What is Agricultural Biotechnology?
Agricultural biotechnology is a range of tools, including traditional breeding techniques,
that alter living organisms, or parts of organisms, to make or modify products; improve
plants or animals; or develop microorgan
isms for specific agricultural uses. Modern
biotechnology today includes the tools of genetic engineering.
How is Agricultural Biotechnology being used?
Crop production
Phytoremediation
Improvements in agriculture not involving plants
Agricultural Biot
echnology
o
Genetically engineered, pest
-
resistant plants
o
Foods with higher protein or vitamin content
o
Drugs developed and grown as plant products
o
Estimated to be a $7 billion market in 2008
What are the new products of agricultural biotechnology?
Insect re
sistant crops commercially available, e.g., Bt corn, cotton, and potatoes
Corn rootworm resistance in 2001
Animal growth hormones, e.g., bST
Herbicide tolerant crops, e.g., Roundup Ready soybeans and corn and Liberty
Link corn
Identity
-
preserved or speci
fic
-
attribute crops (vaccines, higher oil or starch
content, additional amino acids)
Advances in biotechnology may provide consumers with foods that are
nutritionally
-
enriched or longer
-
lasting, or that contain lower levels of certain
naturally occurring t
oxicants present in some food plants.
Developers are using biotechnology to try to reduce saturated fats in cooking
oils, reduce allergens in foods, and increase disease
-
fighting nutrients in foods.
They are also researching ways to use genetically engine
ered crops in the
production of new medicines, which may lead to a new plant
-
made
pharmaceutical industry that could reduce the costs of production using a
sustainable resource
What are the benefits of Agricultural Biotechnology?
The application of biote
chnology in agriculture has resulted in benefits to
farmers, producers, and consumers. Biotechnology has helped to make both
insect pest control and weed management safer and easier while safeguarding
crops against disease.
For example, genetically engine
ered insect
-
resistant cotton has allowed for a
significant reduction in the use of persistent, synthetic pesticides that may
contaminate groundwater and the environment
In terms of improved weed control, herbicide
-
tolerant soybeans, cotton, and
corn enabl
e the use of reduced
-
risk herbicides that break down more quickly in
soil and are non
-
toxic to wildlife and humans. Herbicide
-
tolerant crops are
particularly compatible with no
-
till or reduced tillage agriculture systems that
help preserve topsoil from ero
sion.
Agricultural biotechnology has been used to protect crops from devastating
diseases. The papaya ringspot virus threatened to derail the Hawaiian papaya
industry until papayas resistant to the disease were developed through genetic
engineering. This
saved the U.S. papaya industry. Research on potatoes, squash,
tomatoes, and other crops continues in a similar manner to provide resistance to
viral diseases that otherwise are very difficult to control.
Biotech crops can make farming more profitable by in
creasing crop quality and
may in some cases increase yields. The use of some of these crops can simplify
work and improve safety for farmers. This allows farmers to spend less of their
time managing their crops and more time on other profitable activities.
Biotech crops may provide enhanced quality traits such as increased levels of
beta
-
carotene in rice to aid in reducing vitamin A deficiencies and improved oil
compositions in canola, soybean, and corn. Crops with the ability to grow in salty
soils or bet
ter withstand drought conditions are also in the works.
This genetic information is providing a wealth of opportunities that help
researchers improve the safety of our food supply. The tools of biotechnology
have "unlocked doors" and are also helping in t
he development of improved
animal and plant varieties, both those produced by conventional means as well
as those produced through genetic engineering
Generation of Transgenic Plants
Totipotency
o
Entire plant can be generated from a single, non
-
reproducti
ve cell
o
Single cells can be separated from leaf, stem or root tissue using enzymes
to digest pectin holding cells together (pectinase)
Clones from cuttings in tissue culture
o
Asexual reproduction of plants can occur using fragments of plants
Shoots or stems
or leaves = EXPLANTS
In tissue culture, cells divide from exposed cell
a callus forms
Callus tissue regeneration
o
Callus tissue will develop if cells are grown with proper balance of
nutrients and plant hormones
Magenta boxes or large test tubes, sterile
medium and transfer
instruments
Murishigee and Skoog medium (MS medium)
–
Artificial medium
(agarose, nutrients and hormones)
Under influence of increased cytokinin, shoots will differentiate
Transferred to increased auxins, roots will establish
Eventuall
y transferred to soil
entire plant with reproductive
structures (ovules, pollen)
o
Calluses can be split into many smaller pieces before hormones are added
to increase # of plants
DNA inserted into plants
Transgenic plant
o
Characteristics of transgenic
plants
All cells in the plant are derived from one cell
All cells express the desired genetic information
o
Why make transgenic plants?
