Biotechnology in

Agriculture
An introduction from Genetic
to improvement in Agriculture

19 February

Ryza Aditya

Outline
World Population
Genetic modification consequence
of cultivation
genetic technologies to improved
plants
GM example and principal
Molecular Farming
Conclusion

More than one billion people are

chronically hungry, and more than two
billion people do not get adequate
vitamins or minerals in their diet

www.wfp.org

Children are particularly vulnerable

to dietary deficiencies
Crop failures especially in
vulnerable regions can lead to
famines
War, conflict and corruption can
interfere with food distribution
Poverty contributes to chronic
malnutrition

Image courtesy CDC/ Dr. Lyle Conrad (number 6901) Image courtesy CDC (number 6903)

Vitamin A deficiency is a leading

cause of blindness
100 million
children are
Vitamin A
deficient
Up to 500,000
children become
blind every year
and half of these
die within 12
months of losing
their sight
Occurrence of
Vitamin A
deficiency
(VAD)
b.usuhs.mil/biochem/nutrition/NOTES/

Improving dietary
intake of
carotenes can
reduce child
mortality by 25 %

WHO data

Many people suffer deficiencies of

other vitamins and minerals
Pellagra is a
deficiency of
niacin (vitamin
B3)
Nutritional Rickets is a
condition in which
bones are too soft due
to a deficiency of
vitamin D

Inflamed,
scorbutic
gums caused
by vitamin C
deficiency

Anemia caused by iron deficiency

affects half of all pre-school aged
children
Image courtesy CDC (number 6179) Image courtesy CDC (number 3998) Image courtesy CDC (number 2435)

Weve removed mountains

1984

2009

2 km
The Hobet coal mine in Boone
County, West Virginia spreads
over 10,000 acres (15.6 square
miles)
NASA images by Robert Simmon, based on Landsat 5 data from the USGS Global Visualization Viewer.

...dammed
rivers...

1987

2006
Three Gorges Dam, the world's
largest hydroelectric power
generator. The reservoir just
upstream of the dam is more
than 2 miles (3 km) across

+ View movie of
the dam's constr
uction

Photo credit: NASA/Goddard Space Flight Center Scientific Visualization Studio United States Geological Survey and Le Grand Portage

....caused
extinctions.....
The dodo (Raphus cucullatus) was a
large flightless bird indigenous to
Mauritius. The arrival of humans led
to the dodos extinction by the end of
the 17th century.

Dodo by Edouard Poppig, 1841

...modified other species

in extraordinary ways

... and modified plant genomes for

thousands of years......

Image credits: P. Cos, Cacaphony, USDA, CIMMYT

Plant domestication followed the end

of the most recent glacial period
Glacial retreat
Glacial advance

thousand
years ago

Wild gathering

Predomestication
cultivation

Domestication

Neolithic
Revolutio
n
Allaby, R.G., Fuller, D.Q., and Brown, T.A. (2008) The genetic expectations of a protracted model for the origins of domesticated crops. Proc. Natl. Acad.
Sci. USA 105: 13982-13986, copyright National Academy of Sciences USA

Genetic modification arose as a

consequence of cultivation
Planting seeds from
good plants increased
their representation in
subsequent generations

Natural
variation
within
population

Image courtesy of University of California Museum of Paleontology, Understanding Evolution - www.evolution.berkeley.edu

During maize domestication cob

size increased
Cobs from
archeological
sites in the
Valley of
Tehuacan,
Mexico

7000
years ago

500
years
ago
Photo Robert S. Peabody Museum of Archaeology, Phillips Academy, Andover,
Massachusetts. All Rights Reserved.

The hard casings around many

grains were eliminated
Teosinte, the wild
relative of maize,
has hard
coverings over
each grain.
Humans selected
against these
during maize
domestication.

Photo by Hugh Iltis; Reprinted from Doebley, J.F., Gaut, B.S., and Smith, B.D. (2006). The Molecular
Genetics of Crop Domestication. Cell 127: 1309-1321, with permission from Elsevier.

