AGRICULTURAL

BIOTECHNOLOGY
Agricultural Biotechnology
Tissue culture and plant breeding
Transgenic plants and applications
Animal biotechnology
Livestock and breeding
Cause for concern

AGRICULTURAL BIOTECH
Agricultural biotechnology or Green
Biotechnology is biotechnology applied to
agricultural processes.

Biotechnology Supports
Sustainability
Enhances crop production for sustainable
food, feed, fiber and fuel supplies.
Promotes resource conservation and energy
efficiency.
Reduces environmental footprint of
agriculture.
Improves economic viability for farmers and
communities.
Advances product safety.
Sustainable Agriculture
An integrated system of plant and animal
production practices having a site-specific
application that will over the longer term:
1. Satisfy human food and fiber needs
2. Enhance environmental quality and the natural
resource base upon which the agriculture economy
depends;
3. Make the most efficient use of non-renewable
resources and on farm resources and integrate
where appropriate, natural biological cycles and
controls;
4. Sustain the economic viability of farm operations;
5. Enhance the quality of life for farmers and society as
a whole.
Satisfy human food and fiber
[and fuel] needs

Increased Yield = More Food
Corn: 30 percent increase in yield since 1996
Soybean: 22 percent increase in yield since 1996

Consumer Benefits
Fewer saturated fats and no trans-fats
Allergen free foods
Reduced toxins
Enhance environmental quality
Pesticide Applications :Down 500 million
pounds

No Till Farming : Up 35 percent

Carbon Dioxide Emissions: Down 10.2 million
tons
Efficient Use of
Non-renewable Resources
Water Availability
U.S. cotton farmers saved 93 million
gallons of water in last 6 years.
More crop per drop
Fuel
441 million gallons of fuel saved
Economic Viability of
Farm Operation
Farm Income: Up $27 billion
Production Costs: Down $1.3 billion
Quality of Life for
Farmers and Society
Development
Poverty alleviation
Improved health care
Educational opportunities
Enhanced farmer income translates to viable rural
communities
Sustainable Biofuels
Agricultural Biotechnology provides
enabling technology for:
Yield increases for corn ethanol and
soy biodiesel;
No till cropping for greater residue
collection for cellulosic biomass;
Dedicated energy crops
AGRICULTURAL
BIOTECHNOLOGY
Agricultural biotech can be divided to 2
main areas :
i) Plant biotechnology

ii) Animal biotechnology
PLANT BIOTECHNOLOGY
Can be divided into 2 main areas :
1.Plant Tissue Culture
2.Plant Genetic Engineering
PLANT TISSUE CULTURE
Broadly refers to technique of growing plant cells,
tissues,organs, seeds or other plant parts in a sterile
environment on a nutrient medium.
The first commercial use of plant propagation on artificial
media was in the germination and growth of orchid plants in
the 1920s
It was only after the development of a reliable artificial medium
by
Murashige & Skoog in 1962 that plant tissue culture really took
off
commercially.
Definition
the culture of plant seeds, organs,
explants, tissues, cells, or
protoplasts on nutrient media
under sterile conditions.
Why do plant tissue culture?
Fast commercial propagation of new cultivars
Agriculture
Fast selection for crop improvement nutritional
value,
pest control, hardiness
Cultivation virus free plants
Pharmaceuticals ginseng and taxol
Cloning of rare and endangered plants
Plant cultures easier and safer to export
History
First attempted by Haberlandt (1902) - grew palisade
cells from leaves of various plants but they did not
divide
1934 - White generated continuously growing culture
of
meristematic cells of tomato on medium containing
salts, yeast extract and sucrose and 3 vit B
(pyridoxine,
thiamine, nicotinic acid) established the
importance of
additives
1953 Miller and Skoog, University of Wisconsin
Madison discovered kinetin, a cytokine that plays an
active role in organogenesis
Basis for the cell culture
Plant cells are totipotent
Have the ability to develop into whole plants or plant
organs in vitro when given the correct conditions
Not all plant cells are totipotent. However, there are a
sufficient number of totipotent cells in the plant .
Basis for Plant Tissue Culture
Two Hormones Affect Plant
Differentiation:
Auxin: Stimulates Root Development
Cytokinin: Stimulates Shoot Development
Generally, the ratio of these two hormones
can determine plant development:
Auxin Cytokinin = Root Development
Cytokinin Auxin = Shoot Development
Auxin = Cytokinin = Callus Development
Factors Affecting Plant Tissue Culture
Growth Media
Minerals, Growth factors, Carbon source
Environmental Factors
Light, Temperature, Photoperiod
Explant Source
Types
Usually, the younger, less differentiated the explant, the
better for tissue culture
Genetics
1. Different species show differences in amenability to
tissue
culture.
2. In many cases, different genotypes within a species
will
have variable responses to tissue culture;
Choice of explant
Desirable properties
of an explant
Easily sterilisable
Juvenile
Responsive to culture
Shoot tips
Axillary buds
Seeds
Hypocotyl (from
germinated seed)
Leaves
Callus
Is a natural response of the plant tissue to wounding
A mass of actively dividing undifferentiated cells
produced by plant tissue explant
Callus can be
Resuspended in liquid media to create a
susupension
culture of single totipotent cells
Or differentiated into plant with the appropriate
manipulations of culture conditions
Steps in Plant Tissue Culture
Selection of the explant from a healthy mother plant