Genes from distantly related plant families can be introduced
without need for breeding (some families of plants are
incom
patible)
To improve crop hardiness and characteristics of final plant product
Protein content
Ripening rate
Drought resistance
Procedures for generating transgenic plants
Microinjection
o
DNA constructs injected using fine glass pipettes in combination with
phase contrast microscopy
Electroporation of protoplasts
o
Electric pulses of high field strength
o
Reversibly permeabilize cell membranes
Electric discharge gun
–
Gold beads
o
Firing DNA
-
coated pellets using a modified .22 caliber gun
“Whiskers” of silicon car
bide
–
holes punched, DNA introduced
Agrobacterium tumefaciens
Viral vectors
o
Cauliflower mosaic virus vectors
o
Gemini virus vectors
Liposome
-
mediated transformation of protoplasts
o
Artificial lipid vesicles = Liposomes
Chemically
-
stimulated DNA uptake by p
rotoplasts
o
Polyethylene glycol + CaCl
2
Many gene transfer techniques start with protoplasts
o
Cell wall is digested with cellulase and cells are separated using pectinase
o
Plant cells are maintained in suspension
DNA is introduced, it integrates and express
ion of desired genes is
achieved
Electroporation
Microinjection
Protoplast fusion can also be used to fuse two different plant types together
New Plant Varieties (hybrid plantlet)
o
Fused cell acquires some of the characteristic of both genetic backgroun
ds
and can be regenerated into a plant with some traits from both parental
plants
Fusigenic agents (polyethylene glycol) or electroporation used to
fuse membranes
o
Useful if species are sexually incompatible or cross with difficulty
Commercially important p
lants that can be grown from single somatic (non
-
seed)
cells
o
Asparagus
o
Cabbage
o
Citrus fruits
o
Carrots
o
Alfalfa
o
Millet
o
Tomatoes
o
Potatoes
o
Tobacco
More than 30 different crop plants developed with rDNA techniques are being
tested in field studies
Agrobacterium
tumefaciens
Characteristics
o
Plant parasite that causes Crown Gall Disease
o
Encodes a large (~250kbp) plasmid called Tumor
-
inducing (Ti) plasmid
Portion of the Ti plasmid is transferred between bacterial cells and
plant cells
T
-
DNA (Tumor DNA)
o
T
-
DNA inte
grates stably into plant genome
o
T
-
DNA ss DNA fragment is converted to dsDNA fragment by plant cell
Then integrated into plant genome
2 x 23bp direct repeats play an important role in the excision and
integration process
Tumor formation = hyperplasia
Hormon
e imbalance
Caused by
A. tumefaciens
o
Lives in intercellular spaces of the plant
o
Plasmid contains genes responsible for the disease
Part of plasmid is inserted into plant DNA
Wound = entry point
10
-
14 days later, tumor forms
Observed in many varieties o
f wood plants
o
Grapes
o
Roses
o
Apples
o
Cherries
o
Pecans
Also infects herbaceous plants
o
Daisies
o
Asters
o
Beets
o
Turnips
o
Tomatoes
o
Sunflowers
What is naturally encoded in T
-
DNA?
o
Enzymes for auxin and cytokinin synthesis
Causing hormone imbalance
tumor formation/und
ifferentiated
callus
Mutants in enzymes have been characterized
o
Opine synthesis genes (e.g. octopine or nopaline)
Carbon and nitrogen source for
A. tumefaciens
growth
Insertion genes
Virulence (vir) genes
Allow excision and integration into plant genome
H
ow is T
-
DNA modified to allow genes of interest to be inserted?