Basic research contributes to food

affordability

Higher yields
Less loss to drought,
flooding, temperature
extremes
Less loss to pests and
pathogens
Improved shelf life
Better understanding of
plant biochemistry, nutrient
uptake, photosynthesis,
growth and development
Improved tools for breeding
and genomics

Flooding-tolerant rice

Virus-resistant tomatoes

Broccoli that
stays fresh
longer
Reprinted by permission from Macmillan Publishers Ltd. (NATURE) Xu, K., Xu, X., Fukao, T., Canlas, P., Maghirang-Rodriguez, R., Heuer, S., Ismail, A.M., Bailey-Serres, J., Ronald,
P.C., and Mackill, D.J. (2006).
Sub1A is an ethylene-response-factor-like gene that confers submergence tolerance to rice. Nature 442: 705-708. ; Broccoli photos courtesy Jocelyn Eason, Plant and Food, New Zealand; Anderson, L.K., Lai, A., Stack,
S.M., Rizzon, C. and Gaut, B.S. (2006). Uneven distribution of expressed sequence tag loci on maize pachytene chromosomes. Genome Research. 16: 115-122; William M. Brown Jr., Bugwood.org

Plant Breeding and genetic

technologies

Mendel and Darwin paved the way

for scientific plant breeding

Norman Borlaug was a plant breeder,

and father of the green revolution
One of the most significant
accomplishments of 20th
century science was the
development of lodgingresistant, high-yielding
semi-dwarf grain varieties

Distinguished plant breeder and Nobel

Laureate
Norman Borlaug 1914-2009

Rice breeding at IRRI also brought

huge yield increases
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0

1961

198
2000
World rice yield0 (ton/ha) (FAO)

IR8, released in 1966, was to tropical rices

what the Model T Ford was to automobiles. It
was known as miracle rice because of its
high yields.
Photo courtesy IRRI

Advances in genetic technologies

contribute to improved plants

Marker assisted selection

Genome-wide association
studies
Recombinant DNA technology
and transgenic plants

Photo credit: IRRI

Marker assisted selection

(MAS)

Phenotype:
physical
expression of traits

Genotype: sequence of all

the genes in a genome
Photo credit LemnaTec; Anderson, L.K., Lai, A., Stack, S.M., Rizzon, C. and Gaut, B.S. (2006). Uneven
distribution of expressed sequence tag loci on maize pachytene chromosomes. Genome Research. 16: 115-122.

Marker assisted selection

(MAS)

Selecting for DNA

markers is faster
than selecting for
phenotype

Phenotype:
physical
expression of traits

Genotype: sequence of all

the genes in a genome
Photo credit LemnaTec; Anderson, L.K., Lai, A., Stack, S.M., Rizzon, C. and Gaut, B.S. (2006). Uneven
distribution of expressed sequence tag loci on maize pachytene chromosomes. Genome Research. 16: 115-122.

How markers work: Each generation,

genes reassort or shuffle

Markers let us see

which genes each
individual has
inherited

Example: Introgression of a disease

resistance gene
We want to add a
disease resistance
trait to an elite
tomato plant.

Elite tomato

Poor tomato but

disease resistant
(resistance gene indicated)

Example: Introgression of a disease

resistance gene

We cross the two plants.

Some of their progeny
inherit the disease
resistance trait, some dont
how can we tell the
difference?
Photo by Stephen Ausmus USDA

Example: Introgression of a disease

resistance gene

We can use markers

to look at their DNA
and identify those with
the resistance gene.
Its faster and easier
than infecting them to
see the phenotype

Example: Introgression of a disease

resistance gene

Is this an elite, diseaseresistant tomato? No, half of

its genes are from the poor
tomato

Example: Introgression of a disease

resistance gene

We have to repeatedly
cross back to the elite
tomato, using markers
to identify plants with
the disease resistance
gene

Example: Introgression of a disease

resistance gene
Markers greatly
accelerate
breeding programs

After several
generations,
elite, disease
resistant
tomato

PA CHIAM
SERAUP
BESAR 15

FORTUNA

M ARONG
PAROC

original rice
genome
BPI 76

One of the most

widely grown crops,
indica rice IR64 is
the product of a
complex breeding
program that has
caused extensive
genomic
modification,
mutation, deletion
and rearrangement

