Agrobacterium tumefaciens is a tool for genetic engineering in plants. It is a soil bacterium that can transfer DNA fragments called T-DNA from its tumor-inducing plasmid into the genome of plant cells. The T-DNA can be modified to contain desirable genes for traits like herbicide or pest resistance. The bacterium recognizes wounded plant cells and transfers T-DNA using virulence genes. Integrated T-DNA is then expressed stably in the plant genome. Agrobacterium-mediated transformation is widely used for genetic engineering in plants due to its simplicity, efficiency and ability to transfer large DNA segments.
Agrobacterium mediated gene transfer in plants.ICHHA PURAK
This power point presentation consist of 41 slides. Attempts have been made to illustrate how Agrobacterium behaves us natural genetic engineer. How it can infect a plant through wound and a part of DNA present on Ti plasmid is Tranferred and causes disease as crown gall in the infected plant. In second part of the presentation attempts have been made to describe how Agrobacterium can be utilized for iinsertion of desired gene into the plant,what manipulation are to be made with Agrobacterium.How infection and transfer of desired gene can be made possible.What is the role of plant tissue culture etc.
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.
What are an expression vector? Detailed description of plant gene structure. Plant expression vector systems are generally consists of Ri and Ti plasmids.
The other vectors which are generally used are DNA and RNA viruses.
Agrobacterium mediated gene transfer in plants.ICHHA PURAK
This power point presentation consist of 41 slides. Attempts have been made to illustrate how Agrobacterium behaves us natural genetic engineer. How it can infect a plant through wound and a part of DNA present on Ti plasmid is Tranferred and causes disease as crown gall in the infected plant. In second part of the presentation attempts have been made to describe how Agrobacterium can be utilized for iinsertion of desired gene into the plant,what manipulation are to be made with Agrobacterium.How infection and transfer of desired gene can be made possible.What is the role of plant tissue culture etc.
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.
What are an expression vector? Detailed description of plant gene structure. Plant expression vector systems are generally consists of Ri and Ti plasmids.
The other vectors which are generally used are DNA and RNA viruses.
Introduction
Components of binary vector
Development of binary vector system
Properties of binary vector
Types of binary vector
Plant transformation using binary vector
Advantage of using binary vector
Conclusion
References
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
Agrobacterium tumifaciens
Horizontal gene transfer
Interkingdom gene transfer
Virulence or Vir a b c d e f g genes
Crown gall disease
Regulation of vir genes
Relaxosome
Presented by- MD JAKIR HOSSAIN
Doctoral Research Scholar
Department of Agricultural Genetic Engineering ,
Faculty of Agricultural Sciences and Technologies,
Nigde Omer Halisdemir University, Turkey
E. Mail- mjakirbotru@gmail.com
Introduction
Components of binary vector
Development of binary vector system
Properties of binary vector
Types of binary vector
Plant transformation using binary vector
Advantage of using binary vector
Conclusion
References
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
Agrobacterium tumifaciens
Horizontal gene transfer
Interkingdom gene transfer
Virulence or Vir a b c d e f g genes
Crown gall disease
Regulation of vir genes
Relaxosome
Presented by- MD JAKIR HOSSAIN
Doctoral Research Scholar
Department of Agricultural Genetic Engineering ,
Faculty of Agricultural Sciences and Technologies,
Nigde Omer Halisdemir University, Turkey
E. Mail- mjakirbotru@gmail.com
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The process of transfer, integration and expression of transgene in the host cells is known as genetic transformation. A foreign gene (transgene) encoding the trait must be incorporated into plant cells, along with a "cassette" of extra genetic material to add a desirable trait to a crop. The cassette includes a sequence of DNA called a "promoter", which determines where and when the foreign gene is expressed in the host, and a "marker gene" which allows breeders to determine by screening or selection which plants contain the inserted gene. For example, marker genes may make plants resistant to antibiotics not used routinely (e.g., agrimycin, kanamycin) or tolerant of some herbicides.
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Agrobacterium mediated gene transfer - LIKE NEVER BEFORE!!Shovan Das
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Agrobacterium tumefaciens as a tool for genetic engineering in plants
1. TOPIC: Agrobacterium tumefaciens as a tool for
genetic engineering in plants
By:
Sourabh Sharma
(14MBT003)
Dr. Samantha Vaishnavi
Prof. SBT, SMVDU
2. To increase enormous potential of plants for agriculture, producing more nutritious
and higher-yielding crops that are resistant to environmental stresses (cold and
drought), insect pests and diseases one has to introduce desired/foreign
/recombinant DNA into the plant`s genome.