Establishment of the explant in a culture medium

Multiplication - the explant gives rise to a callus

Differentiation and organogenesis
What is needed?
Explant (some tissues culture better than others)
A suitable growth medium
Aseptic conditions
Growth regulators
Frequent subculturing to ensure adequate nutrition
and to avoid the build up of waste metabolites
Choice of explant
Desirable properties
of an explant
Easily sterilisable
Juvenile
Responsive to culture
Shoot tips
Axillary buds
Seeds
Hypocotyl (from
germinated seed)
Leaves
Media
When you make an explant
like an axillary bud, you
remove it from the sources of
many chemicals and have to
re-supply these to the
explants to allow them to
grow.
Shoot tip - Auxins
and Gibberellins
Roots - water, vitamins
mineral salts and cytokinins
Leaves -
sugars, GAs
Medium constituents
Inorganic salt formulations
Source of carbohydrate
Vitamins
Water
Plant hormones - auxins, cytokinins,
GAs
Solidifying agents
Undefined supplements
Carbohydrates
Plants in culture usually cannot meet
their needs for fixed carbon. Usually
added as sucrose at 2-3% w/v.
Glucose or a mixture of glucose and
fructose is occasionally used.
For large scale cultures, cheaper
sources of sugars (corn syrup) may be
used.
Plant hormones
(Growth regulators)
Auxins
Cytokinins
Gibberellic acids
Ethylene
Abscisic Acid
Plant Growth Regulator-like
compounds
Cytokinins
Absolutely essential (no mutants known)
Single natural compound, Zeatin. Synthetic
analogues Benyzladenine (BA), Kinetin.
Stimulate cell division (with auxins).
Promotes formation of adventitious shoots.
Produced in the root meristem and
transported throughout the plant as the
Zeatin-riboside in the phloem.
Breeding Applications of Tissue Culture
Micropropagation
Germplasm preservation
Somaclonal variation
Embryo culture
Haploid & dihaploid production
In vitro hybridization protoplast
fusion
Plant genetic engineering


Steps of Micropropagation
Stage 0 Selection & preparation of the mother
plant
sterilization of the plant tissue takes place

Stage I - Initiation of culture
explant placed into growth media

Stage II - Multiplication
explant transferred to shoot media; shoots can be
constantly divided

Stage III - Rooting
explant transferred to root media

Stage IV - Transfer to soil
explant returned to soil; hardened off
Through micropropagation, it is now possible to
provide
clean and uniform planting materials in plantations.
Micropropagated plants were found to establish
more quickly, grow more vigorously and taller, have a
shorter and more uniform production cycle, and
produce
higher yields.
Somatic Embryogenesis
The process of initiation and development of
embryos or embryo-like structures from somatic
cells
The production of embryos from somatic or non-
germ cells.
Usually involves a callus intermediate stage which
can result in variation among seedlings
Not a common micro-propagation technique but is
currently being used to produce superior pine
seedlings
Somatic Embryos
Tissue culture maintains the genetic of
the cell or tissue used as an explant
Tissue culture conditions can be
modified to cause to somatic cells to
reprogram into a bipolar structure
These bipolar structures behave like a
true embryo - called somatic embryos
Plant tissue culture belongs to the lower end of the
agricultural biotechnology ladder.
Plant tissue culture is a straightforward technique
and many developing countries have already
mastered it.
Its application only requires a sterile workplace,
nursery, and green house, and trained manpower.
Unfortunately,tissue culture is labor intensive, time
consuming, and can be costly.
Why do Plant Tissue Culture?
A single explant can be multiplied into several
thousand plants in less than a year this allows fast
commercial propagation.
Taking an explant does not usually destroy the
mother plant, so rare and endangered plants can be
cloned safely.
Once established, a plant tissue culture line can
give a continuous supply of young plants
throughout the year.

