International Workshop on Crop Improvement Under Drought: an Integrated approach ABRII, Karaj, IRAN
Introduction to plant transformation technology
Pr. Pr Khaled Masmoudi Centre of Biotechnology of Sfax (CBS), Tunisia
Thousands of years ago…
People first learn to use bacteria to make new and different foods, and to employ yeast and fermentation processes to make wine, beer and leavened bread bread.
1700s
Naturalists begin to identify many kinds of hybrid plants -the offspring of breeding between two varieties of plants plants.
1950
First regeneration of entire plants from an in vitro culture.
In the 1980s I th 1980
Scientists discover how to transfer pieces of genetic information f i f ti from one organism t another, allowing th i to th ll i the expression of desirable traits in the recipient organism. This is i called genetic engineering, or " ll d ti i i "recombinant DNA bi t technology"
1985
Genetically engineered plants resistant to insects, viruses, and bacteria are field tested for the first time time.
1995-1996
The European Union approves the importation and use of Monsanto's Roundup Ready soya beans in foods for people and feed for animals These are beans genetically modified to animals. tolerate spraying of Roundup for weed control while the beans are growing growing.
1998
YieldGard® Corn is approved for import into European Union
2000
Scientists achieve major breakthrough in rice; data to be shared with worldwide research community.
Unlike traditional plant breeding, which involves the crossing of hundreds or thousands of genes plant genes, biotechnology allows for the transfer of only one or a few desirable genes. This more precise science allows plant breeders to develop crops with specific beneficial traits and without undesirable traits. The benefits f l Th b fi of plant bi h l biotechnology, today and i the d d in h future, are nearly limitless. Crop improvements like these can help provide an abundant healthful food supply and protect our abundant, environment for future generations.
G Genetic Engineering? g g
Genetic i i i th b i t l t f bi t h l G ti engineering is the basic tool set of biotechnology
Genetic engineering involves:
Isolating genes fy g g yf Modifying genes so they function better Preparing genes to be inserted into a new species Developing transgenes p g g
Umezawa et al., 2006
Plant regeneration technology underlies the generation of genetically engineered plants
Plant transformation depends on a bacterium d p nd bact ium that transfers its DNA to the plant genome, or on the p g , direct introduction of NA g gene gun DNA using a « g g »
I- Agrobacterium sp. The natural genetic engineering method
History of the Plant Biotechnology
1904, Agrobacterium tumefaciens f
1974, Ti Plasmid, Ghent-Belgium
Marc Van Montagu and Jozef Schell
Overview of gall formation - the big picture
Gelvin, S. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2000. 51:223–56
Ti plasmid p
3 important regions for DNA mobilization and tumour induction T DNA T-DNA
E
T-DNA
LB RB oncogenes and opine synthase
T-DNA borders Vir region
D C G B A
vir
Ti plasmid
ori
opine catabolism t b li
⇒ encodes genes for biosynthesis of opines ⇒ encodes genes for phytohormone biosynthesis (auxin and cytokinin)
T-DNA T DNA
These T-DNA genes have promoter structures and codon g p usage that allows them to be efficiently transcribed in plant cells, but not in bacteria.
‘disarmed’ Ti plasmid
vir region
LB
YFG + kanR RB
MCS
kanR ori No T-DNA
cloning vector plasmid
Selectable marker genes for plants must be efficiently transcribed in the plant nucleus Popular promoters for this purpose are the nos (nopaline synthase gene) and CaMV35S (from the cauliflower y g mosaic virus) promoters
Conventional cloning
Gateway cloning
Site-specific DNA p recombination
Creator cloning
Rapid transient expression assays using Agrobacterium infiltrations
Vectors for RNAi
II- Particle bombardment
DNA delivery into tissues
Helium pressure gauge
Helium metering valve
Vacuum gauge g g
Main chamber containing microcarrier launch assembly
The biolistic PDS-1000/He unit PDS 1000/He
Comparaison of transient GUS expression in wheat scutellum tissues bombarded with: A: Heraeus 0.4-1.2 µm particles B: Bio-Rad submicron 0.6 µm gold particles Coverage of fine expression units and lack of clumping in treatment B
Comparaison of transient GUS expression in wheat inflorescence tissues bombarded under 1550 psi (A) and 650 psi (B). The total area of tissue targeted is clearly increased at the lower acceleration pressure.
