Transcript Genetic Improvement of Crop Plants

David Hildebrand
Plant Physiology, Biochemistry & Molecular Biology
Crop Science
Nutritional Sciences
Agronomy Department
University of Kentucky
Development of Crop Plants - History
• Domestication of crops
• We have been modifying our crops
for 10,000 years through Selection.
• All crops we grow today have
undergone extensive genetic change
from their wild ancestors.
• Crops, strains and genes have moved
around the globe.
Domestication of
corn creating a
new species in the
process – Zea mays
Teosinte
Hybrids
Modern Corn
Thanks to C.S. Prakash for some of the pictures on this and the next couple of slides.
Improving Our Crop Plants
• Developing Modern
Varieties of Crops
– Hybridization
• Crosses with Wild Relatives
• Hybrids
– Mutation
• Irradiation
• Chemicals
– Cell Culture
• Embryo Rescue
• Somaclonal variation
Chihuahuas and Great Danes…
Products of Modern Breeding
Modern Genetic Modification
Inserting one or few genes to achieve
desired traits.
GM Crops
–
–
–
–
Relatively Specific
Changes are Subtle
Allows Flexibility
Expeditious
Are “genetically modified” GM crops
and foods new?
Not in the general sense.
We have been genetically modifying
plants and animals for a very long
time or since the dawn of civilization!
Most all our crop plants are
extensively genetically modified
compared to their wild relatives..
Are “genetically modified” GM
crops and foods new?
Members of the same genus or species of corn
(Zea mays) or soybeans (Glycine max) do not
even exist in the wild.
Modern bread wheat is a mixture of recombined
genes from 3 different wild species and the
strawberry you buy in stores is a mixture of
genes from 2 species.
All of these major crop species are a result
of human directed genetic modification of
the original wild organisms.
The domestication of animals has also been
accompanied by extensive human-directed
genetic modification. This is exemplified with the
domestic dog, Canis familiaris.
Modern genetics including genomics
indicates that all domestic dogs are
originally derived from the wolf,
Canis lupus. Thus the Siberian
Husky and Mexican Chihuahua are
derived from the same original specie
with the only difference being the
extent of the genetic modification!
Traditional Breeding
Crossing individuals with desirable
characteristics (e.g. yield) and selecting among
the progeny. Genes recombine in a random
fashion and finding superior progeny has been
as much art as science. Needs excellent
management since it’s a numbers game and
genotype often masked by environmental
influences.
For specific characteristics such as fatty acid % of oil, naturally
occurring mutants screened for and if not found induced by
chemical mutagenesis or irradiation.
Various new breeding tools have been
developed to assist breeding in the last
50 years. Mutagenesis is one such
technique that has been utilized in the
breeding of many of the food plants
developed in the last decade. Crops
produced this way are considered
GMOs. Modern biotechnology
provides a new tool for breeding
plants and animals with much greater
precision.
Various new breeding tools have been
developed to assist breeding in the last
50 years. Mutagenesis is one such
technique that has been utilized in the
breeding of many of the food plants
currently utilized. Crops produced
this way are considered GMOs.
Modern biotechnology provides a new
tool for breeding plants and animals
with much greater precision.
Various new breeding tools have been
developed to assist breeding in the last
50 years. Mutagenesis is one such
technique that has been utilized in the
breeding of many of the food plants
developed in the last decade. Modern
biotechnology provides a new tool for
breeding plants and animals with
much greater precision.
How do GE crops differ from
conventionally derived varieties
or hybrids?
Conventional:
Wide Hybridization introduces
20,000 to 100,000 potentially
negative genes in order to obtain
one desirable disease resistance
gene.
Induced mutagenesis has been
used for decades to create
genetic variants.
Genetic Engineering:
Introduce one (or a few) foreign
“good” genes into the best accepted
cultivar background.
Genetic Engineering:
Introduce one (or a few) foreign
“good” genes into the best accepted
cultivar background.
The main thing that is new with
genetic engineering is that species
barriers can now readily be bridged.
Genetic Engineering:
Introduce one (or a few) foreign
“good” genes into the best accepted
cultivar background.
The main thing that is new with
genetic engineering is that species
barriers can now readily be bridged.
This opens new opportunities and
depending on how it is used requires
new safeguards.