UNKNOWN

BLUE ROSE
SUPREM E

REXORO

KITCHILI SAM BA

Mutatio

ns

CINA

Breeding tree
of Indica Rice
IR64

SINAWPAGH

UNKNOWN

LATISAIL

TEXAS
PATNA

RSBR

GEB24

Recombinatio
ns

PETA
DGWG

SLO 17

IR86

CP SLO 17

CHOW SUNG

VELLAIKAR
IR400

TSAI Y UAN CHUNG

M UDGO
TETEP

IR1163

IR127

NM S 4

TADUKAN
IR1006

SIGADIS

NAHNG M ON S4

IR262

IR1103

BENONG

Inversions

IR95
IR8

Translocatio
ns
BLUE BONNET

CP231

CO 18

IR238

TN1
IR1416

IR1402

O. nivara
IR1870

IR1641

IR22

IR1614

TKM 6

IR2006

IR579
IR773 A

BPI 121
IR1915 B

Deletions

IR746A

IR747

IR24/ IR661

IR1704

IR1721

GAM PAI
IR1833

IR1916

GAM PAI 15

IR1561

IR833

IR1737
IR2040

IR2146

IR 2055
IR2061

IR5236

IR5338

IR5657

IR18348

IR64

IR 64

Ultimate Landraces
GAM PAI
DEE GEO WOO GEN
CINA
LATISAIL
TADUKAN
KITCHILI SAMBA
PA CHIAM
SERAUPBESAR 15
NAHNG MON S 4
VELLAIKAR

TSAI YUAN CHUNG

BENONG
Unknow n
CHOW SUNG
MUDGO
TETEP
SINAWPAGH
UNKNOWN (JAPANESE)
O. nivara (IRGC 101508)
MARONG PAROC

Slide courtesy of Ingo Potrykus

GM

Agrobacterium tumefaciens
pathogen and useful tool
Agrobacterium tumefaciens
is a plant pathogen that
induces tumors on about
60% of dicotyledonous
angiosperms and
gymnosperms

Cherry

Its tumor-inducing property also

makes it a valuable tool for
introducing genes into plants for
research and agricultural
purposes

Casimiro,I.,Marchant,A.,Bhalerao,R.P.,Beeckman,T.,Dhooge,S.,Swarup,R.,Graham,N.,Inz,D.,Sandberg,G.,Casero,P.J.andBennett,M.
(2001).AuxintransportpromotesArabidopsislateralrootinitiation.PlantCell.13:843852.HerbPilcher

Genetic Modification (GM) is another

breeding method
Recombinant DNA
(or GM) allows a
single gene to be
introduced into a
genome. This
method can be
faster than
conventional
breeding

Elite tomato

Poor tomato but

disease resistant

Elite, disease resistant tomato

Source of gene
(disease-resistant
plant)
Gene of interest

Isolate gene
of interest
using
molecular
biology
methods

Once a gene is
introduced into
the plant
genome it
functions like any
other gene

Recombine into
recipient plant
DNA

Why are GM methods used sometimes

and molecular breeding others?
Molecular breeding

1. Desired trait must

be present in
population

2. Genetic resources
must be available

3. Plant should be
propagated
sexually

Photo credits: Gramene.org

Why are GM methods used sometimes

and molecular breeding others?
Molecular breeding

1. Desired trait must

be present in
population

1. Gene can come

from any source

2. Genetic resources
must be available

GM

2. Genetic resources
not required

3. Plant should be
propagated
sexually

3. Plant can be
propagated
vegetatively
Photo
Photocredits:
credits:Gramene.org
Gramene.org ETH Life International

GM Example: Disease resistant banana

by introduction of a gene from pepper

Resistant

Susceptible

Banana bacterial wilt is destroying

plants in eastern Africa. Transgenic
plants carrying a resistance gene from
pepper are resistant to the disease
Tripathi, L., Mwaka, H., Tripathi, J.N., and Tushemereirwe, W.K. (2010). Expression of sweet pepper Hrap gene in banana
enhances resistance to Xanthomonas campestris pv. musacearum. Molecular Plant Pathology 11: 721-731.

