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Agrobacterium & CaMV-Mediated Gene Transfer –


Agrobacterium and CaMV-Mediated Gene Transfer – Detailed Notes


1. Introduction

Gene transfer in plants is often achieved by exploiting natural genetic mechanisms of Agrobacterium tumefaciens and Cauliflower Mosaic Virus (CaMV). These systems allow stable introduction of foreign genes into plant genomes for transgenic plant development.


2. Agrobacterium-Mediated Gene Transfer


2.1 Definition
Agrobacterium-mediated gene transfer uses the natural ability of Agrobacterium tumefaciens, a soil bacterium, to transfer a part of its DNA (T-DNA) into plant cells.
T-DNA integrates into the plant nuclear genome, enabling stable transformation.


2.2 Mechanism

Recognition and attachment
Agrobacterium detects phenolic compounds secreted by wounded plant cells.
These compounds activate virulence (vir) genes on the Ti (tumor-inducing) plasmid.
Activation of vir genes
VirA (sensor kinase) and VirG (response regulator) induce expression of other vir genes (VirB, VirC, VirD, VirE).
T-DNA processing and transfer
VirD1 and VirD2 excise the T-DNA from the Ti plasmid.
T-DNA forms a T-strand, coated with VirE2 proteins for protection.
Transfer into plant cell
T-strand is transferred via the type IV secretion system (VirB/D4 complex) into the plant cytoplasm.
Integration into plant genome
T-DNA integrates randomly into the plant nuclear DNA via non-homologous recombination.
Expression of foreign gene
Genes inserted in T-DNA, such as selectable markers or traits, are expressed in transformed plant cells.


2.3 Components


Ti plasmid (Tumor-inducing plasmid): Contains T-DNA and vir genes
T-DNA region: DNA segment transferred to plants
Vir genes: Proteins required for excision and transfer

2.4 Advantages

High efficiency in dicot plants
Stable integration into genome
Can transfer large DNA fragments (up to 25 kb)
Well-studied mechanism; widely used in transgenic plant research
2.5 Limitations

Low efficiency in monocot plants (rice, wheat)
T-DNA insertion is random → may disrupt essential genes
Requires wounded plant tissue for infection
2.6 Applications

Transgenic plants with improved traits:
Pest resistance: Bt cotton
Herbicide tolerance: Glyphosate-resistant crops
Nutritional enhancement: Golden rice (Ξ²-carotene)
Functional genomics: Gene overexpression or knockout studies


3. Cauliflower Mosaic Virus (CaMV)-
Mediated Gene Transfer


3.1 Definition
CaMV-mediated gene transfer uses the Cauliflower Mosaic Virus as a plant DNA virus vector to deliver foreign genes into plant cells.
Useful for transient or stable expression in plants.


3.2 Structure of CaMV

Genome: Circular double-stranded DNA (~8 kb)
Genes: Encode replication proteins, movement proteins, and coat protein
Promoter: CaMV 35S promoter is strong and constitutive, widely used in plant transgenic expression
3.3 Mechanism


DNA Delivery
CaMV genome is modified to carry foreign genes.
Infects plant cells through Agrobacterium vectors or direct inoculation.
Replication and Expression
Virus replicates episomally in the nucleus or cytoplasm.
Foreign genes are expressed under viral promoters (e.g., 35S promoter).
Systemic Spread (optional)
Virus can move cell-to-cell, allowing transient expression in multiple tissues.
3.4 Components Used in Gene Transfer


CaMV 35S promoter: Strong constitutive promoter in plants
Modified CaMV genome: Carries foreign gene
Selectable marker genes: For identifying transformed plants

3.5 Advantages
High-level expression of foreign genes
Works in a wide range of plant species
Viral vectors can deliver genes quickly for transient assays
Useful in functional genomics and protein production

3.6 Limitations
Expression may be transient (not always integrated into genome)
Limited size of DNA insert (~5–6 kb)
May induce plant defense responses
Risk of virus spread in field conditions

Application

Stable transgenic plants
Transient expression, functional genomics
Limitation
Low efficiency in monocots, random insertion
Limited insert size, transient expression, plant defense.

