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HBC 410 Agrobacterium Mediated Transformation

The document summarizes the process of plant transformation using Agrobacterium-mediated transformation. It describes that Agrobacterium tumefaciens is used as a vector to transfer foreign DNA into plant genomes. The mechanism involves T-DNA from the tumor-inducing plasmid being transferred into the plant cell and integrating into the plant nuclear genome. Modifications were made to the wild type Ti plasmid to create disarmed vectors like co-integrate and binary vectors that are easier to use. The process involves co-cultivation of plant cells with A. tumefaciens, selection of transformed tissue, and regeneration of transgenic plants.

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Arthur Derman Sith-lee
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59 views20 pages

HBC 410 Agrobacterium Mediated Transformation

The document summarizes the process of plant transformation using Agrobacterium-mediated transformation. It describes that Agrobacterium tumefaciens is used as a vector to transfer foreign DNA into plant genomes. The mechanism involves T-DNA from the tumor-inducing plasmid being transferred into the plant cell and integrating into the plant nuclear genome. Modifications were made to the wild type Ti plasmid to create disarmed vectors like co-integrate and binary vectors that are easier to use. The process involves co-cultivation of plant cells with A. tumefaciens, selection of transformed tissue, and regeneration of transgenic plants.

Uploaded by

Arthur Derman Sith-lee
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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HBC 410

Plant Transformation:
Agrobacterium- mediated
Ms. E. Chirisa
Plant transformation
• “modifying the genomes of plants through
genetic engineering techniques by either adding
a foreign gene from another species (or from
another kingdom) or by removing of a certain
detrimental gene to achieve a desired outcome”
Calcium phosphate
Chemical methods Liposome fusion
PEG

Electroporation
Plant transformation Physical methods
Biolistic
methods Microinjection

Agrobacterium tumefaciens
Biological methods
Virus mediated
Agrobacterium tumefaciens Crown gall disease
A. tumefaciens
• Soil borne, Gram –ve, motile bacillus
• Wounded plants are susceptible
• Plants produce sugar and acetosyringone (phenolic compound) that
attracts bacterium by chemotaxis
• Bacterial genes are transferred to the plant genome via transfer DNA
(T-DNA) from the tumor inducing (Ti) plasmid
• Causes crown gall (tumors) disease in dicotyledons due to the
presence of tumor inducing genes (oncogenes)
A. tumefaciens Mechanism of infection
Agrobacterium as a transformation vector
• Agrobacterium has an extra- chromosomal plasmid (Ti plasmid)
• size ranges between 200 and 800 kbp depending on the class of the Ti
plasmid.
• Ti plasmid has three regions:
✓ transfer DNA (T-DNA) region (~24kbp) which is bordered by the left
and right borders
✓virulence region (encodes proteins required for transfer of T- DNA)
✓opine catabolism region
Wild type Ti plasmid
Wild type Ti plasmid
• The three oncogenes (opine, cytokinin, and auxin biosynthesis genes)
• Opine gene codes for the production of the nutrient, opine, the main
carbon source utilized by the A. tumefaciens
• The opine catabolism region encodes the genes for proteins involved
in opines catabolism.
• The ori allows stable maintenance of the Ti plasmid in the bacterium.
• Oncogenes are replaced with the reporter genes together with the
gene of interest
Wild type Ti Plasmid
• The Ti plasmid is large and would become larger with the genes of
interest and selectable markers. Large-sized plasmids are
cumbersome to handle and
• have low copy numbers in nature. However, this drawback eventually
led to the development of a co-integrative system in combination
with the binary
• vector system which solved the problem for large-sized plasmids.
Modification of Ti plasmid
• Wild type plasmids are difficult to use for transformation due to:
✓Size
✓ Presence of oncogenes
✓low copy number
✓hard to isolate
✓Lack of poly cloning sites or MCS.
✓Inability to replicate in Escherichia coli
• Must be modified and disarmed for transferring genes
• Co integrate and binary vectors developed
Co – integrate vectors
• Disarmed Agrobacterium Ti plasmids
✓ oncogenes are excised and substituted by part of pBR322 vector sequence
✓Left and right border sequences, nopaline synthase gene are conserved.
• Intermediate vectors
• These are small pBR322-based plasmids containing a T-DNA region to allow
recombination to form a co-integrated T-DNA structure.
• Helper vectors (if required)
✓These are small plasmids maintained in E. coli that contain transfer and
mobilization genes to allow the transfer of the conjugation-deficient
intermediate vectors into Agrobacterium.
Co – integrate Ti plasmid vector

the
The co-integrated plasmid assembled might contain: the vir genes, the left and right T-
DNA borders, an exogenous DNA sequence between the two T-DNA borders, and plant
and bacterial selectable markers.
T- binary vector system
• T-binary vector and the vir helper plasmid.

• T- binary vector contains:


✓ T-DNA sequences and the gene of interest

• The vir helper plasmid contains:


✓vir genes encoding for Vir proteins, and serves as a helper for transfer
and integration
Binary vector
A. tumefaciens transformation
Co-cultivation with A. tumefaciens to allow infection
Grown in the presence of bacterial selectable marker e.g ampicilin

Transformed and non-transformed tissue


Grown in the presence of plant selectable marker e.g. kanamycin

Transformed callus
Plant hormones, growth factors

Transformed plantlet

Adult plant
Transformation and
regeneration process
Transformation protocol for rice

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