GM Example: Insect resistance through

introduction of the Bt gene
Wild-type peanut plant

Peanut plant expressing the Bt gene

Photo by Herb Pilcher USDA

Bacillus thuringiensis (Bt) bacteria

produce insecticidal proteins
Bacillus
thuringiensis
expressing Bt
toxin

Bacillus thuringiensis
expressing insecticidal
Bt toxin can be sprayed
onto plants

Plant cell
expressing Bt
toxin

Or the plants can be

engineered to express
the Bt gene coding for
Bt toxin

The effect of Bt toxin is highly

specificIntestine
Bacillus
thuringiensis
expressing Bt
toxin

Processing
Plant cell
expressing Bt
toxin

Intestinal
lumen

Receptor
binding

The Bt toxin affects only some insects

because to be effective it has to be
processed and bind to a specific
receptor protein

The effect of Bt toxin is highly

specificIntestine
Bacillus
thuringiensis
expressing Bt
toxin

Plant cell
expressing Bt
toxin

After binding, the insecticidal proteins

assemble to form a pore in the lining of
the insect intestine which kills the insect

Pore
assembly

GM Example: Herbicide
resistance
Plants compete with other
plants for sunlight and
nutrients. Many farmers
use herbicides to eliminate
weeds (undesired plants)
from their fields.

t corn rows sprayed with herbicide to eliminate competing plants

ht corn being choked by giant foxtail (Setaria faberi)

Photo credit: Doug Buhler, Bugwood.org

Herbicide tolerant plants are

environmentally friendly
Farmers that plant herbicidetolerant crop plants use less
herbicide, herbicides that are less
toxic, and till (plow) less, saving
soil and fuel.

Soybean

*
Cerdeira, A.L. and Duke, S.O. (2006). The Current Status and Environmental Impacts of Glyphosate-Resistant Crops. J.
Environ. Qual. 35: 1633-1658. Photo credit Hunt Sanders, University of Georgia, bugwood.org.

Breeding plants for -carotene (provitamin A) enrichment

Vitamin A deficiency is a leading cause of blindness

Image sources: Petaholmes based on WHO data;

Enhanced -carotene content in food can

prevent vitamin A deficiency
Many staple foods
are poor sources of carotene so many
people do not get
adequate vitamin A in
their diet

-carotene is converted
to vitamin A in the
human body

-carotene

Vitamin A

GM of Synthesis, storage and

breakdown all affect -carotene
content Golden Rice
To increase betacarotene levels in
plants, you need
more synthesis,
more storage or
less catabolism

GGPP
Synthesis phytoene
lycopene
-carotene

Vitamin A

Storage

Chromoplasts
organelles that store
carotenoids

Catabolis
m or
conversion
to other
forms
-carotene
makes the
rice look
golden

Photo credit: University of Wisconsin

Transgenic crops continue to be

developed and adopted
Global area of biotech crops
Million Hectares (1996 2011)

Cotton - Bt cotton (4.5

million acres)
Rice - Resistant to pest,
drought and salinity
Maize - Resistant to
herbicide and pests
Pulses - Pest tolerant chick
peas, pigeon peas
Ground nuts - Resistant to
peanut clump virus
Potatoes with high protein
content.
Tomatoes - Fungal
resistance
Sugarcane - Resistant to
the fungal disease red rot
Source: Clive James, ISAAA

What risk assessments are

performed on GM crops?
Before release into the environment, GM crops are subject to riskassessment and risk-management measures to evaluate:
Risks to human health (including toxicity and allergenicity)
Risks of evolution of resistance in target pathogens or pests
Risks to non-target organisms
Risks from movement of transgenes

Molecular Farming

Plant molecular farming (Biopharming)?

The use of modified plants to produce
therapeutically active proteins or industrial
products
The end product of crop harvests is not food,
feed or fibers, but plant-made pharmaceuticals
(PMPs)
Biopharming industry may be worth $100 billion
by 2015
50,000 /40 kg

Plant
Products
I.

Plant-derived pharmaceuticals (non-GE)

Over 120 pharmaceutical products currently in

use are derived from plants, mainly from tropical
forest species
II. Plant-made pharmaceuticals and industrial products
(GE)
1. Pharmaceuticals

Recombinant human proteins

Therapeutic proteins

Enzymes

Antibodies (plantibodies)

Vaccines
2. Industrial products

Proteins

Enzymes

Modified starches

Oils

Waxes

Plastics

Why use plants?

