There are three main methods to produce transgenic plants: microprojectile bombardment, electroporation, and Agrobacterium tumefaciens-mediated transformation. A. tumefaciens is a soil bacterium that causes crown gall disease in plants by transferring tumor-inducing (T-DNA) segments from its Ti plasmid into plant cells. The T-DNA can be modified to contain foreign genes and used to create transgenic plants by replacing tumor-causing genes with selectable markers like kanamycin resistance. However, Ti plasmids also have limitations for making transgenic plants, such as preventing regeneration due to phytohormone production and large plasmid size restricting cloning in E. coli.
An overview of the Agrobacterium-mediated gene transfer process. Moreover, studied different kinds of Agrobacterium species are involved in this mechanism.
Agrobacterium is a rod-shaped, Gram-negative bacteria found mostly in the soil. It is a plant pathogen that is responsible for causing crown gall disease in them. This bacteria is also known as the natural genetic engineer because of it's the ability to integrate its plasmid Gene into the plant genome.
Agrobacterium tumefaciens transfer of their genetic material T-DNA of Ti-plasmid into the plant cell: A: Agrobacterium tumefaciens; B: Agrobacterium genome; C: Ti Plasmid : a: T-DNA , b: Vir genes , c: Replication origin , d: Opines catabolism genes; D: Plant cell
A Ti-Plasmid (tumor-inducing plasmid) is a ds, circular DNA that often, but not always. It's a piece of genetic equipment that transfers genetic material from bacterial cells means Agrobacterium tumefaciens into plant cells used to induce tumors in the plant. The Ti-plasmid is damage when Agrobacterium is grown above 28 °C. Such cured bacteria don't induce crown gall disease in the plant due to they are avirulent. The Ti-Plasmid are classified into two types on the basis of opine genes are present in T-DNA.
The Plasmid has 196 genes that code for 195 proteins. There is no one structural RNA. The plasmid is 206.479 nucleotides long. the GC content is 56% and 81% of the genetic material is coding genes.
The modification of this plasmid is a very important source in the production of transgenic plants.
The T-DNA must be cut out of the circular plasmid. A VirD1/D2 complex nicks the DNA at the left and right border sequences. The VirD2 protein is covalently attached to the 5' end. VirD2 contains a motif that leads to the nucleoprotein complex being targeted to the type IV secretion system (T4SS).
In the cytoplasm of the recipient cell, the T-DNA complex becomes coated with VirE2 proteins, which are exported through the T4SS independently from the T-DNA complex. Nuclear localization signals, or NLS, located on the VirE2 and VirD2 are recognized by the importin alpha protein, which then associates with importin beta and the nuclear pore complex to transfer the T-DNA into the nucleus. So that the T-DNA can integrate into the host genome.
We inoculate Agrobacterium containing our genes of interest, onto wounded plant tissue explants. The Agrobacterium then transfers the gene of interest into the DNA of the plant tissue.
An overview of the Agrobacterium-mediated gene transfer process. Moreover, studied different kinds of Agrobacterium species are involved in this mechanism.
Agrobacterium is a rod-shaped, Gram-negative bacteria found mostly in the soil. It is a plant pathogen that is responsible for causing crown gall disease in them. This bacteria is also known as the natural genetic engineer because of it's the ability to integrate its plasmid Gene into the plant genome.
Agrobacterium tumefaciens transfer of their genetic material T-DNA of Ti-plasmid into the plant cell: A: Agrobacterium tumefaciens; B: Agrobacterium genome; C: Ti Plasmid : a: T-DNA , b: Vir genes , c: Replication origin , d: Opines catabolism genes; D: Plant cell
A Ti-Plasmid (tumor-inducing plasmid) is a ds, circular DNA that often, but not always. It's a piece of genetic equipment that transfers genetic material from bacterial cells means Agrobacterium tumefaciens into plant cells used to induce tumors in the plant. The Ti-plasmid is damage when Agrobacterium is grown above 28 °C. Such cured bacteria don't induce crown gall disease in the plant due to they are avirulent. The Ti-Plasmid are classified into two types on the basis of opine genes are present in T-DNA.