In plants prone to virus diseases, virus free explants
(new meristem tissue is usually virus free) can be
cultivated to provide virus free plants
Plant tissue banks can be frozen, then regenerated
through tissue culture.
Plant cultures in approved media are easier to export
than are soil-grown plants.
Agricultural biotechnology also uses genetic engineering which is a process of
inserting a foreign gene into a plant/animal cell and cloning that cell into a genetically
engineered crop/animal.
Agricultural Biotechnology:
PLANT GENETIC
ENGINEERING
Classical Plant Breeding:
Genetic modification following
introduction of large amounts of
undefined DNA from a genetically
similar source.
Plant Biotechnology:
Genetic modification of a plant
cell by introduction of defined
DNA(s) from a genetically
different source.
Cloning

Have you ever eaten/used cloned plants?
in Plants
Cotton
Poplar
Xmas Cactus Fern
Soybean Ohio Buckeye
What is a transgenic?

Transgene the genetically engineered
gene added to a species
Transgenic an organism containing a
transgene introduced by technological
(not breeding) methods
GMOs - Genetically modified organisms
GMO - an organism that expresses traits
that result from the introduction of foreign
DNA

Advantages of Plants for
Genetic Engineering
Can reproduce asexually and produce
large numbers offspring.
Clone themselves
Can be grown in tissue culture
What kinds of features are desirable to
genetically engineer into plants?
I) Herbicide resistance
What are herbicides ?
Compounds that kills plants!
Weed control
II) Insect resistance
III) Bio Pharming
IV) Improvement of nutritional value
V) Delayed ripening

Agrobacterium Tumefaciens : Natures
plant genetic engineer
Causes Crown Gall disease in
dicotyledenous plants.
Infected plants grow "Galls" or tumors
R. Shanthini
11 March 2010
Bt toxin bred GM crop
Bacillus thuringiensis (Bt) is a common
soil bacterium that produces crystals
containing proteins that are toxic to certain
insects.
The Bt gene was successfully inserted into the
genome of several crops.
The insertion of the Bt gene directly into the genome of crops allowed the crops to
constantly produce Bt toxin crystals in all tissues of the plant. No application of any
pesticide is required to protect the crop from a large number of pests.
Crop productivity could be increased by introducing such
qualities as disease resistance and increased drought
tolerance to the crops.


Genes from naturally drought-resistant plants can be
used to increase drought tolerance in many crop
varieties growing in dry climates so that crops shall use
water as efficiently as possible.
Source: http://www.ctahr.hawaii.edu/gmo/risks/benefits.asp
Advantages : Agricultural Biotechnology


Increased crop productivity

Advantages : Agricultural Biotechnology

Decreased application of pesticide /
herbicide.
Perennial production of crops non
seasonal dependence.
Shorter life cycle for crops.




Agriculture Transgenics On
the Market

Insect resistant cotton Bt toxin kills the
cotton boll worm

transgene = Bt protein
Insect resistant corn Bt toxin kills the
European corn borer

transgene = Bt protein
Virus resistance - papya resistant to
papaya ringspot virus
transgene = virus coat protein
Source: Monsanto
Herbicide resistant crops
Now: soybean, corn, canola
Coming: sugarbeet, lettuce, strawberry
alfalfa, potato, wheat (2005?)
transgene = modified EPSP synthase or
phosphinothricin-N-acetyltransferase
Biotech chymosin; the enzyme used
to curdle milk products
transgene = genetically engineered enzyme
bST; bovin somatotropin; used to increase
milk production
transgene = genetically engineered enzyme
Source: Rent Mother Nature
Source: Chr. Hansen
Next Generation of Ag Biotech Products
Source: Minnesota
Microscopy Society
Golden Rice increased Vitamin A content
(but not without controversy)
transgene = three pathway enzymes
Sunflower white mold resistance
transgene = oxalate oxidase from wheat
Turfgrass herbicide resistance;
slower growing (= reduced mowing)
Bio Steel spider silk expressed in goats; used to
make soft-body bullet proof vests (Nexia)
First biotech plant
product Flavr
Savr tomato
Bt Corn Reduces:
Insecticide Mycotoxin
Application Production
X
Monarch larvae on
Butterfly weed
Animal Biotechnology
Transgenics are genetically modified organisms
with DNA from another source inserted into their
genome
A large number of transgenic animals have been
created
Mice Cows Pigs Sheep Goats Fish Frogs
Insects
Currently, no transgenic animal or animal
product is approved by the FDA or USDA for
human consumption
Some of the goals of transgenic animal
creation are:
Research into animal and human disease
Improve livestock animals
Use of animals as bioreactors
Transgenic Animal Creation
Microinjection
into the germ line -> transgenic animal
Gene injected into the male
pronuclei
Eggs are infected prior to fertilization
Virus integrates into one of the chromosomes
Recombinant Defective Retrovirus
Some of the drawbacks of these methods are:
The inserted DNA randomly integrates into the
genome
The eggs must be harvested & fertilized in
vitro
More than one copy of the gene may get into
the genome
Transgenic Animal Generation
Examples of Transgenic Animals
Transgenic Cattle



