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7. Molecular Biotechnology in Agriculture7. Molecular Biotechnology in Agriculture
Bi7430 Molecular Biotechnology
Definition of green biotechnology
Genetic engineering of plants
Genetic engineering of animals
Biopharming
GMO benefits and controversies
Outline
Green (agricultural) biotechnology
green biotechnology applied to agricultural processes
environmentally-friendly solutions as alternative to traditional
agriculture, horticulture, and animal breeding processes
modification of plants and animals increasing value in agriculture
traditiona l agricultu r e – selectiv e crossbreed in g and hybridi za t ion
modern molecular biotechno lo g y – transgenes is (rDNA)
transgenic organism - altered by addition of exogenous DNA
transgene – DNA that is introduced
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> 150 different plant species in 50 countries worlwide
DNA sequence of A. thaliana (2000), rice (2005), cotton (2006),
corn (2009), potato (2011), tomato (2012) …
transgenic plants engineered to
overcome biotic and abiotic stress
• p e s t i c i d e s ( h e r b i c i d e s )
• p e s t s a n d d i s e a s e s ( i n s e c t s , v i r u s e s , b a c t e r i a , f u n gi )
• e n v i r o n m e n t a l s t r e s s ( s a l t , c o l d a n d d r o u g h t )
improved crop quality
• i m p r o v e d n u t r i t i o n a l q u a l i t y
• e n h a n c e t a s t e , a p p e a ra n c e a n d f ra g ra n c e
• i n c r e a s e s h e l f- l i f e
biopharming
• p l a n t s a s b i o r e a c t o r s f o r p r o d u c t i o n o f u s e f u l c o m po u n d s
( e . g ., t h e ra p e u t i c s , va c c i n e s , a n t i b o d i e s )
phytoremed ia t ion
Genetic engineering of plants
plant transgenesis procedure
1 . c o n s t r u c t i o n o f v e c t o r / p l a s m i d
( r e s t r i c t i o n d i g e s t s , l i g a t i o n )
2 . p r o p a g a t i o n i n E . c o l i
3 . t r a n s f o r m a t i o n
4 . c u l t u r e a n d s e l e c t i o n
totipotency - entire plant generat e d from a single, non-reproduc t iv e cell
Genetic engineering of plants
DIRECT METHODS
protoplast polyethylene gl yco l (PEG) method
• f i r s t t e c h n i q u e f o r p l a n t t ra n s g e n e s i s
• P E G i n d u c e s r e v e r s i b l e p e r m e a b i l i z a t i o n o f t h e p l a s ma m e m b ra n e
protoplast electroporat ion
• i n t e n s i v e e l e c t r i c a l f i e l d l e a d s t o p o r e s o n p l a s m a me m b ra n e
silicon carbide fibers
• f i b e r s p u n c h h o l e s t h r o u g h p l a n t c e l l s d u r i n g v o r t e x i n g
protoplast microinjection
Methods of plant transformation
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DIRECT METHODS
particle bombardm en t
• m o s t c o m m o n t e c h n i q u e f o r d i r e c t t ra n s f o r m a t i o n
• „ p a r t i c l e g u n ” o r „ g e n e g u n ”
• D N A p r e c i p i t a t e d o n t o t u n g s t e n o r g o l d p a r t i c l e s
• p a r t i c l e s s h o t i n t o t h e p l a n t t i s s u e / c e l l s
Methods of plant transformation
INDIRECT METHODS (VECTORED)
Agrobacter iu m- me di a te d transforma t ion
• A . t u m e f a c i e n s p l a n t p a t h o g e n i c b a c t e r i a c a u s e s C r o w n g a l l ( t u m o r s )
• t u m o r i n d u c i n g ( T i ) p l a s m i d
• T - D N A t ra n s f e r e d a n d i n t e g ra t e d i n t o p l a n t c e l l
Methods of plant transformation
transformation frequency is low
(less than 3%)
without selective advantage transformed
cells overgrown by non-transformed
selection markers
antibiot i cs resistence
( Ka n a m y c i n , G e n e t i c i n )
herbicid es resistenase
( P h o s p h i n o t h r i c i n )
reporter genes
GUS (β-glucuron id as e )
GFP (green flourescent protein)
LUC (luciferas e)
Markers and selection
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pest and disease resistence
toxin gene from Bacillus thuringiensi s
B t - c o r n r e s i s t a n t t o e u r o p e a n c o r n b o r e r
B t - c o t t o n r e s i s t a n t t o c o t t o n b o l l w o r m