1. Plants are the cheapest, most abundant source of
protein on the planet: low cost
2. Plant provide low upstream production costs : low cost
3. Plants, as eukaryotes, can express and process most
prokaryotic and eukaryotic proteins: stability
4. Plant provide scalable production capacity and
flexibility: easy scale up
5. Plant seed permit stockpiling of inexpensive inventory:
good storage
6. Plants are free from animal and human pathogens:
safety
Golgi complex

Plant Golgi
-1,2-Mannosidase I

1,2-mannosidase I

UDP-GlcNAc
Transporter

GnT1

N-acetyl-glucosaminyltransferase I (GnT-I)

UDP-GlcNAc
Transporter

-mannosidase II

GnT-II
UDP-Gal
Transporter

GalT

CMP-NeuAc
synthetase

SAT

50,000 /40 kg

Complex Type glycan

-Mannosidase II
Fuc-Transferase
F

GnT-II

Xyl-Transferase

F X N-acetyl-Glucosaminidase
F X
Complex Type

Comparison of pharmaceutical
expression systems
Expression
system

Advantages

Disadvantages

Applications

Established regulatory track; wellunderstood genetics; cheap and easy to

grow

Proteins not usually secreted;

contain endotoxins; no
posttranslational modifications

Insulin (E. coli; Eli Lilly);

growth hormone (Genentech);
growth factor; interferon

Yeast

Recognized as safe; long

history of use; fast; inexpensive;
posttranslational modifications

Beer fermentation;
recombinant vaccines;
hepatitis B viral vaccine;
human insulin

Insect
cells

Posttranslational modifications;
properly folded proteins; fairly high
expression levels

Overglycosylation can ruin

bioactivity; safety; potency;
clearance; contains
immunogens/antigens
Minimal regulatory track; slow
growth; expensive media;
baculovirus infection (extra
step); mammalian virus can infect
cells
Expensive media; slow growth;
may contain allergens/
contaminants; complicated
purification
Little regulatory experience;
potential for viral contamination;
long time scales; isolation/GMPs
on the farm
Potential for new contaminants
(soil fungi, bacteria, pesticides);
posttranslational modifications;
contains possible allergens

Bacteria

Mammalian
cells

Usually fold proteins properly; correct

posttranslation modifications; good
regulatory track record; only choice
for largest proteins

Transgenic
animals

Complex protein processing; very high

expression levels; easy scale up; lowcost production

Transgenic
plants

Shorter development cycles; easy seed

storage/scaling; good expression levels;
no plant viruses known to infect humans

Note. Data from BioPharm International (2003).

Cost per
gram

$50100

Relatively new medium;

Novavax produces virus-like
particles
Tissue plasminogen activator;
factor VIII (glycoprotein);
monoclonal antibodies
(Hercepin)
Lipase (sheep, rabbits; PPL
Therapeutics); growth
hormone (goats; Genzyme);
factor VIII (cattle)
Cholera vaccine (tobacco;
Chlorogen, Inc.); gastric lipase
(corn; Meristem); hepatitis B
(potatoes; Boyce Thompson)

$500
5,000

$2050

$1020

Status of Biotech Pharmaceutical

Production

Erythropoietein
Human Growth Factor
-Interferon
Malaria epitopes
Streptococcus Surface
Antigen IgA (Dental
Caries)
Carcinoembryonic
Antigen
Colon Cancer Antigens
Interleukin 10 (Chrohns
Disease; Inflammatory
Bowel Syndrome)
Glucocerebrosidase
(Gauchers Disease)
Interleukin 4
Urokinase (Breaks Blood
Clots)
Human Serum Albumin
Rabies Antigens
Hepatitis B Surface
Antigen
Rotavirus VP6
Labile Eneterotoxin
(Botulism)

Measles

Aprotinin (Protease
Inhibitor)

-Galactosidase (Fabrys
Disease)

Interferon 2a and 2b
Personalized NonHodgkins Lymphoma

Vaccines

Papilloma Virus Vaccines

Lysosomal Acid Lipase

(Womans Disease;
Atherosclerotic Plaques)

Personalized Vaccines for

Follicular Lymphoma

Trichosanthin (HIV)