Dairy cows carrying extra copies of two types of casein
genes produce 13% more milk protein
Not only will this make the milk more nutritious, it would
allow for less milk to make more cheese
Currently the milk from these animals is under FDA review
The important difference between this & other transgenics
is that the DNA added is not foreign
EnviroPig
TM

Transgenic pigs express phytase in their salivary glands
Phytic acid in the pig meal is degraded releasing phosphorus
The phosphorus is absorbed by the pig
Normally the phytic acid/phosphorus complex passes
through the pig and is excreted as waste
Pig waste is a major pollutant & can cause eutrophication of
lakes & streams
http://www.nature.com/news/2005/050221/images/nbt0305-283-I1.jpg
Transgenic Fish
Tilapia
Salmon/trout
Catfish
Can grow up to 6 times faster than wildtype fish
Most have extra copies of growth hormone (GH) gene
Transgenic
Wildtype
http://www.nature.com/nbt/journal/v19/n6/images/nbt0601_500a_I1.jpg
Concerns if these supersized transgenic fish got loose
Transgenic fish are farm-raised, isolated from wild stocks
But even during farming of wildtype fish, escapes happen
frequently (~14 million/yr)
What would happen if a large number of transgenic escaped
& started breeding with wild fish?
http://pewagbiotech.org/buzz/index.php3?IssueID=10
In experiments, transgenic males mated 3x
more frequently than the smaller wild males
Offspring of transgenic males lived <70% as
long as wild males
Could lead to a decline of the wild fish
population & endanger a species as whole
http://pubs.acs.org/hotartcl/chemtech/99/jun/fletcher.html
+Antifreeze
wild
transgenic
Antifreeze Proteins (AFP)
AFPs lower the freezing temperature of blood & fluids
Trout normally do not survive in water below 0.6C
Transgenic trout containing an AFP gene & promoter can
survive in waters as cold as 1.2C
Animal Bioreactors
Pharming
http://nolswf.bbc.net.uk/science/genes/gene_safari/pharm/a_pharming.shtml
1997, Tracy the sheep, the first transgenic animal to
produce a recombinant protein drug in her milk
alpha-1-antitrypsin (AAT) treatment for emphysema &
cystic fibrosis
Created by PPL Therapeutics & The Roslin Institute
Webster and Peter
Nexia Biotechnologies transfered the silk gene from Orb
spiders into goats
The resulting male goats were used to sire silk-producing
female goats
Each goat produces several grams of silk protein in her
milk
The silk is extracted, dried to a white powder, and spun
into fibers
The fibers are stronger and more flexible than steel
Transgenic male kids
carrying silk gene
GTC Biotherapeutics has received approval to sell human
anti-thrombin (ATryn) purified from goats milk in Europe
Technology is not restricted to cows, goats, & sheep
There is interest in using rabbits since housing costs are
significantly less & generation time is faster
Chickens which produce recombinant drugs in their eggs
have been produced by The Roslin Institute
Other Types of Transgenic Animals
Transgene ->
Gene coding
for a growth
hormone