B t - p e a n u t r e s i s t a n t t o c o r n s t a l k b o r e r
Papaya ringspot virus resistenc e
i n s e r t i n g g e n e f r o m p a t h o g e n i n t o c r o p
a f f o r d s t h e c r o p p l a n t r e s i s t a n c e
Application of transgenic plants
herbicide resistence
herbicid e target modific at i on
herbicid e target overprodu ct i on
herbicid e detoxifi c at i on (enzymat ic )
EXAMPLES:
s u l f o n y l u r e a r e s i s t a n c e
b l o c k i n g t h e e n z y m e f o r s y n t h e s i s Va l , L e u , i s o L e u
m u t a t e d g e n e t ra n s f e r e d f r o m r e s i s t e n t t a b a c o
b r o m o x y n i l r e s i s t a n c e
t ra n s g e n e e n c o d i n g e n z y m e b r o m o x y n i l n i t r i l a s e
g l y p h o s i n a t e r e s i s t e n c e
b a c t e r i a l t ra n s g e n e p r o t e i n i n a c t i va t i n g h e r b i c i d e
Application of transgenic plants
improved crop quality
higher nutrition value
g o l d e n r i c e ( b e t a – c a r o t e n e g e n e s )
o 1 2 0 m i l l i o n c h i l d r e n s u f f e r s f r o m v i t a m i n A d e f i c i e n cy
o h e a l t h y v i s i o n a n d p r e v e n t s n i g h t b l i n d n e s s
b l a c k t o m a t o e ( a n t h o c ya n i n a n t i o x i d a n t g e n e )
o p r e v e n t h e a r t d i s e a s e , d i a b e t e s a n d c a n c e r
improve shelf life
o d e l a y e d f r u i t r i p p e n i n g ( F l a v r S a v r t o m a t o e )
a n t i s e n s e g e n e b l o c k i n g p e c t i n a s e
improved appearance
o d e l p h i n i d i n g e n e f r o m p a n s y c l o n e d t o r o s e
biopharming
Application of transgenic plants
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selective breeding
t i m e c o n s u m i n g a n d c o s t l y
l i m i t e d n u m b e r o f p r o p e r t i e s a va i l a b l e
d i f f i c u l t t o i n t r o d u c e n e w g e n e t i c t ra i t s t o e s t a b l i s h e d l i n e
transgenic animals
fast generat ion lines carrying desired properties
i n c r e a s e d g r o w t h
i n c r e a s e d m u s c l e m a s s
i m p r o v e d d i s e a s e r e s i s t a n c e
i m p r o v e d n u t r i t i o n a l q u a l i t y
i n c r e a s e d w o o l q u a l i t y
model animals for human disease research
biopharming - production of useful molecules
biosensors for environmenta l polution
Genetic engineering of animals
direct microinjection (pronucleus method)
injection of desired DNA to male pronucleus
most popular, commerci a l availa b l e
success range from 10 to 30%
transfer of large genes possible,
no theoretic a l limit for gene construct size
random insertion of the transgen
Methods to produce GE animals
retrovirus mediated gene transfer
retrovirus es used as vectors (gene therapy)
virus gene is replace d by transgene
replica t ion defectiv e virus infect host cells
(e.g., ES cells, embryo cells)
efficient mechanism of transgen integrat i on
transfer of genes < 8 kb possible
random insertion of the transgen
Methods to produce GE animals
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embryonic stem cell method
transfection of gene construct into in vitro
culture of embryonic stem (ES) cells
ES recombinan t cells incorpora t e d into embryo
at blastocys t stage
targete d insertion or knockout of gene(s)
prior to microinj ec t ion
ES cell lines not availa b l e in farm animals
has revolution iz e d genetics, develop m ent ,
immunology and cancer research in mice
Methods to produce GE animals
disease-resistant livestock
i n v i v o i m m u n i z a t i o n : o v e r e x p r e s s g e n e s e n c o d i n g m o n o c l o n a l a n t i b o d i e s
e l i m i n a t e p r o d u c t i o n o f h o s t c e l l c o m p o n e n t s t h a t in t e ra c t w i t h i n f e c t i o u s a g e n t
improving milk quality
i n c r e a s e c a s e i n c o n t e n t s l e t t o i n c r e a s e c h e e s e p r o d u c t i o n
d e c r e a s e l a c t o s e c o n t e n t b y o v e r e x p r e s s l a c t a s e
a b o l i s h l a c t o g l o b u l i n e x p r e s s i o n ( f o r m i l k a l l e r g i c c o n s um e r )