Wheat plants with developing spikes
Immature embryos and spikes
Induced callus placed on high osmoticum media
Acclimated transgenic plantlet
Selection and regeneration of transgenic wheat
Biolistic transformation
Wheat transformation procedure using the PDS-He1000
Plant cell DNA-delivery methods
Method Ti plasmid-mediated gene transfer Microprojectile bombardment Viral vectors Direct gene transfer into plant protoplasts t l t Microinjection ec opo o Electroporation Liposome fusion Comment Excellent and highly effective, but limited to dicots Easy and effective; used with a wide range of plants Not very effective Only certain protoplasts can be regenerated i t whole plants t d into h l l t Tedious and slow Limited to p o op s s that c be ed o protoplasts can regenerated into whole plants Limited to protoplasts that can be regenerated into whole plants
Comparison between classical plant improving and genetic engineering methods
lignée donneuse d
x
cross
lignée élite receveuse
Identification et clonage
x
25%
50%
50%
plasmide
rétrocroisement
Genetic transformation
75%
x
biolistique
rétrocroisement
Agrobacterium
12,5% 12 5%
insertion de nombreux gènes
87,5% 87 5%
+ 4 insertion of one gene
rétrocroisements
Fujita et al., (2006)
Bohnert et al., 2006
Mittler, 2006
What to Expect from the Gene Revolution
• Gene cloning, genome sequencing, DNA g, g q g, fingerprinting technologies, are bringing unprecedented opportunities for
plant breeding the understanding and molecular description of the processes of plant growth and development
• 25 years of plant gene engineering in the laboratory and 10 years of transgenic plants in the field demonstrated the importance of GMO’s • Plants with a better tolerance to drought, salinity, temperature can now be made in the laboratory
More Than 10 Years of Plant Biotechnology
FACTS • 10 years of commercial experience on >1 bn acres
400 350 300 250
M. Ac. >10% increase every year Increased >50 fold since 1996 canola cotton corn soy
• In 2006 >100 mln ha and >10 mln growers used GM crops (90% of which are resource poor) • GM crops have p p provided proven economic and environmental benefits • Promising future benefits from new technologies
2003 3
200 150 100 50 0
1998 1996 6 1997 7 9 1999 2000 0 2001 2002 2 2004 4 2005 5 2006 6 2010 0F
Source: ISAAA (International Service for the Acquisition of Agri-Biotech Applications) & Monsanto estimates
Plant Biotechnology – future trend
ISAAA
2006 # of Biotech Countries # of Farmers Planting Biotech Crops Global Biotech Area
Source: Clive James, 2006 Source: Clive James, 2006
2015 ~40 ~20 million
22 10.3 million
102 mill. has ~200 mill. has
( (252 mill. acres) )
( (500 mill. acres) )
Ag Biotech Driven By Lengthy Product Development Cycle And Large Investment Process
On Average:
Time to first market: 8-10 years Total expense: ~$100M (Probability of Success in %) y $15-30M
(75%)
$20-40M $20 40M
(90%)
• Regulatory submission • Seed bulk-up • Pre-marketing g
$10-15M
(50%)
• Trait development • Pre-regulatory data •L Large scale l transformation
Sp pending
• Trait integration • Field testing • Regulatory data generation
$5-10M
(25%)
• Gene optimization • Crop transformation
$2-5M
(5%)
• High throughput screening • Model crop testing
Year 0
1
Discovery
2
3
Phase I
4
5
Phase II
Early Development
6
7
Phase III
Advanced Development
8
9
Phase IV
Regulatory Submission
10
Gene/Trait Identification
Proof of Concept
* Numbers (time duration, spending, and probability of success) are all estimates. The actual for individual projects could vary.
Slide courtesy of Rob Fraley, Monsanto
Corn hybrid with a Bt gene (left) and a hybrid susceptible to European corn borer (right).