Diagnostics for Ovarian

Cancer
Diagnostics for

Ecclampsia

Biomarkers for Alzheimers

Disease

Zonna pellucida ZB3 Protein

(Contraceptive)
Gastric Lipase (Cystic Fibrosis)
Creatine Kinase
Protein C (Anticoagulant)
Neutropenia (Granulocyte
Macrophage Colony Stimulating
Factor)
Epidermal Growth Factor
and Hemoglobin
Angiotensin Converting Enzyme
(Hypertension)
Insulin Like Growth Factor
(Diabetes)
Tissue Necrosis Factor
(Rheumatoid Arthritis)
HIV-1 Peptide
Lactoferrin
Substance P (Neuropeptide)
Animal Pharmaceuticals
(Vaccines)
Feline Parvovirus (Panleukopenia)
Canine Parvovirus
Bovine Foot and Mouth

Commercial Pharma/IndustrialCrop
Products
Product

Trade Name

Pharma
Crop

Source of
Genes

Commercial
Purpose

Company
Producing

Company Selling
(product number)

Aprotinin

AproliZean

Corn

Cow

Research and
manufacturing

ProdiGene

Not available

Aprotinin

Apronexin

Tobacco

Cow

Research and
manufacturing

LargeScaleBi
ology

Sigma Chemical Company

(A6103)

Avidin

Recombinant
avidin

Corn

Chicken

Research and
diagnostic reagent

ProdiGene

Sigma ChemicalCompany
(A8706)

B-glucuronidase
(GUS)

Not available

Corn

Bacteria

Research and
diagnostic reagent

ProdiGene

Not available

Trypsin

TrypZean

Corn

Cow

Research
andmanufacturing

ProdiGene

Sigma Chemical Company

(T3568 andT3449)

Lactoferrin

Not available

Rice

Human

Research

Ventria
Bioscience

Sigma ChemicalCompany
(L4040)

Lysozyme

Not available

Rice

Human

Research

Ventria
Bioscience

Sigma Chemical Company

(L1667)

Notes
(1) Information on specific commercial uses of pharmaceutical and industrialcrop products is not available to the public.
Some have potential applications for human or veterinary medicine but none has been approved by the Food and Drug
Administration, which regulates drugs.
(2) Most of these products have been commercialized through Sigma Chemical Company, which specializes in products for
research and diagnostic purposes. AproliZean and B-glucuronidase have been commercialized according to ProdiGene
documents, but their current availability is unknown.
(3) Unlike pharmaceutical corn, tobacco itself is not genetically engineered. Rather, a tobacco mosaic virus is engineered to
contain a gene for aprotinin. The engineered virus then produces aprotinin in tobacco plants infected with the virus.

The process of molecular farming

Golgi complex
1,2-mannosidase I

UDP-GlcNAc
Transporter

GnT1

-mannosidase II

GnT-II
UDP-Gal
Transporter

GalT

CMP-NeuAc
synthetase

SAT

Complex Type glycan

Host development

Recombinant DNA
Transformation
technology

Production of PMPs

regeneration

Purification

&Cultivation

Cell culture and harve

High anthocyanin tomatoes are also

being developed

WT

del/ros
(line N)

del/ros

del/ros

(line C)

(line N)
Cancer-prone mice fed
high-anthocyanin
tomatoes lived longer
than control mice

Butelli, E., Titta, L., Giorgio, M., Mock, H.-P., Matros, A., Peterek, S., Schijlen, E.G.W.M., Hall, R.D., Bovy, A.G., Luo, J. and Martin, C. (2008).
Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nat. Biotech. 26: 1301-1308.

Conclusion

-carotene
makes the
rice look
golden

There is no
inherently right
or wrong way to
enhance plant
nutritional
quality
-carotene

Vitamin A
The -carotene
enriched foods
shown here
have been
produced using
GM and non-GM
approaches
Photo credit: Golden rice humanitarian board

Are GM crops safe to eat?

All GM plants are subject to
extensive testing and
regulatory oversight and no
detrimental health effects
have been identified

Bt corn is less prone

contamination by fungi
which produce toxins
linked to cancer and birth
defects

YES

GM biofortification can
ensure that all children
get adequate levels of
protein, vitamins and
mineral nutrients.

GM is a safe and
beneficial tool in the
quest to sustainably
feed the growing
population
Photo credit: Neil Palmer/ CIAT

Thank you

Reference / Sources
Teaching tool in Plant Biology www.theplantcell.org
Molecular farming slides: PBTL, Gyeongsang Nat. Univ.
And some other online resources