The Plasmid has 196 genes that code for 195 proteins. There is no one structural RNA. The plasmid is 206.479 nucleotides long. the GC content is 56% and 81% of the genetic material is coding genes.
The modification of this plasmid is a very important source in the production of transgenic plants.
The T-DNA must be cut out of the circular plasmid. A VirD1/D2 complex nicks the DNA at the left and right border sequences. The VirD2 protein is covalently attached to the 5' end. VirD2 contains a motif that leads to the nucleoprotein complex being targeted to the type IV secretion system (T4SS).
In the cytoplasm of the recipient cell, the T-DNA complex becomes coated with VirE2 proteins, which are exported through the T4SS independently from the T-DNA complex. Nuclear localization signals, or NLS, located on the VirE2 and VirD2 are recognized by the importin alpha protein, which then associates with importin beta and the nuclear pore complex to transfer the T-DNA into the nucleus. So that the T-DNA can integrate into the host genome.
We inoculate Agrobacterium containing our genes of interest, onto wounded plant tissue explants. The Agrobacterium then transfers the gene of interest into the DNA of the plant tissue.
description of Ti plasmid derivatives as vector and explanation of T DNA and cointegrate vectors and binary vectors and its uses and advantages and disadvantages
Introduction
Ti plasmid
Agrobacterium tumefaciens
Ti plasmid structure
Overview of infection process
Ti plasmid derived vector systems
Cointegrate vectors
Binary vectors
Agrobacterium mediated transformation of explants
Conclusions
References
It is a rod-shaped, Gram-negative, Peritrichous flagella, Soil bacterium. Agrobacterium is well known for its ability to transfer DNA between itself become an important tool for genetic engineering.
Dr.S.KARTHIKUMAR
Associate Professor
Department of Biotechnology
Kamaraj College of Engineering and Technology, K.Vellakulam-625701, TN, India
Email: skarthikumar@gmail.com
Agrobacterium mediated gene transfer, Ti-plasmid, cloning vectors based on Ti-plasmid, advantages disadvantages regarding cloning vectors based on Ti-plasmid are major areas covered in this Presentation.
This bacterium has a large plasmid that induces tumor, and for this reason, it was named tumor-inducing (Ti) plasmid.
This is process of altering the genetic makeup of an organism using Recombinant DNA Technology.
In this Presentation i have collected some basic information about that how Bacteria causes diseases in plants....
crown gall disease is discussed.
Contact 0311 9469029
description of Ti plasmid derivatives as vector and explanation of T DNA and cointegrate vectors and binary vectors and its uses and advantages and disadvantages
Introduction
Ti plasmid
Agrobacterium tumefaciens
Ti plasmid structure
Overview of infection process
Ti plasmid derived vector systems
Cointegrate vectors
Binary vectors
Agrobacterium mediated transformation of explants
Conclusions
References
It is a rod-shaped, Gram-negative, Peritrichous flagella, Soil bacterium. Agrobacterium is well known for its ability to transfer DNA between itself become an important tool for genetic engineering.
Dr.S.KARTHIKUMAR
Associate Professor
Department of Biotechnology
Kamaraj College of Engineering and Technology, K.Vellakulam-625701, TN, India
Email: skarthikumar@gmail.com
Agrobacterium mediated gene transfer, Ti-plasmid, cloning vectors based on Ti-plasmid, advantages disadvantages regarding cloning vectors based on Ti-plasmid are major areas covered in this Presentation.
This bacterium has a large plasmid that induces tumor, and for this reason, it was named tumor-inducing (Ti) plasmid.
This is process of altering the genetic makeup of an organism using Recombinant DNA Technology.
In this Presentation i have collected some basic information about that how Bacteria causes diseases in plants....
crown gall disease is discussed.
Contact 0311 9469029
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3. Methods of production of Transgenic
plants
• Microprojectile bombardment: shooting
DNA-coated tungsten or gold particles into
plant cells.
• Electroporation: use of a short burst of
electricity to put the DNA into cells.