It is well known that plants can propagate vegetatively and some plant cells are
totipotent, having the potentiality of regeneration and differention to give complete
plant. (Animal cells donot have this capacity)
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Agrobacterium mediated Gene Transfer in
plant
INTRODUCTION
4. Common Features of Agrobacterium tumefaciens
Soil borne, gram negative, rod shaped, motile found in rhizosphere.
Causative agents of “Crown gall” disease of dicotyledons.
Have ability to transfer bacterial genes to plant genome.
Attracted to wound site via chemotaxis in response to chemicals
(sugar and Phenolic molecules: acetosyringone) released from
damaged plant cells.
Contains Ti plasmid which can transfer its T-DNA region into genome
of host plants .
5. Tumor inducing (Ti) Plasmid
Vir region is organised into 8 Operons.
And has approx. 25 genes.
Vir region mediates the transfer of T-DNA to the plant genome.
Conjugal transfer reg.
25 bp repeat
25 bp repeat
6. Ti plasmid features…..
Two strains of Ti plasmid:
Octopine strains- contains two T-DNA regions TL (14 kb) and
TR ( 7 kb).
Nopaline strains- contain one T-DNA region(20 kb)
Size is about 200 kb.
Has a central role in ‘Crown-Gall’ formation.
Contains one or more T-DNA region that is integrated in the
genome of host plant.
Contains a vir region (40 kb) with atleast 8-11 kb vir
genes that transfers T-DNA to plant cell.
Has origin of replication.
Contains a region enabling conjugal transfer.
Has genes for the metabolism of opines.
7. Vir genes Functions
Vir A Kinase; Encodes acetosyringone receptor protein, also activates vir G by
phosphorylation leading to constitutive expression of all genes.
Vir B Encodes membrane protein, involved in conjugal tube formation through which
the T- DNA is transport.
Vir C Encodes Helicase enzymes; unwinding of T-DNA.
Vir D Topoisomerase activity; vir D2 is an endonuclease.
Vir E Single strand binding protein (SSBP); binds to T-DNA during transfer.
Vir F Activity not known.
Vir G Master controller DNA binding protein; vir A activates vir G by phosphorylation,
vir G dimerises and activates constitutive exp. of all vir genes.
Vir H Activity not known.
Vir genes and their
Functions....
9. Acetosyringone and α-hydroxyacetosyringone binds with Vir A
protein (located in the inner membrane) and activates it. It start
functioning as autokinase to phosphorylate itself by ATP.
Phosphorylated Vir A protein then phosphorylates Vir G protein which
then dimerises.
Phosphorylated Vir G protein has DNA binding function. It induces
expression of rest of Vir operons.
Vir D1 protein has topoisomerase and endonuclease activity.
It binds to right border sequence of T-DNA and facilitate the action of
Vir D2 protein which is also endonuclease and nicks at the right
border and remains bound to 5’end so generated.
4/29/2019 9
Agrobacterium mediated Gene Transfer in
plant
Mechanism of Transfer
10. The 3’end produced at the site of nick serves as a primer for DNA synthesis in 5’--
--3’ direction as a result of which one strand of
T-DNA is displaced from the DNA duplex.
The T-DNA strand is again nicked at the left border to generate a single strand
copy of T-DNA.
To this single strand copy Vir E 2 protein (single strand DNA binding proteins )
bind for its protection against exonucleases
Vir B operon consisting of 11 genes encode membrane bound Vir B proteins.
These along with Vir D4 proteins participate in conjugal tube formation between
bacterial and plant cells for transfer of T-DNA
Vir D2 which remains bound to 5’end of T DNA has a signal sequence which
drives it into the nucleus of plant cell.
4/29/2019 10
Agrobacterium mediated Gene Transfer in
plant
11. Generation of the T-strand.
overdrive
Right
BorderLeft Border
T-DNA
virD/virC
VirD nicks the lower strand (T-strand) at the right border
sequence and binds to the 5’ end.
5’
14. INTEGRATION OF T-DNA INTO PLANT GENOME
T-DNA enters plant cell as a single stranded structure which is immediately converted
into double stranded form.
Vir E2 also has nuclear localization sequence and is responsible for transfer of T DNA
into plant cell nucleus
Double stranded T-DNA integrate at random sites in the host plant genome.
For integration 23-79 base pair deletion takes place at the integration or target site
After integration of T-DNA into plant genome ,the genes for auxins, cytokinins and
opines express themselves which result in uncontrolled growth in the form of tumor
4/29/2019 14
Agrobacterium mediated Gene Transfer in
plant
16. HARNESSING AGROBACTERIUM IN TRANSFERRING FOREIGN
GENE INTO PLANT…….
Agrobacterium’s ability to introduce its DNA into plant genome with efficiency makes Ti
plasmid an attractive vector for gene transfer into plants.