Ostrinia nubialis, Sesamia cretica and Sesamia nonagrioides are major Corn Pests in Egypt g j gyp
At the same larvae stage, Sesamia is always much bigger than Ostrinia nubialis At the last larvae stage, Sesamia size is of 40 stage mm, Ostrinia nubialis size of 20 mm Sesamia cretica and S S i i d Sesamia nonagrioides are i i id coloured pale pink, with a more yellow trend for S. nonagrioides Ostrinia nubialis is coloured pale yellow to light brown, with a darker line on the back
Open Field Trials to Illustrate Yield Value of TM Bollgard II in Burkina Faso (2006)
The Golden Rice:
accumulating provitamin A (β-carotene ) in the grain (β
Biofortified rice as a contribution to the alleviation of life-threatening micronutrient deficiencies in developing countries: Zinc, high quality protein, and vitamin E
Green Revolution Promises and Constraints
The Green Revolution demonstrated the power of Plant Breeding Tripling W ld P l ti T i li World Population created the need for intensive agriculture The extensive use of irrigation, soil fertilization and chemical pest p control brought many sustainability problems
Soy field with Brazil Nut tree, Amazon y
CONSEQUENCES
INTENSIVE USE OF PESTICIDES:
(140 kg ha-1 yr-1 in Asia from 0 in 1960)
• HIGHER COSTS • POOR FARMERS CANNOT AFFORD • ENVIRONMENTAL IMPACT • HEALTH RISK,
ESPECIALLY FOR WOMEN AND CHILDREN 25 million people suffer acute poisoning in developing countries per annum
CONSEQUENCES
GREEN REVOLUTION TECHNOLOGY DEPENDENT UPON IRRIGATION
NOT A SUSTAINABLE SOLUTION
Tripling world population created the eed o e s e agriculture need for intensive ag cu u e
‘Blue revolution’
• Drought has become the single most limiting factor to crop production worldwide • Irrigation lead to saline soils • Crop plants with improved water-use efficiency
Drought Tolerance Field test releases in USA
Organism Institution Acknowledge/ Acreage Issued Corn Cotton Rice Soybean Monsanto Syngenta Monsanto Rice Tech, Inc Monsanto 93(26 ) 93(26*) 8 8 2 1(1*) 12301(5550 ) 12301(5550*) 2441 186 2 2480
(*) : currently in effect
Plants as source of new materials, chemicals, bio-fuel
• Dupont/Pioneer • Monsanto •D Dow Ch i l Chemical • BASF • Bayer y • Toyota
Energy from plants is as an essential element for building a ti l l t f b ildi sustainable economy
Sugar-cane
Dendê
Castor beans (Mamona)
Possibilities to produce plant-derived biomolecules
Wild or cultivated plants Chemical synthesis Plant cell and tissue cultures GM-plants with new pathways
Terpenoid indole alkaloids
Catharanthus roseus produces vinblastine & vinchristine i bl ti i h i ti Use: Hodgkin's disease, acute leukemia, breast cancer Content in plants: In leaves ~ 0.0003 % (500 kg plant material is needed to obtain 1 g vincristine) Price: ~13 000 € / g
Rischer et al., PNAS (2006)
Changing Lexis in Science and Society Dialogue
Increase food production in impoverish regions p g Greener agriculture g Healthier food Improve the mechanisms of dialogue with society.
Major Public Concerns
Safety Issues
• Human and Animal Health No adverse effect reported with the approved GM-crops • Environmental Already a long list of beneficial effects No alarming scenario was confirmed Long term ecological effects can be lower than those of traditional agriculture
Lessons from Molecular Evolution
The living world is one large g g gene-pool of functional and pseudogenes This gene-pool is permanently evolving, this is the base of evolution Nature is one big g g genetic laboratory It is very misleading to talk about human gene, pig gene, rat gene etc.
The violent opposition to GM-plants
1. Science harms “nature” 2. Multinationals control our food chain 3. Whatever positive scientists say about GM-plants, «we are against». It is society not scientists who decide on moral and ethical issues
You have to decide, this door or this this…