The Underlying Science of
Genetic Engineering
Restriction Enzymes & Recombinant DNA
Gene Discovery, Isolation and Cloning
Move Foreign Gene or Transgene from Any
Organism to Any Other Organism
KEY ELEMENT OF
BIOTECHNOLOGY/
GENETIC ENGINEERING
Can Use Recombinant DNA
Methods To Move A Gene From
Any Organism To Any Other
Organism
What is
Recombinant
DNA?
DNA_Extraction.swf
Gene Cloning
Chromosomal
DNA
Ti plasmid
T-DNA
Chromosome
T-DNA
Crown
Gall
A. tumefaciens
Agrobacterium
tumefaciens
Plant chromosomes
containing T-DNA
Plant crown gall tissue
naturally transgenic
How Do You Introduce
a Foreign Gene into
a Recipient Organism?
Overview
Methods Used to Date for
Plant Transformation
Agrobacterium tumefaciens and rhizogenes
Gene Gun Microprojectiles (PDS)
Electroporation of protoplasts
Microinjection
Pollen Tube Pathway
Silica Carbide fibers
Microlaser
Viruses [some native genes replaced]
plasmid
Recombinant
plasmid
Desired DNA
Plant cell
inoculated with
A. tumefaciens
Regenerant
Plant cell containing Desired DNA
Agrobacterium
tumefaciens
containing Wt
Ti plasmid
Adult plant expressing
desired trait (DNA)
A. tumefaciens containing
engineered Ti plasmid
Cultured plant cells
Inserting foreign genes into plant cells. A plasmid containing DNA is cut with a restriction enzyme & DNA of
desired gene (red) inserted. Desired gene then inserted into Ti (tumor-inducing) plasmid naturally found in A.
tumefaciens. Plant cell inoculated with A. tumefaciens containing engineered Ti plasmid + the desired DNA
transfers desired DNA + t-DNA into plant chromosomes. Plantlets with desired trait then regenerated.
The Gene Gun
Helium chamber
Rupture disk
Macrocarrier
DNA coated
gold particle
Stopping screen
Focusing device
PDS1000 Microparticle Delivery System
Gene gun
Target tissue
From Collins lab
Biolistic Transformation
Before impact
DNA coated gold particle
During impact
Plant Cell Wall
?
After impact
How do Genes do Their Job?
GENE EXPRESSION
DNA
Transcription
mRNA
Translation
Protein
Structural Gene
Enzyme (Protein)
Product A
Product B
Roundup Ready Crops
Monsanto web page:
Roundup (Glyphosate) is a very strong inhibitor of EPSP1
Synthase.
PEP
Shikimate
3-phosphate
O-
OC
O
O-
C
O
C
CH2
C
EPSP
O
+ Pi
+
Pi
O
Pi
OH
O
O-
OH
Pi
C
O
OH
O
NH
Glyphosate
CH2
Pi
1EPSP =
5-ENOLPYRUVYLSHIKIMATE 3-PHOSPHATE
Phe, Trp, Tyr
CH2
O-
C
C
O
Sulfonylurea Tolerant Soybeans - STS These cultivars are resistant to certain sulfonyl ureas (SUs), a
family of herbicides which are most effective against broadleaf
weeds. STS herbicides used over soybean varieties that have the
STS gene offer the benefit of using broad spectrum sulfonylurea
broadleaf herbicides without injuring young soybean plants.
Labeled sulfonylurea herbicides include Synchrony STS¹, Reliance
STS¹, Classic¹, Pinnacle¹, Canopy¹, Canopy XL¹ and Concert¹.
The STS gene was incorporated into soybean germplasm using
conventional breeding methods.
SU tolerant gene induced by EMS mutagenesis (US patent #
5,084,082).
Bx breeding
Mechanism of action of SUs
SUs inhibit the essential plant enzyme acetolactate
synthase or ALS. Animals do not have ALS.
O
-keto butyrate
HC
O-
O
HC
O
HC
CH2
H3C
CH3
CH
CO2
O
HC
O
HC
O
CH3
H2
C C
acetolactate
OOH
O
O
CH3
-
pyruvate
H3C
isoleucine
HC
O-
HC
NH2
CH
CH2
CH3
Bt crops
Grower Advantages
GM crops on the market today do not directly increase
& can decrease costs to farmers.