T-DNA is modified as needed, before actual transfer of desired gene. Genetically
engineered Agrobacterium were produced with disarmed T-DNA region in which
oncogenes and opine biosynthesis genes were replaced by desired (Foreign ) gene,
selectable marker gene and a unique restriction site. The natural Ti plasmids are unsuitable
to be used directly as vectors for invitro manipulation due to following reasons;
1) Large size 2) Tumor induction (Oncogenic ) property 3)Absence of unique restriction
enzyme site.
Disarmed T-DNA , left and right borders along with genes of Vir region are essential
elements for designing of transformation vectors. The strategies for harnessing
Agrobacterium for introduction of new/desired/foreign genes into plants are; 16
17. Prototype disarmed Ti-vectors
T-DNA has been disarmed by making it nononcogenic by deleting all of its
oncogenes.
Zambryski et al. (1983) substituted pBR322 sequences for almost all of the T-
DNA of pTiC58, leaving only the left and right border regions and the nos gene
and resulting construct was called pGV3850.
Opine production was exploited as a screenable phenotype, and the ocs and nos
genes have been widely used as screenable markers.
To achieve high-level expression in plants, a very active promoter is required. In
dicots, promoters from the Agrobacterium nopaline synthase (nos), octopine
synthase (ocs) and mannopine synthase (mas) genes have been widely used.
Dominant selectable markers have been inserted into the T-DNA, so that
transformed plant cells can be easily selected on the basis of drug or herbicide
resistance.
21. Intermediate vectors have been widely used, the large cointegrates are not easy for
transformation. The vir-genes of the Ti plasmid function in trans and can act on any T-
DNA sequence present in the same cell.
Therefore, the vir genes and the disarmed T-DNA containing the transgene can be
supplied on separate plasmids, and this is the principle of binary vector systems
(Hoekma et al. 1983, Bevan 1984). The T-DNA can be subcloned on a small E. coli
plasmid for ease of manipulation.
This plasmid, called mini-Ti or micro-Ti, can be introduced into an Agrobacterium
strain carrying a Ti plasmid from which the T-DNA has been removed. The vir
functions are supplied in trans, causing transfer of the recombinant T-DNA to the plant
genome.
Binary Ti-Vectors
23. Glufosinate herbicide
Sethoxydim herbicide
Bromoxynil herbicide
Glyphosate herbicide
Sulphonylurea herbicide
Bt Gene
Modified Fatty Acid
Gene
Flower Colour
Flower Life
Delayed Fruit
Ripening Gene
Virus Resistance
Improved Genetic Traits in Plants using A. tumefaciens
r
r
r
r
r
24. TRANSFORMATION TECHNIQUE USING AGROBACTERIUM
•Agrobacterium gene transfer can be achieved in the following two
ways
Co culture with tissue explants.
In planta transfusion.
4/29/2019 24
Agrobacterium mediated Gene Transfer in
plant
25. Co- culture with tissue explants
For transformation explants like protoplast, callus , tissue slices, leaf disc,
stem or floral tissue etc. can be co-cultured with genetically engineered
Agrobacterium with recombinant vectors for about 2 days .
The explants can then be transferred to shoot inducing (regeneration
medium ) containing kanamycin and carbenicillin.
Kanamycin allows only transformed plant cells to divide and regenerate
shoots in about 3-4 weeks, while carbenicillin kills Agrobacterium cells.
The shoots are separated and transferred to root inducing medium and
finally after few weeks are transferred to soil.
4/29/2019 25
Agrobacterium mediated Gene Transfer in
plant
26.
27. In planta transformation
Transformation can also be achieved by imbibition of
seeds in fresh cultures of Agrobacterium. T-DNA
becomes integrated into plant genome. It appears that
Agrobacterium cells enter the seedling during
germination, are retained within the plants, when
flowers develop, the zygote or cells become
transformed.
28. Agrobacterium mediated gene transfer is most frequenly used indirect
method, it has many advantages;
Advantages of Agrobacterium mediated gene transfer
• Simple and comparatively less expensive.
• High transformation efficiency.
• Transgenic crops obtained have better fertility percentage.
• Protocols for both dicotyledons and monocotyledon are available.
• Relatively large length DNA segment can be transferred.
29. Disadvantages of Using
Agrobacterium as Vectors
• Agrobacterium can only be used to clone
genes in dicots like potato, tomato,
tobacco, peas and beans etc. but not
monocots like wheat, rice , barley
etc.which are most important crop
plants.due to ; lack of production of
Acetosyringone or α-
hydroxyacetosyringone like phenolics that
the bacteria appear to respond to simple
molecules, such as sugars and amino acids.
• Time consuming.