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Transcript
Plant Genetic Engineering
(Plant Transformation)
Nono Carsono, PhD.
Dr. rer. nat Suseno Amien
Anas, PhD.
Genetic transformation/modification of plant?
• Introduction of exogenous DNA into a plant
cell
- transient: no incorporation of exogenous DNA
(transgenes) into the genome
- stable: incorporation into genome
Transgene - the genetically engineered gene added to a
(plant) species
Stable expression of GFP
Product - Transgenic plant: plant containing transgenes
introduced by genetic engineering/modification/
transformation (not classical breeding)
• Transformation of multicellular organisms:
- can not directly transform every cell transformation involves one cell which then
regenerates an entire organism
Transient expression of GFP
Why use Genetic Transformation?
• Accelerate the breeding process
– Introduce/enhance desired trait in an established
genetic background
• Extend the gene pool
– Select genes from any Kingdom (with care, especially if
potential for entry into the food chain)
Diff. between conv. breeding and genetic engineering
Parameter
Conventional
Breeding
Genetic
engineering
Level
Whole plant
Cell/organelle
Precise
Many genes
One gene or some
Taxonomy barrier
Within species/genus No restriction
Certainty
Genetic change- hard Genetic change
to estimate
quite easy to
estimate
Bt Corn Reduces:
Insecticide
Mycotoxin
Application
Production
What are the Uses of GM Plants?
• Research
– Largest number of transgenic plants are currently
created for research purposes
• Knock-outs, over-expression, modified proteins
stress-inducible
promoter driving
drought- and
cold-responsive
transcription factor
wild type
K. Yamaguchi-Shinozaki, JIRCAS, Japan
What are uses of GM Plants?
• Commercial
Applications
– Altered agronomic traits
time
•
•
•
•
•
•
Disease/insect resistance
Virus resistance
Herbicide resistance
Salt/drought tolerance
Cold tolerance
2007
Enhanced yields, other
quantitative traits
• Phytoremediation
• Production of vaccine
Application of Roundup herbicide
Field following application
Other uses of GM Plants?
• Bioreactors / Molecular farming
– Therapeutic proteins
• Human lactoferrin to treat
iron deficiencies
• Antibodies
– Vaccine production
• Antigen expression
– HepC, HIV
Dow AgroSciences Achieves World’s First Registration for Plant-Made Vaccines
Indianapolis, IN - January 31, 2006
Dow AgroSciences LLC, a wholly owned subsidiary of The Dow Chemical Company, (NYSE: DOW), announced today that it
has received the world's first regulatory approval for a plant-made vaccine from the United States Department of Agriculture
(USDA) Center for Veterinary Biologics. This approval represents an innovative milestone for the company and the industry...
Other uses of GM Plants?
• Functional foods (humans
and livestock)
– Today: Golden rice
• Vitamin A enriched
– Future directions:
• Boosted antioxidants
• Elevated content of specific
minerals
• Removal of food allergens,
carcinogens
Greater public acceptance when the
technology is shown to more greatly
benefit consumers?
Golden Rice
Scientists from Swiss and German universities
have engineered two genes from daffodil and
one bacterial gene into rice to produce
provitamin A.
Provitamin A biosynthesis pathway
GGPP
Phytoene synthase (psy)
Phytoene
(daffodil)
Phytoene desaturase (crtl)
Lycopene
(bacteria)
Funding: Rockefeller Foundation,
Swiss Federal Institute Of Technology,
European Community Biotech Program
Lycopene ß-cyclase (lcy)
beta-Carotene
= provitamin A
(daffodil)
An Overview of the Genetic Engineering/modification cycle
Prerequisites for genetic transformation
• Available gene of interest (incl. selectable marker and other
regulatory sequences)- isolation and construct design
• Efficient method for genetic transformation- gene transfer
• Efficient method for regeneration (whole plant)- plant
regeneration
?
Promoter
Bt coding
sequence
Termination
sequence
How the direction of transcription? Right or left?
