol for its stable genetic transformation, a new tool for studying carnivory.

PENGEMBANGAN TEKNOLOGI SELEKSI IN VITRO BERULANG (REPEAT CYCLING–IN VITRO SELECTION) TOLERAN STRES KEKERINGAN UNTUK PERBAIKAN MUTU GENETIK DAN PRODUKSI TANAMAN KEDELAI BERMIKORIZA

2017 ◽  
Vol 1 (1) ◽  
pp. 74-84
Author(s):  
Ahmad Riduan ◽  
Rainiyati Rainiyati ◽  
Yulia Alia
Keyword(s):  
Arbuscular Mycorrhizae ◽  
In Vitro Selection ◽  
Soil Samples ◽  
Single Spore ◽  
Isolation And Characterization ◽  
Single Spore Culture ◽  
Spore Culture ◽  
Glomus Sp

Every plant rhizospheres in any ecosystem there are various living microorganisms including Arbuscular Mycorrhizae Fungi (AMF).  An isolation and characterization is required to investigate the species or type of the AMF. This research was aimed at studying the isolation and characterization of AMF sporulation in soybean rhizospheres in Jambi Province. The results of evaluation on soil samples before trapping showed that there are spores from three genus of AMF twelve types Glomus , two types Acaulospora and one type of Enthrophospora.  Following single spore culture in soybean rhizosphere, 5 spore types were obtained:  Glomus sp-1, Glomus sp-4, Glomus sp-7, Glomus sp-8 Glomus sp-10.

scholarly journals Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

2021 ◽  
Vol 9 (5) ◽  
pp. 1107
Author(s):  
Wonho Choi ◽  
Yoshihiro Yamaguchi ◽  
Ji-Young Park ◽  
Sang-Hyun Park ◽  
Hyeok-Won Lee ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Physiological Function ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Cell Growth Inhibition ◽  
The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

scholarly journals In Vitro Characterization of the Enzyme Properties of the Phospholipid N-Methyltransferase PmtA from Agrobacterium tumefaciens

2009 ◽  
Vol 191 (7) ◽  
pp. 2033-2041 ◽  
Author(s):  
Meriyem Aktas ◽  
Franz Narberhaus
Keyword(s):  
Agrobacterium Tumefaciens ◽  
In Vitro Assay ◽  
Enzyme Properties ◽  
Vitro Assay ◽  
Plant Infection ◽  
In Vitro Characterization ◽  
End Products ◽  
And Control

ABSTRACT Agrobacterium tumefaciens requires phosphatidylcholine (PC) in its membranes for plant infection. The phospholipid N-methyltransferase PmtA catalyzes all three transmethylation reactions of phosphatidylethanolamine (PE) to PC via the intermediates monomethylphosphatidylethanolamine (MMPE) and dimethylphosphatidylethanolamine (DMPE). The enzyme uses S-adenosylmethionine (SAM) as the methyl donor, converting it to S-adenosylhomocysteine (SAH). Little is known about the activity of bacterial Pmt enzymes, since PC biosynthesis in prokaryotes is rare. In this article, we present the purification and in vitro characterization of A. tumefaciens PmtA, which is a monomeric protein. It binds to PE, the intermediates MMPE and DMPE, the end product PC, and phosphatidylglycerol (PG) and phosphatidylinositol. Binding of the phospholipid substrates precedes binding of SAM. We used a coupled in vitro assay system to demonstrate the enzymatic activity of PmtA and to show that PmtA is inhibited by the end products PC and SAH and the antibiotic sinefungin. The presence of PG stimulates PmtA activity. Our study provides insights into the catalysis and control of a bacterial phospholipid N-methyltransferase.

scholarly journals In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor

2003 ◽  
Vol 77 (6) ◽  
pp. 3669-3679 ◽  
Author(s):  
Caterina Trozzi ◽  
Linda Bartholomew ◽  
Alessandra Ceccacci ◽  
Gabriella Biasiol ◽  
Laura Pacini ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Serine Protease ◽  
In Vitro Selection ◽  
Three Dimensional ◽  
Host Cells ◽  
Dimensional Structure ◽  
In Vitro Systems

ABSTRACT The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.

In Vitro Selection and Characterization of DNA Aptamers to a Small Molecule Target

2017 ◽  
Vol 9 (4) ◽  
pp. 233-268 ◽  
Author(s):  
Annamaria Ruscito ◽  
Erin M. McConnell ◽  
Anna Koudrina ◽  
Ranganathan Velu ◽  
Christopher Mattice ◽  
...  
Keyword(s):  
Small Molecule ◽  
In Vitro Selection ◽  
Dna Aptamers

In vitro Selection and Characterization of Drought Tolerant Somaclones of Tropical Maize (Zea mays L.)

2008 ◽  
Vol 7 (4) ◽  
pp. 641-650 ◽  
Author(s):  
J.M. Matheka ◽  
E. Magiri ◽  
A.O. Rasha ◽  
J. Machuka
Keyword(s):  
Zea Mays ◽  
In Vitro Selection ◽  
Zea Mays L ◽  
Tropical Maize ◽  
Drought Tolerant
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