o
In vitro
modification of Ti plasmid
T
-
DNA tumor causing genes are deleted and replaced with
desirable genes (under proper regulatory control)
Insertion genes are retained (vir genes)
Selectabl
e marker gene added to track plant cells successfully
rendered transgenic [antibiotic resistance gene
geneticin (G418)
or hygromycin]
Ti plasmid is reintroduced into
A. tumefaciens
A. tumefaciens
is co
-
cultured with plant leaf disks under hormone
condi
tions favoring callus development (undifferentiated)
Antibacterial agents (e.g. chloramphenicol) added to kill
A.
tumefaciens
G418 or hygromycin added to kill non
-
transgenic plant cells
Surviving cells = transgenic plant cells
Techniques to transform plan
t cells by
A. tumefaciens
o
Wounding and direct inoculation
o
Inoculation of explants
in vitro
o
Transformation of leaf
-
disks
o
Co
-
cultivation of
Agrobacterium
with protoplasts
Examples of Crop Improvement Measures
Nitrogen fixation
To enable plants to fix atmos
pheric N
2
so that it can be converted into NH
3
, NO
3
-
,
and NO
2
-
providing a nitrogen source for nucleic acid and amino acid
synthesis
o
Thereby eliminating need to fertilize crops with nitrogen
Exploit N
2
fixation metabolic machinery of bacteria and fungi
o
Some live freely in soil and water
o
Some live in symbiosis
Rhizobium spp
. live in symbiosis with leguminous species of plants
in root nodules (e.g. soy, peas, beans, alfalfa, clover)
Frost Resistance
Ice
-
minus bacteria
o
Ice nucleation on plant surfaces caus
ed by bacteria that aid in protein
-
water coalescence
forming ice crystals @ 0
o
C (32
0
F)
o
Ice
-
minus
Pseudomonas syringae
Modified by removing genes responsible for crystal formation
Sprayed onto plants
Displaces wild type strains
Protected to 23
o
F
Dew fr
eezes beyond this point
Extends growth season
First deliberate release experiment
–
Steven Lindow
–
1987
-
sprayed potatoes
Frost Ban
o
Different strain of bacteria
–
Julie Lindemann led different project
–
1987
o
Strawberries in California
Resistance to biolo
gical agents
Anti
-
Insect Strategy
-
Insecticides
o
From
Bacillus thuringensis
Toxic crystals found during sporulation
Alkaline protein degrades gut wall of lepidopteran larvae
Corn borer catepillars
Cotton bollworm catepillars
Tobacco hornworm catepilla
rs
Gypsy moth larvae
Sprayed onto plants
–
but will wash off
Monsanto Chemical Company
–
1991Trials
o
BT
into cotton plants using
A. tumefaciens
vector
o
Cott
on bollworms
protection in 6 loc
a
tions, 5 different states,
consistent results
o
First crops
–
199
6
Corn
Cotton
Seed potatoes
Soybean
Others
Cloned BT toxin gene into a different bacterium that lives
harmlessly in corn plants
Pressure applied to introduce modified bacterium into seeds
Corn stalks protected from corn borers
BT in poplar and white spruc
e
catepillar resistance
o
BT
-
resistant strains are beginning to emerge in some catepillars
Anti
-
Viral Strategy
o
TMV
-
coat protein inserted into tobacco and tomato plant cells using Ti
plasmid
Viral capsids inhibit viral replication of TMV when infected
o
Gra
pe fan
-
leaf virus (GFLV)
Causes yellowing and deformation of grape leaves
Transmitted in soil by nematodes
Viral capsid genes introduced into champagne grape vines using T
plasmid
Resistance to virus acquired
o
Other trials using capsid proteins: potato lea
f
-
roll virus, cantaloupe
mosaic virus, rice strip virus
o
Concerns that recombination events may lead to new plant virus strains
Anti
-
Bacterial Strategies
o
Resistance to
Xanthomonas oryzae
(rice wilting)
o
Conferred by cloning resistance genes from wild rice st
rains
Anti
-
Worm Strategies (Animal pest)
o
Nematode resistance gene from wild beet plants
o
To protect sugar beet
Resistance to herbicides
o
Glyphosate resistance
o
Glyphosate = “Roundup”, “Tumbleweed” = Systemic herbicide
o
Glyphosate inhibits EPSP synthase (S
-
e
no
l
p
yruvl
s
hikimate
-
3
p
hosphate
–
involved in chloroplast amino acid synthesis)
o
Escherichia coli
EPSP synthase = mutant form
less sensitive to
glyphosate
Cloned via Ti plasmid into soybeans, tobacco, petunias
Increased crop yields of crops treated with he
rbicides
o
Bromoxynil
= bromine
-
based herbicide
Bromoxynil resistant cotton
Concern over movement of resistance genes into weeds
making
compounds useless
Bioengineered foods
Flavr
-
Savr tomato
o
“Rot
-
Resistant Tomato”
o
Calgen, Inc.