• Agrobacterium tumefaciens-mediated
transformation
4. A. tumefaciens
• A. tumefaciens is a soil bacterium responsible
for crown gall disease of dicotyledonous
plants. The galls or tumors are formed at the
junction between the root and the stem of
infected plants.
7. • The ability of A. tumefaciens to induce crown galls in plants
is controlled by genetic information carried on Ti plasmid
(tumor-inducing plasmid).
• Ti plasmid has two components the T-DNA (Transferred
DNA) and
• the vir region, which are essential for the transformation of
plant cells.
• During the transformation process, the T-DNA is excised
from the Ti plasmid, transferred to a plant cell, and
integrated into the DNA of the plant cell.
• The integration of the T-DNA occurs at random
chromosomal sites. In some cases, multiple T-DNA
integration events occur in the same cell.
8.
9.
10. Structure of Ti Plasmid
• T DNA: In nopaline-type Ti plasmids the T-DNA is a
23,000-nucleotide-pair segment
• It carries 13 known genes including genes encoding
enzymes that catalyze the synthesis of phytohormones
(the auxin indoleacetic acid and the cytokinin
isopentenyl adenosine).
• These phytohormones are responsible for the
tumorous growth of cells in crown galls.
• The T-DNA region is bordered by 25-nucleotide-pair
imperfect repeats
• One of this must be present in cis for T-DNA excision
and transfer.
11. Structure of Ti Plasmid
The vir (virulence) region
• It contains the genes required for the T-DNA transfer
process.
• These genes encode the DNA processing enzymes
required for excision, transfer, and integration of the T-
DNA segment.
• They are expressed at very low levels in A. tumefaciens
cells growing in soil.
• Exposure of the bacteria to wounded plant cells or
exudates from plant cells induces enhanced levels of
expression of the vir genes.
• This induction process is very slow for bacteria, taking
10 to 15 hours to reach maximum levels of expression.
• Phenolic compounds such as acetosyringone act as
inducers of the vir genes.
12. Structure of the nopaline Ti plasmid pTi
C58, showing
ori, origin of replication; Tum, genes
responsible for tumor formation; Nos,
genes involved in nopaline biosynthesis;
Noc, genes involved in the catabolism of
nopaline; vir, virulence genes required
for T-DNA transfer. The nucleotide-pair
sequences of the left and right terminal
repeats are shown at the top; the
asterisks mark the four base pairs that
differ in the two border sequences
13.
14.
15.
16.
17. Ti plasmid vector for creating
transgenic plants
• Foreign genes could be inserted into the T-
DNA and then transferred to the plant.
• In the modified Ti plasmid the genes
responsible for tumor formation are deleted
• Selectable markeris added along with
appropriate regulatory elements.
18. Ti plasmid vector for creating
transgenic plants
• The kanr gene from the E. coli transposon Tn5 has been
used extensively as a selectable marker.
• it encodes an enzyme called neomycin
phosphotransferase type II (NPTII), it detoxify the
kanamycin.
• The NPTII coding sequence are provided with a plant
promoter and plant termination and polyadenylation
signals.
• Such constructions with prokaryotic coding sequences
flanked by eukaryotic regulatory sequences are called
chimeric selectable marker genes.
19. • One widely used chimeric selectable marker gene
contains the cauliflower mosaic virus (CaMV) 35S
promoter, the NPTII coding sequence, and the Ti
nopaline synthase (nos) termination sequence;
this chimeric gene is usually symbolized
35S/NPTII/nos.
• The Ti vectors used to transfer genes into plants
have the tumor-inducing genes of the plasmid
replaced with a chimeric selectable marker gene
such as 35S/NPTII/nos.
20.
21.
22. Limitations as routine Ti plasmid
vectors
• The production of phytohormones by transformed cells
prevents them from being regenerated into mature
plants.
• A gene encoding opine synthesis is not useful to a
transgenic plant and may lower the final plant yield
• Ti plasmids are large (approximately 200 to 800 kb).
• Ti plasmid does not replicate in Escherichia coli,
therefore it cannot be cloned in E. coli.
• Transfer of the T-DNA, which begins from the right
border, does not always end at the left border. Rather,
vector DNA sequences past the left border are often
transferred.
