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8. Molecular Biotechnology in Agriculture8. Molecular Biotechnology in Agriculture
Bi7430 Molecular Biotechnology
Definition of green biotechnology
Genetic engineering of plants
Genetic engineering of animals
Biopharming
Outline
Green (agricultural) biotechnology
green biotechnol ogy applied to agricul tural processes
environmentally-friendly solutions as alternative to traditional
agriculture, horticulture, and animal breeding processes
modificati on of plants and animals increasing value in agriculture
traditiona l agricultu r e – selectiv e crossbreedin g and hybridiza t ion
modern molecular biotechno lo gy – transgenes is (rDNA)
transgeni c organis m - 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) …
transgeni c plants engineered to
overcome biotic and abiotic stress
• p e st ic id e s (h e r b ic id e s)
• p e st s a n d d ise a se s (in se c t s, v ir u se s, b a c t e r ia , f u n g i)
• e n v ir on m e n t a l st r e ss (sa lt , c old a n d d r ou g h t )
improved crop quality
• im p r ov e d n u t r it ion a l q u a lit y
• e n h a n c e t a st e , a p p e a ra n c e a n d f ra g ra n c e
• in c r e a se sh e lf- lif e
biopharming
• p la n t s a s b ior e a c t or s f or p r od u c t ion of u se f u l c om p ou n d s
(e . g ., t h e ra p e u t ic s, va c c in e s, a n t ib od ie s)
phytoremed ia t ion
Genetic engineering of plants
plant transgenesis procedure
1. c o nstr uc ti o n of v e c t or /p la sm id
(r e st r ic t ion d ig e st s, lig a t ion )
2. p r o p a g a ti o n in E. c oli
3. tr a nsfo r m a ti o n
4. c ul tur e a nd se l e c ti o n
totipotency - entire plant generat ed from a single, non-reproduc t iv e cell
Genetic engineering of plants
DIRECT METHODS
protoplast polyethylene gl yco l (PEG) method
• f ir st t e c h n iq u e f or p la n t t ra n sg e n e sis
• P E G in d u c e s r e v e r sib le p e r m e a b iliza t ion of t h e p la sm a m e m b ra n e
protoplast electroporat ion
• in t e n siv e e le c t r ic a l f ie ld le a d s t o p or e s on p la sm a m e m b ra n e
silicon carbide fibers
• f ib e r s p u n c h h ole s t h r ou g h p la n t c e lls d u r in g v or t e x in g
protoplast microinjection
Methods of plant transformation
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DIRECT METHODS
particle bombardm en t
• m ost c om m on t e c h n iq u e f or d ir e c t t ra n sf or m a t ion
• „ p a r t ic le g u n ” or „ g e n e g u n ”
• D NA p r e c ip it a t e d on t o t u n g st e n or g old p a r t ic le s
• p a r t ic le s sh ot in t o t h e p la n t t issu e /c e lls
Methods of plant transformation
INDIRECT METHODS (VECTORED)
Agrobacter iu m- me di a te d transforma t ion
• A . tum efac i ens p la n t p a t h og e n ic b a c t e r ia c a u se s Cr ow n g a ll (t u m or s)
• tum o r i nd uc i ng (T i) p la sm id
• T - DN A t ra n sf e r e d a n d in t e g ra t e d in t o p la n t c e ll
Methods of plant transformation
transformation frequency is low
(max 3% of all cells)
without selective advantage transformed
cells overgrown by non-transformed
selection markers
antibiot ics resistence
(Ka n a m y c in , Ge n e t ic in )
herbicides resistenase
(P h osp h in ot h r ic in )
reporter genes
GUS (β-glucuron idas e)
GFP (green flourescent protein)
LUC (luciferas e)
Markers and selection
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pest and disease resistence
tox in gen e f rom Bacillus thuringiensi s
Bt- c o r n r e sist a n t t o e u r op e a n c or n b or e r
Bt- c o tto n r e sist a n t t o c ot t on b ollw or m
Bt- peanut r e sist a n t t o c or n st a lk b or e r
Papay a r in gs pot v ir u s r es is ten c e
in se r t in g g e n e f r om p a t h og e n in t o c r op
a f f or d s t h e c r op p la n t r e sist a n c e
Application of transgenic plants
herbicide resistence
herbicide target modific at ion
herbicide target overprodu ct ion
herbicide detoxific at ion (enzymat ic )
EXAMPLES:
sul fo nyl ur e a r e si sta nc e
b loc k in g t h e e n zy m e f or sy n t h e sis Va l, L e u , isoL e u
m u t a t e d g e n e t ra n sf e r e d f r om r e sist e n t t a b a c o
br o m o xyni l r e si sta nc e
t ra n sg e n e e n c od in g e n zy m e b r om ox y n il n it r ila se
g l yp ho si na te r e si ste nc e
b a c t e r ia l t ra n sg e n e p r ot e in in a c t iva t in g h e r b ic id 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 ot e n e g e n e s)
o 120 m illion c h ild r e n su f f e r s f r om v it a m in A d e f ic ie n c y