o
Anti
-
sense gene
comple
mentary to polygalacturonase (PG)
PG = pectinase
accelerates plant decay/rotting
Laurate canola oil
o
Canola plant modified with thioesterase gene obtained from California bay
laurel tree
Enzyme produces lauric acid (up to 40% in oil from genetically
modi
fied (GM) canola seeds)
Low saturated fat content
Heat tolerant
o
Does not break down
o
Excellent for high temperature cooking processes
Biopharming
What is Biofarming
Drug production in genetically modified plants
o
Tobacco
o
Alfalfa
o
Potatoes
o
Corn
o
Soybeans
o
Whea
t
o
Rice
o
Oilseed rape
o
Ethiopian mustard
Drugs = Biopharmaceuticals
o
Drugs synthesized organically
Many drugs are made naturally in plants
Aspirin (originally isolated from willow bark)
Vincristine and vinblastine (periwinkle)
Taxol (Pacific yew)
Digitalis (fo
xglove)
Recombinant DNA techniques enable many more drugs to be made
artificially in plants
Human proteins in plants = xenogenic proteins
Why Farm for Pharmaceuticals in Plants?
o
Cheaper than producing pharmaceutical proteins in cell culture
Could reduce th
e cost of medicine
Example:
Newest factories producing GM proteins in mammalian cell
culture costs ~$100 million/300 kg, costing ~$1000/g
Biopharming producing GM proteins in plants costs ~$10
million capital investment/300 kg, costing ~$200/g
(according
to Monsanto’s Integrated Protein Technologies)
o
However, costs of extracting and purifying biopharmaceuticals can be high
and processing strategies need to be improved
Fewer complications than producing proteins in animals (e.g. cell
culture or milk from “
pharm” animals)
Possible transmission of animal viruses
–
zoonoses
Plant viruses cannot infect animals
Plants do not serve as hosts for infectious agents such as
HIV, HepB, prions
Ethical considerations (animal welfare concerns)
o
Plants effectively transc
ribe, translate and assemble proteins derived from
eukaryotic sources
o
Improved quality of life
o
Produce beneficial pharmaceuticals in tobacco rather than cigarettes
“If we can actually find a medical use for tobacco that saves lives,
what a turnaround for t
he much
-
maligned tobacco plant.”
o
Tobacco is favored for many reasons
Easy to genetically engineer (Agrobacterium
-
mediated
transformation)
Excellent biomass producer
~1 million seeds can be isolated from a single plant (scale
-
up
benefits)
Number one cash cr
op in Virginia
Examples of Biopharmaceuticals
o
Hepatitis B and other subunit vaccines
o
Urokinase (clot dissolving drug)
o
Human serum albumin (liver cirrhosis treatment)
o
Hemoglobin
o
Human erythropoietin
o
Glucocerebrosidase (Gaucher’s disease)
o
Blood coagulants
o
Pr
oteases (e.g. trypsin)
o
Protease inhibitors (e.g. aprotinin
-
used by surgeons)
o
Growth promoters
o
HIV viral coat protein (HIV therapy)
o
Nutraceuticals (Vitamin A and E, amino acids)
o
Neurologically active agents (human enkephalins)
o
Protein based sweetener (Br
azzein)
o
Avidin
o
Beta
-
glucoronidase
o
Indirect thrombin inhibitor (Hirudin
–
anticoagulant originally isolated from
the leech
Hirudo medicinalis
)
o
Human epidermal growth factor
o
Human interferon
-
alpha (Hepatitis B and C treatment)
o
Bacterial enterotoxins
o
Human
insulin
o
Norwalk virus capsid protein
o
“Natural” plastic (plastic
-
like polymers) (Biopol)
o
Human GM
-
CSF
o
Human alpha
-
1 antitrypsin (cystic fibrosis/liver treatment)
o
Angiotensin
-
1
-
converting enzyme (hypertension)
Edible Vaccines
–
Ongoing Research Areas
o
Hepati
tis B
o
Dental caries
-
Anti
-
tooth decay Ab (CaroRxTM) (anti
-
Streptococcus