improving animal production traits
t r a n s g e n i c f i s h : e n h a n c e d g r o w t h 3 - 5 t i m e s ( g r o w t h h o r m o n e )
t r a n s g e n i c p i g : p r o d u c t i o n o f o m e g a - 3 - f a t t y a c i d s ( r o u n d w a r m g e n e )
t r a n s g e n i c p o l t r y : l o w e r c h o l e s t e r o l a n d f a t i n e g g s
biopharming
Application of transgenic animals
use of plants or animals for the production of useful molecules
industrial products
proteins (enzymes)
fats and oils
polymers and waxes
pharmaceuticals
recombinan t human proteins
therapeut i c proteins and pharmaceut i c a ls
vaccines and antibodi es
Biopharming
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INDUSTRIAL PRODUCTS FROM PLANTS
cheap and easy to produce
free of animal viruses
risk of food supply contamination
environmental contamination
EXAMPLES (transgeni c corn, Sigma):
avidin
o m e d i c a l d i a g n o s t i c s
β-glycuronidas e
o v i s u a l m a r k e r i n r e s e a r c h l a b s
trypsin
o t ra d i t i o n a l l y i s o l a t e d f r o m b o v i n e p a n c r e a s
o f i r s t l a r g e s c a l e t ra n s g e n i c p l a n t p r o d u c t
o w o r l d w i d e m a r k e t = U S $1 2 0 m i l l i o n
Biopharming
EDIBLE VACCINES FROM PLANTS
no purification required
no hazards associated with injections
may be grown locally, where needed most
no transportation costs
no need for refrigeration or special storage
EXAMPLES:
HIV-suppre ss ing protein in spinach
rabies virus G protein in tomato
human vaccine for hepatitis B in potato
Biopharming
PLANT-MADE ANTIBODIES
plantibodies - monoclonal antibodies produced in plants
free from potential contamination of mammalian viruses
plants used include tobacco, corn, potatoes, soya and rice
EXAMPLES: cancer, herpes simplex virus, respira to r y syncytia l virus
PLANT-MADE PHARMACEUTICALS
therapeutic proteins and intermediates
EXAMPLES: proteins to treat CF, HIV, hypertension , hepatitis B
Biopharming
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PRODUCTION OF PHARMACEUTICALS IN MILK
easy to purify - few other proteins in milk
dairy cattle produce 10,000 liters of milk/year (35 g protein/liter)
only few transgenic cows can meet worldwide demand
risk of food supply contamination
EXAMPLES:
COW: human serum albumin, human lactoferrin
SHEEP: alpha-1-an ti t ry psi n
GOAT: human antithrombi n III (FDA approved),
tissue plasminogen activato r, malaria antigen
PRODUCTION OF MATERIALS IN MILK
BioSteel from spider silk (Nexia Biotech)
Biopharming
CROPS
enhanced taste and quality, reduced maturation time
increased nutrients, yields, and stress toleranc e
improved resistance to disease, pests, and herbicid es
ANIMALS
better yields of meat, eggs, and milk
improved animal health, resistance, productiv it y, and feed efficienc y
ENVIRONMENT
more efficient processing
conservat ion of soil, water, and energy
better natural waste managem ent
SOCIETY
increased food security for growing populations
GMO benefits
SAFETY
human health : allerg ens, antibiot i c resistance, unknown effects
environment: unintended transfer through cross-poll in a t ion, unknown
effects on other organisms, loss of biodiv ers it y
ACCESS AND INTELLECTUAL PROPERTY
dominati on of world food production by few companies
increasing dependenc e on industria l iz e d nations by develo p ing countries
ETHICS
violat ion of natural organism´s intrinsic values
tamperin g with nature by mixing genes among species
objecti ons to consuming animal genes in plants and vice versa
stress for animals
GMO controversies
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GMO future
GMO crop first commercia l i z e d in 1996
17.3 million farmers grew biotech crops on 170 million hectares
90% of new users are small resource-po or farmers in develo pin g countries
EU-funded risk research on GMOs over the past two decades unable to detect
any risks that have not yet been known from conventiona l agricultur e*
* EU Commission (2012): A Decade of EU-funded GMO Impacts Research.