5. Applications of Agrobacterium and CaMV Systems

Agrobacterium:
Stable transgenic crops: Bt cotton, Golden rice
Functional genomics: Gene knockouts/overexpression
CaMV / 35S promoter:
High-level protein expression in plants
Transient assays for promoter studies and protein localization
Production of vaccines and pharmaceuticals in plants
These notes are sufficient for a detailed 30-mark answer, including mechanism, components, advantages, limitations, and applications.


.
Agrobacterium & CaMV-Mediated Gene Transfer – 50 MCQs


Agrobacterium tumefaciens is a:
a) Virus
b) Bacterium ✅
c) Fungus
d) Protozoa
The DNA transferred from Agrobacterium is called:
a) Viral DNA
b) Ti DNA
c) T-DNA ✅
d) Plasmid DNA
T-DNA is part of which plasmid?
a) Ri plasmid
b) Ti plasmid ✅
c) F plasmid
d) R plasmid
Agrobacterium-mediated transformation occurs naturally in:
a) Monocots
b) Dicots ✅
c) Algae
d) Fungi
Which gene region encodes proteins for T-DNA transfer?
a) T-DNA
b) vir genes ✅
c) Coat protein genes
d) 35S promoter
Vir genes are activated by:
a) Plant hormones
b) Phenolic compounds from wounded plants ✅
c) Light
d) Calcium ions
VirA and VirG are:
a) T-DNA segments
b) Virulence regulators ✅
c) Selectable markers
d) Plant genes
Which proteins coat the T-DNA during transfer?
a) VirA
b) VirB
c) VirE2 ✅
d) Coat protein
T-DNA integrates into plant genome via:
a) Homologous recombination
b) Non-homologous recombination ✅
c) Viral replication
d) RNA interference
Agrobacterium-mediated gene transfer is most efficient in:
a) Monocots
b) Dicots ✅
c) Fungi
d) Animals
The main advantage of Agrobacterium-mediated transformation is:
a) Transient expression
b) Stable integration ✅
c) Random mutations only
d) Small DNA insert only
Limitation of Agrobacterium-mediated transfer:
a) Works only in monocots
b) Random T-DNA insertion ✅
c) Cannot transfer large DNA fragments
d) Works only in bacteria
Which plant tissue is commonly used for Agrobacterium infection?
a) Leaf discs ✅
b) Seeds only
c) Root hairs
d) Flowers
Selectable markers in T-DNA help:
a) Enhance plant growth
b) Identify transformed cells ✅
c) Promote viral replication
d) None of the above
Agrobacterium can transfer DNA up to:
a) 5 kb
b) 10 kb
c) 25 kb ✅
d) 50 kb
Cauliflower Mosaic Virus (CaMV) is a:
a) RNA virus
b) DNA virus ✅
c) Bacterium
d) Fungus
CaMV genome is:
a) Single-stranded RNA
b) Circular double-stranded DNA ✅
c) Linear RNA
d) Single-stranded DNA
Which CaMV promoter is widely used in plant biotechnology?
a) 19S promoter
b) 35S promoter ✅
c) T7 promoter
d) CMV promoter
CaMV 35S promoter is:
a) Tissue-specific
b) Constitutive and strong ✅
c) Weak and transient
d) Viral capsid protein
CaMV-mediated gene transfer is often:
a) Stable
b) Transient ✅
c) Only in bacteria
d) Only in monocots
CaMV vector can carry DNA up to:
a) 1 kb
b) 5–6 kb ✅
c) 20 kb
d) 50 kb
CaMV is primarily used for:
a) Stable transgenic plants
b) High-level expression in transient assays ✅
c) Bacterial transformation
d) Monocot transformation
Agrobacterium T-DNA integrates into:
a) Chloroplast genome
b) Nuclear genome ✅
c) Mitochondrial genome
d) Cytoplasm
Agrobacterium transformation requires:
a) Healthy tissue
b) Wounded plant tissue ✅
c) Seeds only
d) Roots only
Which Agrobacterium component forms a channel for T-DNA transfer?
a) VirD2