mutans
)
o
Autoimmune diabetes
o
Cholera
o
Rabies
o
HIV
o
Rhinovirus
o
Foot and Mouth
o
Enteritis virus
o
Malaria
o
Influenza
o
Cancer
EHEC Edible Vaccine
o
Foodborne Pathogen
o
Vaccine exists, cost prohibiti
ve delivery
o
Plant
-
based vaccination can be cost effective
o
Improved safety of the food supply
o
Safety evaluation of the vaccine protein
Dr. Carole Cramer
–
while at Virginia Tech
o
Engineered rDNA so that protein is only expressed when the tobacco
leaves are
cut
Drug is only produced when plant is wounded
o
Currently chief scientific officer of CropTech
Developing ~20 human proteins
Including human protein C (blood clotting regulator)
Lysosomal enzyme
-
glucocerebrosidase
Tobacco plants produce proteins after
leaves are shredded
Clinical trials must be initiated, and approval by FDA still lie ahead
Planet Biotechnology
o
Clinical trials involving anti
-
tooth decay antibody
o
Monoclonal antibody that binds to bacteria (Viridans Streptococci)
associated with tooth de
cay
o
Interferes with adhesion of bacteria to tooth enamel
Potentially Harmful Effects
Contamination by pesticides
Co
-
purification of plant chemicals (e.g. nicotine)
Different glycos
ylation in plants versus animal
o
Interference with norma
l function of protei
n in animal
o
Stimulation of hypersensitivity reactions in animals (allergies)
o
Research is underway to engineer tobacco to synthesize “human
-
compatible” glycans
Environmental Risks
Pharmaceutical products may inadvertently be introduced into the general
fo
od supply
Cross
-
pollination
o
Pollen from a drug
-
containing crop fertilizes a neighboring related crop
(or wild relatives) used for animal consumption
Wind
Insects
Consumption of GM plant by insects
Food chain
o
Accumulation in birds
–
extinction? (e.g. DDT
and bald eagle)
o
Deleterious effects on non
-
target organisms (NTO’s)
NTO’s = organisms in the environment that are affected by the
product unintentionally
Insects, arthropods
Risk to NTO’s
Depends on recombinant protein involved
Risk assessment carried out
case
-
by
-
case
o
Misrouting of crops seeds during processing
o
Alteration in soil microbes
o
Leaching of drug into the soil from the roots (soil contamination)
General Risk Assessment
o
Most biopharmaceuticals are proteins which have little biological
activity (e.g
. monoclonal antibodies, subunit vaccines)j
Digestible, little hazard of toxicity
o
Some biopharmaceuticals may be toxic in higher doses (e.g.
anticoagulants, hormones, enzymes)
o
Persistence in environment (lipophillic)
Management Strategies
o
Inducible genes p
ost harvest
o
Product activation post purification
o
Terminator technology to prevent pollen development
o
Government permits for field trials of drug
-
producing plants
o
Double distance between crops to prevent cross
-
pollination
–
Buffer
zones
o
Regulate planting o
f drug
-
containing crops indefinitely
o
Secluded or enclosed fields
o
Transgene tracking tools
o
Marker proteins to label specific biopharmaceutical plants
Rhizosecretion
o
Soil contamination has already been observed in GM plants producing
the
Bacillus thuringensi
s
toxin (Bt)
Biologically active Bt isolated 9 months after transgenic plant
was harvested
o
Taking advantage of rhizosecretion:
Roots of transgenic plants are submerged in hydroponic
solutions
Continuous secretion of recombinant proteins
Economical alternat
ive to downstream processing and
chemical extraction of active compounds
Attractive
–
but what about consequences and
regulation?
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