According to Kline & Company corn pesticides can be reduced
as much as 70% when growers use GM corn. This can save
growers > $200 million/yr in pesticide costs. Also EPA
EPA: "The average net benefit of $3.31 per acre on 19.75 million
acres of Bt corn planted in 1999 (a year of low corn borer pressure)
leads to the national estimate of $65.4 million." The net benefit to
sweet corn producers was calculated at $5.38 per acre in 1999.
Plant Incorporated Pesticides (PIP’s)
Scientists in industry and academia have been developing corn and
soybean lines that produce their own natural insecticides to resist
insect losses. Since 1996, six types of Bt corn have been
commercialized to protect against attack by European and
southwestern corn borer. Other types of Bt corn are being
evaluated that prevent losses to corn rootworm, black cutworm,
and fall armyworm.
Transgenic virus resistant papaya:
Main hope for controlling papaya
ringspot virus in Hawaii
From a DuPont Webpage
Zygotic embryo
Somatic Embryo
(SE)
Blue SEs expressing introduced GUS gene
Soybean
Engineering
h
CO2
CH3(CH2)nCOOR
N2
H2O
Fatty Acid Biosynthesis
C4
C2
KAS III
TG
18:3
3
wD
AT
S
KAS I
18:2
C18:1
AT
C18 CoA
S
plastid
C16 CoA
2D
C18 DS
D-9
TE
D -1
TE
C16
KAS II
oil
body
18:1
TE
18:1 CoA
(PC)
ER
A much higher linolenic acid (18:3) content would
enhance the drying characteristics of soybean oil such
as in printing inks.
New molecular techniques have provided the most
efficient method to accomplish this.
As Expected, Developing Flax seeds have much higher
w3 Desaturase Activity than Soybean or Arabidopsis.
In vivo w3 Desaturase Activity Measurement
14 C-18:3
14 C-18:2
14 C-
18:2
Arabidopsis
Flax
2 mg
Flax
4 mg
Soy
26 mg
Soy
90 mg
Significance of Epoxy FA
u Plant defense
u Physical,
u Industrial
chemical?
Soybean Seed
Uses
40% Protein
u Epoxy
coatings
u Plasticizers
u oil-based
paints
Animal Feed, etc.
u adhesives
u high
20% Oil
performance composites, etc.
Edible Oil Products,
Limited Industrial Use
Engineering Soybeans for Epoxy Fatty
Acid Accumulation
Take genes from wild plant(s) with high epoxy fatty acid
accumulation
in seed oil and put in commercial oilseed such as soybeans.
Stokesia laevis
Vernonia galamensis
Some GE Plants in Field Trials
Maize
Cantaloupe
Wheat
Carnation
Plum Trees
Chrysanthemum
Potato
Flax
Mellon
Eggplant
Grape
Cranberry
Tomato
Chicory
Petunia
Strawberry
Spruce
Oilseed Rape
Soybean
Rice
Cucumber
Barley
Cabbage
Sweet Potato
Cotton
Poplar
Apple
Peanut
Trefoil
Fodder Rape
Mustard
Squash
Cauliflower
Eucalyptus
Kiwi
Tobacco
Sunflower
Sugarcane
Carrot
Rose
Sugar Beet
Rapeseed
Sweetgum
Serviceberry
Papaya
Alfalfa
Birch
Walnut
Pepper
Asparagus
Canola
Lettuce
Raspberry
Fescue
Watermelon
From Collins lab
What’s Being Tested
(US field releases, cumulative since 1987
Herbicide
Resistance
30%
Viral
Resistance
14%
Fungal
Resistance
3%
Product
Quality
24%
Other
8%
Insect
Resistance
21%
SOURCE: US DEPARTMENT OF AGRICULTURE
From Collins lab
Our new century is predicted to be the “Century of Biology” as
the last century was the “Century of Chemistry & Physics”.
Until ~ 100 years ago most building materials, everyday
materials in the home, fuels and clothing came from our farms
and forests. In the last century we have seen a major shift to
such materials coming from petrochemicals rather than plants
and animals.
Petrochemicals come from what were once plants and animals
but are not a readily renewable resource. Now with modern
technology, particularly biotechnology, we can derive more and
more of the materials humankind needs in the future from
plants and animals produced by our farmers. Thus in the
future, farmers should play an increasingly vital role in our
economic prosperity in addition to their essential role in food
production.
From our state government leaders to our state
universities, Kentucky is working to be a significant
player in this “new economy”. Biotechnology has
already spawned a number of new companies in KY
(see URL below for a list):
http://www.biokentucky.com/
http://www.ca.uky.edu/agbiotechnology/
What has happened to the
farmer’s share of the food dollar?