Building the Transgenes
ON/OFF Switch
PROMOTER
INTRON
Makes Protein
CODING SEQUENCE
Plant Transgene
Plant Selectable
Marker Gene
Plasmid DNA
Construct
bacterial genes
•antibiotic marker
•replication origin
stop sign
poly A signal
Transformation Cassettes
Contains
1. Gene of interest
• The coding region and its controlling elements
2. Selectable marker
• Distinguishes transformed/untransformed plants
3. Insertion sequences
• Aids Agrobacterium insertion
Transformation Steps
Prepare tissue for transformation
• Tissue must be capable of developing into normal plants
• Leaf, germinating seed, immature embryos
Introduce DNA
• Agrobacterium or gene gun
Culture plant tissue
• Develop shoots
• Root the shoots
Field test the plants
• Multiple sites, multiple years
Delivering the gene to the plant genome:
Gene transfer methods
• Direct transfer of DNA
- PEG-polyethylene glycol
- Electrophoration
• Transfer of DNA via carrier
- Microinjection
- Particle bombardment
• Transfer of DNA via vector
- Agrobacterium tumefaciens
- Viral vector
Cloned Gene in Vector DNA Molecule
Biolistic bombardment
(gene gun)
Transformation of
Agrobacterium
Protoplast transformation
followed by cell wall
regeneration
Agrobacterium-mediated
transformation of plant
cell
Migration and integration of
gene into nucleus
Plant cells
grown in
tissue culture
Regeneration of
genetically
modified plant
from tissue
culture
Biolistic / Gene Gun
Plasmid vector
Donor DNA
Selectable
antibiotic
resistance
marker
Donor DNA cut
with EcoRI
Donor DNA fragments
Vector cut
with EcoRI
Add DNA ligase
Plasmids
Introduce into
E. coli
Recombinant DNA
Transformed cell
Tetracycline-resistant
Bacterial colony from
transformed cell
The Next Test Is The Field
Herbicide Resistance
Non-transgenics
Transgenic plant
Example: Final Test of the Transgenic
Consumer Acceptance
RoundUp Ready Corn
Before
After
Roadmap Bioengineering Plants for the Future
Plant production platforms
Health food and
quality
- Amino acids
- Oil
- Starch
Efficient
agriculture
- Bt technology
- Herbicide
resistance etc
1997
2005
Plant protection
-
Viruses
Nematodes
Fungi
Insects
2015
-
Vitamines
Fatty acids
Enzymes
Bio-polymers
Pigments
Pharmaceutical products
Fibers
Stress resistance
- Cold
- Drought
- Salinization
2025
Nature Biotechnology 25: 271 (2007)
Nature Biotechnology 25: 271 (2007)
Nature Biotechnology 25: 271 (2007)
Transgenic Confirmation
(Genotypic analysis)
PCR for rapid screening
Southern blot for precise gene detection
Northern blot for transcription analysis
Western blot for translation analysis, together with
Ab-binding or enzymatic analysis
• Mendelian analysis for insertion locus and linkage
analysis
• In situ hybridization for precise insertion locus analysis
• DNA methylation analysis for silencing potential
analysis
•
•
•
•
Regulatory principles of transgenic crops:
1. Scientifically based, based on information of organism,
used technology and effects to humans and environment
2. Product-based approach, use existing product-based
legislation
3. Familiarity and substantial equivalence, experience with the
use of that species. The determination is based on scientific
literature and practical experience with the plant and
similar plant varieties.
4.
Case-by case, allow the development of knowledge that
could inform criteria and requirement over time.
Regulatory principles:
5.
6.
7.
8.
Step-wise fashion, products should be assessed throughout
the chain of development : From laboratory to greenhouse
and finally large-scale field trial
Transparency
Precautionary principle/approach, derived from Rio
Declaration, regulatory groups can make decisions about
products based on scientific uncertainty.
Harmonization, sharing of or acceptance of another group’s
review
Questions
Do not forget..next week student
presentation!