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
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
Stable 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 to estimate
Genetic change quite easy to estimate
Bt Corn Reduces:Insecticide MycotoxinApplication 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
K. Yamaguchi-Shinozaki, JIRCAS, Japan
stress-induciblepromoter drivingdrought- andcold-responsivetranscription factor
wild type
What are uses of GM Plants?• Commercial
Applications– Altered agronomic traits
• Disease/insect resistance• Virus resistance• Herbicide resistance• Salt/drought tolerance• Cold tolerance• Enhanced yields, other
quantitative traits• Phytoremediation• Production of vaccine
Application of Roundup herbicide
Field following application
time 2007
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 VaccinesIndianapolis, IN - January 31, 2006Dow 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 greatlybenefit 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.
GGPP
Phytoene
Lycopene
beta-Carotene= provitamin A
Phytoene synthase (psy)
Phytoene desaturase (crtl)
Lycopene ß-cyclase (lcy)
(daffodil)
(daffodil)
(bacteria)
Provitamin A biosynthesis pathway
Funding: Rockefeller Foundation,Swiss Federal Institute Of Technology,European Community Biotech Program
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 Termination sequence
Bt coding sequence
How the direction of transcription? Right or left?
?
CODING SEQUENCEINTRON poly A signalPROMOTER
Building the Transgenes
Plant Transgene
bacterial genes• antibiotic marker• replication origin
Plant Selectable Marker Gene
Plasmid DNA Construct
ON/OFF Switch Makes Protein stop sign
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
Introduce DNA
Culture plant tissue
• Develop shoots• Root the shoots
Field test the plants
• Leaf, germinating seed, immature embryos
• Tissue must be capable of developing into normal plants
• Agrobacterium or gene gun
• 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
Biolistic bombardment(gene gun)
Transformation of Agrobacterium
Cloned Gene in Vector DNA Molecule
Protoplast transformationfollowed by cell wall regeneration
Agrobacterium-mediatedtransformation of plantcell
Migration and integration of gene into nucleus
Plant cells grown in
tissue culture
Regeneration ofgeneticallymodified plantfrom tissueculture
Biolistic / Gene Gun
Plasmid vector
Vector cutwith EcoRI
Donor DNA
Donor DNA cutwith EcoRI
Donor DNA fragments
Add DNA ligase
Introduce intoE. coli
Tetracycline-resistantBacterial colony fromtransformed cell
Transformed cell
Plasmids
Recombinant DNA
Selectable antibiotic resistance marker
The Next Test Is The Field
Non-transgenics
Transgenic plant
Herbicide Resistance
Example: Final Test of the TransgenicConsumer Acceptance
RoundUp Ready Corn
Before After
Roadmap Bioengineering Plants for the Future
1997 2005 2015 2025
Efficient agriculture- Bt technology- Herbicide
resistance etc
Health food and quality- Amino acids- Oil- Starch
Plant protection- Viruses- Nematodes- Fungi- Insects
Plant production platforms- Vitamines- Fatty acids- Enzymes- Bio-polymers- Pigments- Pharmaceutical products- Fibers
Stress resistance- Cold- Drought- Salinization
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. Step-wise fashion, products should be assessed
throughout the chain of development : From laboratory to greenhouse and finally large-scale field trial
6. Transparency7. Precautionary principle/approach, derived from
Rio Declaration, regulatory groups can make decisions about products based on scientific uncertainty.
8. Harmonization, sharing of or acceptance of another group’s review
Questions
Do not forget..next week student presentation!
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