1999: 20.6%, 2000: 19.9%, 2001: 21%, 2002e: 20%
Adding value to crops can reduce or reverse this trend
What is the Future of Biotechnology?
Value Added
Extended Uses
Genomics
Bioinformatics
Diagnostics
Sustainability/Environment
Acceptance
Biotechnology Industrial Areas
Agriculture
Pharmaceuticals
Health Care
Food
Energy
Environment
Harvesting Tobacco Biomass for
Pharmaceutical Production
See: The Kentucky Tobacco Research
and Development Center
http://www.uky.edu/KTRDC
Tobacco Plants Produce Cancer Vaccine
Non-Hodgkin’s B-cell lymphoma is the most prevalent form of lymphoma, a
malignancy which affects the lymph system and is the sixth most common cause of cancerrelated deaths in the US. Researchers have now established that a therapeutic vaccine protein
produced by modified tobacco plants is effective in preventing the growth of non-Hodgkin’s
lymphoma cells - at least in laboratory mice. Eighty percent of the mice receiving the vaccine
survived the lymphoma, while untreated mice died within three weeks after contracting the
disease.
As reported in the Proceedings of the National Academy of Sciences, the work was a
collaborative effort between scientists at Stanford and Biosource Technologies, based in
Vacaville, Calif. The researchers removed malignant B cells from laboratory mice, and then
isolated the gene coding for the surface markers specific to those cells. They inserted the gene
into a tobacco mosaic virus, and exposed tobacco plants to the virus. As the virus spread
through the tobacco leaves, the plants produced the desired B-cell protein, which was extracted
and injected into mice which had received lethal dosages of tumor cells.
Biosource hopes to begin clinical trials of the vaccine in humans within a year, if the
vaccine passes necessary safety tests. To speed development, the company launched a new
division called Biosource Antigenics to focus initially on the vaccine. Robert Erwin, CEO of
Biosource, says that the plant-based expression system has proven itself to be faster than other
technologies for producing effective vaccines. He says the Antigenics division will use highthroughput robotics to provide human and animal health vaccines companies with a means to
rapidly prototype lead vaccine designs.
Source: AgBiotech Reporter - February 1999
New Ways to Protect Drought-Stricken Plants
Anne Simon Moffat. Science 296:1226-1229, May 17 2002.
With drought an ever-present threat, researchers are identifying genes that can help plants
tolerate arid conditions in hopes of using them to produce hardier crops.
Tomato plants carrying a foreign gene that
protects their cells from salt-induced
dehydration thrive in a 200-mM salt solution,
whereas unaltered plants wither.
CREDITS: E. BLUMWALD/UNIVERSITY OF
CALIFORNIA, DAVIS
Fields of ... brown. Improving the drought
tolerance of corn could make dried-out
crops like this one a thing of the past.
CREDIT: RICHARD HAMILTON SMITH/CORBISS
Studies have shown that ethanol:
Reduces tailpipe carbon monoxide emissions by as much as 30%
Reduces exhaust VOC (volatile organic compounds) emissions by 12%
Reduces toxic emissions by 30%
Reduces particulate matter (PM) emissions by more than 25% (Particulate matter has been found to penetrate deeply
into human lungs.)
Corn can be genetically engineered to improve the efficiency
of ethanol production.
+
+
From Collins lab
fbi.gov/programs/lab/labhome.htm
From Collins lab
Welcome Page
The Hazardous Substance Research Center/South and Southwest is a
competitively awarded, peer-reviewed research consortium led by
Louisiana State University with the cooperation of the
Georgia Institute of Technology and Rice University to
address critical hazardous substance problems, especially
as they relate to contaminated sediments.
The South & Southwest Center was established in October
Researchers test a pilot-scale
1991 under Section 311(d) of CERCLA to conduct research
system for using vegetation in soil
and technology transfer designed to promote risk-based
remediation.
management and control of hazardous substances for the nation and
regions 4 and 6 the Environmental Protection Agency (EPA).
The contaminated sediments and dredged materials of concern contain
organics, metals, and conventional pollutants. These environmental
contaminants are either suspended in the water column or stored on the
bottom of rivers, bayous, lakes, harbors, estuaries, freshwater wetlands,
and in ocean waters of the continental shelf.
