Transgenic Plants Production

by
Dr. Sheeba Naz
 Transgenic plants
• Those plants which carry the gene from the other organism in its genome and can carry
to their successive generations.
• The transgenic plants are developed for various reasons, such as:
i. To improve nutritional quality (Golden rice)
ii. For the resistance against the insect pest (Bt cotton)
iii. For herbicide tolerance (transgenic soyabean-against glyphosate)
iv. For virus resistance (transgenic tobacco-against TMV)
v. For the resistance against diseases (transgenic potato resistant to late blight)
and so on.
 Transformation
• The process of altering the genetic constituents in a plant of interest by introducing
DNA segments into the plant genome to achieve desired gene expression.

• Categorized under two groups: indirect or direct gene transfer.

Indirect gene transfer (also known as vector-mediated gene transfer) involves the
introduction of exogenous DNA into the plant genome via biological vectors.

Direct gene transfer methods involve the introduction of exogenous DNA directly into
plant genome through physical or chemical reactions
How the gene from one organism to other is transferred??

1. Direct methods: it includes the physical methods (biolistic,

electroporation, laser mediated, etc..) and chemical methods (calcium
phosphate mediated and PEG mediated).

2. Indirect methods: it includes the viral mediated and Agrobacterium

mediated.
APPLICATION
✓ Herbicide resistance ✓ Pollen control

✓ Insect resistance ✓ Enhanced shelf life

✓ Virus resistance ✓ Pharmaceutical & edible vaccines

✓ Altered oil content ✓ Biotic &Abiotic stress tolerance

✓ Delayed fruit ripening ✓ Nutritional quality

✓ Drought, cold, salinity resistance

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Agrobacterium tumefaciens—Nature’s Smallest
Genetic Engineer
Crown gall disease
• Crown gall is plant tumour, a lump of undifferentiated tissue
• It is produced by a gram-negative soil bacterium Agrobacterium
tumefaciens in gymnosperms and dicot plants

• The bacterium transfer a genetic element called ‘Ti plasmid’ , mainly

responsible for tumour

• T-DNA from ti-plasmid remained in plant tissues

Ti-plasmid derivatives are plant vector
• T-DNA, responsible for oncogenic activity

• Agrobacterium transferred the modified T-DNA to plant tissue

• Selectable marker also constructed in T- DNA such as kanamycin and

hygromicine resistance
The Ri plasmid
• Over the years there has also been interest in developing plant cloning vectors
based on the Ri plasmid of Agrobacterium rhizogenes.

• Ri and Ti plasmids are very similar, the main difference being that transfer of the
T-DNA from an Ri plasmid to a plant results not in a crown gall but in hairy root
disease, typified by a massive proliferation of a highly branched root system.

• The possibility of growing transformed roots at high density in liquid culture has
been explored for obtaining large amounts of proteins from genes cloned in plants
Limitations of cloning with Agrobacterium
plasmids
• The main difficulty stems from the fact that in nature A. tumefaciens
and infect only dicot plants; A. rhizogenes in monocots

• Transformation with an Agrobacterium vector normally involves

regeneration of an intact plant from a transformed protoplast, cell, or
callus culture.

• Plant can be regenerated depends on the particular species involved.

• The most difficult plants are the monocots

Acetosyringone
• Acetosyringone is a phenolic natural product, and is a chemical compound related
to acetophenone and 2,6-dimethoxyphenol.
• It was first described in relation to lignan/phenylpropanoid-type phytochemicals,
with isolation from a variety of plant sources, in particular, in relation to wounding
and other physiologic changes.
• Its role as a signal attracting and transforming unique, oncogenic bacteria in
genus Agrobacterium.
• The virA gene on the Ti plasmid of Agrobacterium tumefaciens and the Ri plasmid
of Agrobacterium rhizogenes is used by these soil bacteria to infect plants
• Via its encoding for a receptor for acetosyringone and other phenolic
phytochemicals exuded by plant wounds.
Direct gene transfer to protoplast
• Removal of the cell wall from the plant cells making the resulting protoplasts
amenable to transformation by DNA

• Electroporation involves a pulse of high voltage applied to protoplasts/cells/

tissues to make transient (temporary) pores in the plasma membrane which
facilitates the uptake of foreign DNA.

• Chemical mediated gene transfer e.g. chemicals like polyethylene glycol (PEG)
and dextran sulphate induce DNA uptake into plant protoplasts.