b) VirB/D4 complex ✅
c) VirA
d) T-DNA
The role of VirD2 protein is:
a) Cut T-DNA from plasmid ✅
b) Transport T-DNA
c) Coat T-DNA
d) Express selectable marker
Agrobacterium-mediated transformation is inefficient in:
a) Dicots
b) Monocots ✅
c) Arabidopsis
d) Tobacco
Biotechnological use of CaMV 35S promoter includes:
a) Driving foreign gene expression in plants ✅
b) Bacterial antibiotic resistance
c) Animal cell expression
d) Viral capsid formation
Agrobacterium-mediated transformation can be used to create:
a) Transgenic plants ✅
b) Transgenic animals only
c) RNA viruses
d) Bacterial plasmids
CaMV viral vectors can move:
a) Within plant tissue cell-to-cell ✅
b) Only in protoplasts
c) Only in bacteria
d) Cannot move
Advantages of CaMV-mediated transfer include:
a) High-level expression ✅
b) Wide host range
c) Quick transient assays
d) All of the above ✅
Limitations of CaMV vectors include:
a) Limited insert size ✅
b) May trigger plant defense
c) Often transient expression
d) All of the above ✅
Agrobacterium Ti plasmid carries:
a) vir genes ✅
b) T-DNA ✅
c) Both ✅
d) None
Agrobacterium-mediated transfer is widely used in:
a) Rice and wheat
b) Tobacco and tomato ✅
c) Bacteria only
d) Animals
Selectable marker genes in T-DNA include:
a) GFP
b) Antibiotic resistance ✅
c) Plant hormones
d) Viral proteins
Integration of T-DNA into genome is:
a) Site-specific
b) Random ✅
c) Viral-mediated
d) Episomal
The CaMV promoter drives gene expression in:
a) Roots only
b) Leaves only
c) All tissues ✅
d) Flowers only
Agrobacterium-mediated gene transfer uses which type of plasmid?
a) Ti plasmid ✅
b) Ri plasmid
c) F plasmid
d) R plasmid
VirE2 protein functions to:
a) Cut DNA
b) Coat T-DNA for protection ✅
c) Integrate DNA
d) Express selectable marker
Agrobacterium T-DNA can be modified to carry:
a) Reporter genes ✅
b) Resistance genes ✅
c) Both ✅
d) None
Agrobacterium-mediated transformation requires which condition?
a) Dark incubation only
b) Wounded cells ✅
c) High salt only
d) None
CaMV viral vector can be used to:
a) Study gene function quickly ✅
b) Generate stable transgenic plants
c) Transform bacteria
d) Only produce capsid
High-level expression from CaMV 35S promoter is seen in:
a) All plant tissues ✅
b) Bacteria
c) Animals
d) Fungi
VirD1/D2 complex in Agrobacterium functions to:
a) Excise T-DNA ✅
b) Coat T-DNA
c) Transport T-DNA
d) Express plant genes
Agrobacterium-mediated gene transfer is often combined with:
a) Particle bombardment
b) Tissue culture techniques ✅
c) Lipofection
d) Electroporation only
CaMV-mediated transient expression is useful for:
a) Promoter analysis ✅
b) Stable genome modification
c) Bacterial cloning
d) Protein purification only
Agrobacterium-mediated transfer is less efficient in:
a) Tobacco
b) Monocots like rice ✅
c) Tomato
d) Arabidopsis
Agrobacterium vir genes are located on:
a) T-DNA
b) Plasmid outside T-DNA ✅
c) Chromosome
d) Viral vector
Applications of CaMV vectors include:
a) Vaccine protein production ✅
b) Functional genomics ✅
c) Promoter studies ✅
d) All of the above ✅
Overall advantage of Agrobacterium and CaMV systems:
a) Random DNA transfer only
b) Stable or high-level expression of foreign genes ✅
c) Only bacterial transformation
d) Cannot be used in plants

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