Research theme areas include:
www.hsrc.org/hsrc/html/ssw/
From Collins lab
Issue: Impact on non-target
organisms
There has been much media attention given
to the potential impact of GM crops on nontarget organisms. An ideal pest control tactic
would be one that controls the pest, but does
not harm other non-target organisms in any
way. Non-target organisms include all
organisms except for the pest to be
controlled. Examples of non-target
organisms would be mammals, fish, birds,
reptiles and other insects.
Lady Beetle
Lacewing
Monarch
From Ric Bessin
Issue: GM Crops
Compliment
Biological Control
One group of non-target organisms that need to be encouraged is
the natural enemies of our crop pests. Natural enemies are
composed of a wide array of parasitic and predatory insects and
other arthropods. Control of crop pests by natural enemies is
referred to as biological control. Plant pesticides produced by
genetically modified crops are more selective than pesticide sprays,
thus not harming natural pest predators. In this way, genetically
modified crops that produce their own plant pesticides are more
compatible with biological control.
From Ric Bessin
Bt crops -- EPA Assessments
"Bt Biopesticides Registration Action Document," the EPA's
review contains 283 pages and can be viewed in its entirety at:
www.epa.gov/scipoly/sap.
“the Bt protein behaves as would be expected of a dietary protein,
is not structurally related to any known food allergen or protein
toxin, and does not display any oral toxicity when administered at
high doses."
"(Data) provide a weight of evidence assessment indicating no
unreasonable adverse effects of Bt Cry proteins expressed in plants
to non-target wildlife or beneficial invertebrates,whether they are
earthworms, springtails, parasites, predators, pollinators or soil
microbial and invertebrate flora."
“...widespread cultivation of Bt crops may have
huge benefits for monarch butterfly survival."
From Ric Bessin
Concerns
Like all new technologies there are concerns with the
commercial application of biotechnology. These concerns
can be divided into three areas [although in many cases the
concern crosses more than one area]:
•Social and Economic Considerations
•Ethical Concerns
•Environmental Impact
Also since biotechnology impacts our food supply a
fourth concern has been voiced
•Food Safety
Extensive studies indicate that biotech-derived foods are
as safe or safer than conventional foods. Also although
regulatory oversight is evolving, it is more extensive for
biotech foods than any others.
In balance biotech crops are considered to be beneficial to
the environment. We should work to ensure that biotech
has maximum benefit on sustainable agricultural
development.
U.S. Labeling Policy for
Food Biotechnology
• FDA safety standards are consistent for all foods.
– Must label the presence of common allergens not
normally found in certain foods
– Must demonstrate scientifically that allergens are not
present in modified food.
– If nutritional content or composition has been
changed, product must be labeled accordingly.
Social and Economic Considerations
Among the major social and economic concerns that
have been raised are:
Is biotechnology scale-neutral or does it mainly benefit the
larger and wealthier farmers?
Can it provide a market advantage for large multinational
corporations?
Do just a few corporations control much of the intellectual
property associated with agricultural biotechnology?
Might biotechnology increase farmer’s reliance on seed
companies and cause them to be less likely to use seeds of
varieties they have traditionally used?
Can it exacerbate over-production making farming even less
profitable than it already is?
Is it a “tool of capitalism”?
Ethical Concerns
In many ways the social and economic concerns are also
ethical considerations. Also in some ways adoption of
biotechnology represents a break from tradition.
Some of the major ethical concerns that have
been voiced are:
•Should we mess with Mother Nature [or are we]?
•Are genetically engineered plants and animals
“natural”?
•Should anyone be allowed to own or patent a living
organism [or does this actually occur]?
Bioethics
Health and Food
Safety
Farm Impact
Research and
Science
About Us
News
Environmental
Topics
FAQs
Glossary
Just for Kids /
Teachers
BREI is a multi-disciplinary team of research, extension and
teaching professionals from the College of Agriculture.
Sharing information about agricultural biotechnology
through:
•Publications
•Visual Media
•Education Curriculum
•Web-based Information
•In-service Training
Comments and suggestions are welcome, send to BREI.
Copyright © 2000, University of Kentucky, College of Agriculture
www.ca.uky.edu/BREI
Charles Darwin...
“It is not the
strongest species
that survive, nor the
most intelligent, but
the ones most
responsive to change”
“I’m all for progress; it’s change I
don’t like” - Mark Twain
Slide from C.S. Prakash
Thanks