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!