Agricultural

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?