o h e a lt h y v ision a n d p r e v e n t s n ig h t b lin d n e ss
bl a c k to m a to e (a n t h oc ya n in a n t iox id a n t g e n e )
o p r e v e n t h e a r t d ise a se , d ia b e t e s a n d c a n c e r
improve shelf life
o d e la y e d f r u it r ip p e n in g (F la v r S a v r t om a t oe )
a n t ise n se g e n e b loc k in g p e c t in a se
improved appearance
o d e lp h in id in g e n e f r om p a n sy c lon e d t o r ose
biopharmi ng
Application of transgenic plants
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selective breeding
t im e c o nsum i ng a n d c o stl y
l i m i te d n u m b e r of p r op e r t ie s a va ila b le
d i ffi c ul t t o in t r od u c e n e w g e n e t ic t ra it s t o e st a b lish e d lin e
transgeni c animals
fast generat ion lines carrying desired properties
i n c r e a s e d g r o w t h
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 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 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 microinjecti on (pronucl eus method)
injection of desired DNA to male pronucleus
most popular, commercia l availa ble
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
retrovi rus mediated gene transfer
retrovirus es used as vectors (gene therapy)
virus gene is replaced by transgene
replica t ion defectiv e virus infect host cells
(e.g., ES cells, embryo cells)
efficient mechanism of transgen integrat ion
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 ed into embryo
at blastocys t stage
targeted insertion or knockout of gene(s)
prior to microinj ec t ion
ES cell lines not availa ble in farm animals
has revolution iz ed genetics, developm ent ,
immunology and cancer research in mice
Methods to produce GE animals
disease-resis tant livestock
i n v i v o i m m uni z a ti o n: ov e r e x p r e ss g e n e s e n c od in g m on oc lon a l a n t ib od ie s
e lim in a t e p r od u c t ion of h ost c e ll c om p on e n t s t h a t in t e ra c t w it h in f e c t iou s a g e n t
improving milk quality
i nc r e a se c a se i n c on t e n t s le t t o in c r e a se c h e e se p r od u c t ion
d e c r e a se l a c to se c on t e n t b y ov e r e x p r e ss la c t a se
a b olish la c t og lob u lin e x p r e ssion (f or m ilk a lle r g ic c on su m e r )
improving animal production traits
tr a nsg e ni c fi sh: e n h a n c e d g r ow t h 3- 5 t im e s (g r ow t h h or m on e )
tr a nsg e ni c p i g : p r od u c t ion of om e g a - 3- f a t t y a c id s (r ou n d w a r m g e n e )
tr a nsg e ni c p o l tr y: low e r c h ole st e r ol a n d f a t in e g g s
biopharmi ng
xenotrans pl antati on
p r od u c t ion of d on or c om p le m e n t a r y or g a n s, t issu e s a n d c e lls
Application of transgenic animals
use of plants or animals for the production of useful molecules
industrial products
proteins (enzymes)
fats, oils and waxes
pharmaceuti cals
recombinan t human proteins
therapeut ic proteins and pharmaceut ic a ls
vaccines and antibodies
Biopharming
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INDUSTRIAL PRODUCTS FROM PLANTS
cheap and easy to produce
free of animal viruses
risk of food supply contami nation
environmental contamination
EXAMPLES (transgenic corn, Sigma):
av idin
o m e d ic a l d ia g n ost ic s
ββββ - gly cu r on idas e
o v isu a l m a r k e r in r e se a r c h la b s
tr y ps in
o t ra d it ion a lly isola t e d f r om b ov in e p a n c r e a s
o f ir st la r g e sc a le t ra n sg e n ic p la n t p r od u c t
o w or ld w id e m a r k e t = U S $ 120 m illion
Biopharming
EDIBLE VACCINES FROM PLANTS
no purification required
no hazards associated with injections
may be grown locally, where needed most
no transportati on costs
no need for refrigeration or special storage
EXAMPLES:
HI V-sup p re ss i ng p rote i n i n sp i na c h
ra b i e s vi rus G p rote i n i n tom a to
hum a n va c c i ne f or he p a ti ti s B i n p ota to
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
therapeuti c 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
no harm for transgeni c animal, no change to physiology
dairy cattle produce 10,000 liters of milk/year (35 g protein/liter)
only few transgeni c cows can meet worldwide demand
risk of food supply contami nation
EXAMPLES:
COW: hum a n se rum a l b um i n, hum a n l a c tof e rri n
SHEEP : Al p ha -1 -a n t i tr yp s i n
G OAT: hum a n a nti throm b i n I I I (F DA a p p rove d ),
ti ssue p l a sm i nog e n a c ti va to r, m a l a ri a a nti g e n,
Bi oSte e l f rom sp i d e r si l k (N e x i a Bi ote c h)
Biopharming