ANDi, the first transgenic primate born in January, 2000
224 unfertilized rhesus eggs were infected with a GFP virus
~Half of the fertilized eggs grew and divided
40 were implanted into twenty surrogate mothers
five males were born,two were stillborn
ANDi was the only live monkey carrying the GFP(Green Fluorescent Protein ) gene
http://www.ohsu.edu/unparchive/2001/011001andi.shtml
Alba, the EGFP (enhanced GFP) bunny
Created in 2000 as a transgenic artwork
http://www.ekac.org/gfpbunny.html#gfpbunnyanchor
http://news.aol.com/story/_a/glowing-pig-passes-genes-to-piglets/20080109143909990001?ncid=NWS00010000000001
Transgenic Pigs Pass on the Transgene
GloFish, originally developed in Singapore as a way to monitor water pollution
The normally black-and-silver zebrafish was turned green or red by inserting
various versions of the GFP gene
Glofish are on sale throughout the US except in California
Glofish retail for about $5 per fish. Normal zebrafish cost around one tenth of the
price
http://www.nus.edu.sg/corporate/research/gallery/research12.htm
Mouse Knock-out Technology
Gene Targeting
A knockout mouse is a laboratory
mouse in which researchers have
inactivated, or "knocked out," an
existing gene by replacing it or
disrupting it with an artificial piece of
DNA.
The loss of gene activity often causes
changes in a mouse's phenotype.
Knock-out technology allows for the specific loss of
a gene in mice
Allows for the function of the KOd gene to be
deduced from the defects seen in the mice can be
used to mimick some disease
Unlike traditional transgenics the trangene is
targeted to a specific site in the DNA of the mouse
http://tasq.uq.edu.au/blasto.html
Blastocyst Injection
Blastocyst ES cells
http://bunseiserver.pharm.hokudai.ac.jp/gihou/knockout.html
Chimeric mouse
The brown fur comes from ES cells injected into the blastocyst
of an albino mouse
Some Examples of Knockout Mice
Many of these knockout mouse models are named
after the gene that has been inactivated. For
example, the p53 knockout mouse is named after the
p53 gene which codes for a protein that normally
suppresses the growth of tumors by arresting cell
division.
p27 knockout mouse is bigger than the control
This is not due to obesity, but the skeletal structure is increased in size
(everything about the mouse is larger)
http://www.bioreg.kyushu-u.ac.jp/saibouE.html
p27 knockout mouse
http://www.bbc.co.uk/science/genes/gene_safari/wild_west/bigger_and_better02.shtml
GDF8 (Myostatin) knockout mouse
Myostatin inhibits muscle differentiation and growth.
Over twice the muscle mass of a wildtype mouse
normal knockout
Naturally Occurring GDF8 Mutants
FGF5 knockout mouse has long, angora-like hair
Clones and Cloning
Dolly as a lamb with her
surrogate mother
Dolly, First Mammal Cloned From an Adult Cell
Dolly, as an adult
Somatic Cell
Nuclear Transfer
What Has Been Cloned So Far?

Somatic Cell Nuclear Transfer
Sheep, Goat, Mouse, Rabbit, Cattle (domestic & wild),
Pig, Horse, Mule, Dog, Cat (domestic & wild), Deer

Embryo Splitting (Twinning)
Sheep, Cattle, Primate (Rhesus)
Cat Clone
Donor Surrogate mother with clone (CC)
Out of 87 implants only CC survived to birth

































































































































































































http://www.usatoday.com/news/science/2003-01-21-cloned-cats_x.htm
Donor & Clone
Rainbow & CC
Cloned transgenic cat containing red fluorescent protein
http://news.aol.com/story/_a/glowing-pig-passes-genes-to-piglets/20080109143909990001?ncid=NWS00010000000001
Transgenic Clones
http://news.nationalgeographic.com/news/2003/05/0529_030529_muleclone.html
Idaho Gem, first cloned mule
1
st
try 134 implants 2 pregnancies, both failed
2
nd
try 113 implantations 14 pregnancies, one birth
Surrogate
mother
(horse)
In addition to cloning pets or prized livestock,
researchers are looking to clone transgenic animals
This would allow for more uniform expression of
transgenic genes
Not all transgenic animals express their transgenic
genes at equal levels
Also allows for the rapid expansion to large flocks
or herds of transgenic animals


http://www.newrivervalley.com/biotech/day1.html
Piglets clones created by PPL Therapeutics in 2000
The piglets carry a silenced copy of alpha 1,3 galactosyl
transferase, or GT, an enzyme involved in organ rejection
In order to guarantee compatibility a second GT gene must
also be silenced
Conservation Cloning
Many endangered or extinct animals are being cloned or
considered for cloning
Gaur
Bucardo mountain goat
Mammoth
Quagga
Banteng
Mammoth Quagga
Bucardo Gaur
Noah, a Banteng clone created by Advanced Cell Technologies
Banteng are endangered wild bovine from Southeast Asian
This clone was created from frozen tissue of an animal that
died in 1980
3 Pig clones, born in 2002, died of heart attacks due to adult
clone sudden death syndrome within days of each other by
the time they were 6 months old.
Dolly had a weight problem, telomeres 20% shorter than
normal, she suffered from arthritis, and finally lung cancer
due to an infection for which she was finally euthanized at
age 6yrs.
The success rate ranges from 1 to 3% this contrasts to in
vitro fertilization which has a success rate of 50 to 20%
Problems with Cloning
http://fig.cox.miami.edu/~cmallery/150/gene/cloning.success.jpg
Nearly all clones show some genetic anomalies
Some suffer from placental defects others cardiac defects
Many suffer from large offspring syndrome (LOS)
Normal mouse pup Cloned mouse pup
suffering from LOS