• Calcium phosphate is also used to transfer DNA into cultured cells.

Electroporation
Biolistic gene transfer

The gene gun is part of a method called the biolistic (also known as
bioballistic) method, and under certain conditions

DNA (or RNA) become “sticky,” adhering to biologically inert particles

such as metal atoms (usually tungsten or gold).
 Molecular Analyses
• DNA analysis
• Southern blot analysis
• Polymerase Chain reaction (PCR)
• RNA analysis
• Northern blot
• Real-time reverse transcriptase (RT)-PCR
• Protein analysis
• Western blot analysis
• Enzyme linked immunosorbent assay (ELISA)
• Phenotypic Analysis
Techniques to analyse transgenic plants

1. PCR
DNAlevel
2. Southern blotting
3. Northern blotting RNAlevel

4. Western blotting
5. ELISA Protein level
6. Strip test
Phenotypic Analysis
• After screening and molecular characterization, transgenic plants should be grown
and their phenotypes assessed to determine whether they differ from wild-type.

• The phenotype is the genetic make-up (genotype) as influenced by the

environment.

• A transgenic plant may also have an altered phenotype with respect to:
• Seedling emergence, growth habit, days to flower, days to maturity, seed color,
disease resistance, and other parameters, in comparison to wild-type
 Why the analysis??
• The analysis of the transgenic plants gives a confirmation that gene of interest is present in
the transgenic plant.
• If present, analysis confirms the transgene integration with the host genome-stable vs
transient transformation.
• It determines the no. of sites at which the transgene is integrated.
• It determines no. of copies of the transgene.
• Production and accumulation of the transgene encoded protein.

• Tansient systems, foreign DNA, unable to replicate independently from the host's DNA,
persists only for a few days.
• Stable transfection, foreign DNA is integrated into the genome, replicated alongside it, and,
more importantly, passed down to the progeny.
Summary:

Is my plant transgenic? Is my plant expressing the

transgene?
• PCR
• Southern blot analysis • Northern blot analysis
• Western blot analysis
• ELISA
• RT-PCR
• Strip test
Field Testing of Transgenic Plants
Questions – Field Analysis

• What factors would be needed for the risk assessment of transgenic plant?

• How much testing or risk assessment is necessary for a new transgenic crop to be
considered “safe”?
 What is Being Regulated? Why?

• Presence of the transgene…How does it affect the plant? Phenotype? Performance?

• Transgenic event
• Biosafety concerns– human and environmental welfare
• “Protect” organic agriculture
• “Precautionary principle”
 Ecological Risks

• Non-target effects– killing the good insects by accident

• Transgene persistence in the environment– gene flow
• Resistance management– insects and weeds
• Horizontal gene flow
 Environmental Risk Assessment

Scientific Method: Observe, create hypothesis, perform experiments, collect data, report:
1. Initial evaluation
2. Problem formulation
3. Tiered risk assessment
4. Controlled experiments and gathering of information
5. Risk evaluation
Tiered approach—mainly non-targets
 Tier 1: Lab Based Experiments
Examples of insect bioassays

www.ars.usda.gov/.../photos/nov00/k9122-1i.jpg

Bioassays to determine the resistance of the two-spotted spider mite to various

chemicals
 Tier 2: Tier 3: Field Studies
Semi-Field/Greenhouse

Photo courtesy of C. Rose

Greenhouse Study: Transgenic Tobacco

Field Trials: Transgenic Canola

 Goals of Field Research

1. Hypothesis testing
2. Assess potential ecological and biosafety risks
(must be environmentally benign)
3. Determine performance under real agronomic
conditions (economic benefits)
 Ecological Concerns

• Damage to non-target organisms

• Acquired resistance to insecticidal protein
• Intraspecific hybridization
• Crop volunteers
• Interspecific hybridization
• Increased hybrid fitness and competitiveness
• Hybrid invasiveness
 Experimental Endpoints
• Hypothesis testing
• Tiered experiments– lab, greenhouse, field
• Critical P value
• Relevancy
• Comparisons– ideal vs pragmatic world

Hypotheses must be made—we cannot simply take data and look for problems!
Features of good risk assessment experiments
• Gene and gene expression (dose)
• Relevant genes
• Relevant exposure
• Whole plants
• Proper controls for plants
• Choose species
• Environmental effects
• Experimental design and replicates
Thank You