Bi7430 Molecular Biotechnology
11. Molecular Biotechnology in Agriculture
Outline
 Definition of green biotechnology
 Genetic engineering of plants
 Genetic engineering of animals
 Biopharming
 GMO benefits and controversies
Green (agricultural) biotechnology
 green biotechnology applied to agricultural processes
 environmentally-friendly solutions as alternative
to traditional agriculture, horticulture, and animal breeding
 modification of plants and animals increasing value in agriculture
 traditional agriculture – selective crossbreeding and hybridization
 modern molecular biotechnology – transgenesis (rDNA)
 transgenic organism - altered by addition of exogenous DNA
 transgene – DNA that is introduced
Genetic engineering of plants
 > 150 different plant species in 50 countries worldwide
 DNA sequence of A. thaliana (2000), rice (2005), cotton (2006), corn
(2009), potato (2011), tomato (2012), etc.
 transgenic plants engineered to
 overcome biotic and abiotic stress
o pesticides (herbicides)
o pests and diseases (insects, viruses, bacteria, fungi)
o environmental stress (salt, temperature, cold and drought)
 improved crop quality
o improved nutritional quality
o enhance taste, appearance and fragrance
o increase shelf-life
 biopharming
o plants as bioreactors for production of useful compounds
(e.g., therapeutics, vaccines, antibodies)
 phytoremediation
Genetic engineering of plants
 plant transgenesis procedure
1. construction of vector/plasmid
(restriction digests, ligation)
2. propagation in E.coli
3. transformation
4. culture and selection
 totipotency - entire plant generated from a single, non-reproductive cell
Methods of plant transformation
 direct methods
 protoplast polyethylene glycol (PEG) method
o first technique for plant transgenesis
o PEG induces reversible permeabilization of the plasma membrane
 protoplast electroporation
o intensive electrical field leads to pores on plasma membrane
 silicon carbide fibers
o fibers punch holes through plant cells during vortexing
 protoplast microinjection
Methods of plant transformation
 direct methods
 particle bombardment
o most common technique for direct transformation
o „particle gun” or „gene gun”
o DNA precipitated onto tungsten or gold particles
o particles shot into the plant tissue/cells
Methods of plant transformation
 indirect methods (vectored)
 Agrobacterium-mediated transformation
o A. tumefaciens plant pathogenic bacteria causes Crown gall (tumors)
o tumor inducing (Ti) plasmid
o T-DNA transferred and integrated into plant cell
Markers and selection
 transformation frequency is low (less than 3%)
 without selective advantage transformed
cells overgrown by non-transformed
 selection markers
 antibiotics resistance
(Kanamycin, Geneticin)
 herbicides resistance
(Phosphinothricin)
 reporter genes
 GUS (β-glucuronidase)
 GFP (green fluorescent protein)
 LUC (luciferase)
Application of transgenic plants
 pest and disease resistance
 toxin gene from Bacillus thuringiensis
o Bt-corn resistant to European corn borer
o Bt-cotton resistant to cotton bollworm
o Bt-peanut resistant to cornstalk borer
 Papaya ringspot virus resistance
inserting gene from pathogen into crop
affords the crop plant resistance
Application of transgenic plants
 herbicide resistance
 herbicide target modification
 herbicide target overproduction
 herbicide detoxification (enzymatic)
 EXAMPLES
 sulfonylurea resistance
blocking the enzyme for synthesis Val, Leu, isoLeu
mutated gene transferred from resistant tabaco
 bromoxynil resistance
transgene encoding enzyme bromoxynil nitrilase
 glyphosinate resistance
bacterial transgene protein inactivating herbicide
Application of transgenic plants
 resistance to environmental stress
 marginal land or climate change induced drought
 crucial ways of securing the world's food supply
 drought tolerance
- gene from Xerophyta viscosa - unique protein in cell membrane
- gene for production of protective waxy cuticle on leaves
- gene for expression of trehalose (stabilization of biomolecules)
 salt tolerance
- gene for enhanced glycinebetaine production
Application of transgenic plants
 improved crop quality
 higher nutrition value
o golden rice (beta – carotene genes)
120 million children suffers from vitamin A deficiency
healthy vision and prevents night blindness
o black tomato (anthocyanin antioxidant gene)
prevent heart disease, diabetes and cancer
 improve shelf life
o delayed fruit ripening (FlavrSavr tomato)
antisense gene blocking pectinase
 improved appearance
o delphinine gene from pansy cloned to rose
 biopharming
Break 5 min
Genetic engineering of animals
 selective breeding
 time consuming and costly
 limited number of properties available
 difficult to introduce new genetic traits / lines
 transgenic animals
 fast generation lines carrying desired properties
o increased growth
o improved disease resistance
o improved nutritional quality
o increased wool quality
 model animals for human disease research
 biopharming - production of useful molecules
 biosensors for environmental pollution
Genome targeting technologies
 direct microinjection (pronucleus method)
 injection of desired DNA to male pronucleus
 most popular, commercial available
 success range from 10 to 30%
 transfer of large genes possible
 no theoretical limit for gene construct size
 random insertion of the transgene
(affecting other genes and expression patterns)
 retrovirus mediated gene transfer
 retroviruses used as vectors (gene therapy)
 virus gene is replaced by transgene
 replication defective virus infect host cells
(e.g., ES cells, embryo cells)
 efficient mechanism of transgene integration
 transfer of genes < 8 kb only possible
 random insertion of the transgene
Genome targeting technologies
 embryonic stem cell method
 transfection of gene construct into in vitro
culture of embryonic stem (ES) cells
 ES recombinant cells incorporated into embryo
at blastocyst stage
 1 in a million incorporated at desired position
 ES cell lines not available in farm animals
 random insertion of the transgene
Genome targeting technologies
Genome targeting technologies
 engineered nucleases, „molecular scissors"
 site-specific double stranded breaks
 Zinc finger nucleases (ZFNs)
 transcription-activator like effector nucleases (TALEN)
 RNA-guided DNA endonuclease (CRISPR-Cas9)
Genome targeting technologies
 CRISPR-Cas9
 synthetic guide RNA (gRNA)
 delivering Cas9 nuclease complexed with gRNA into a cell
 in vivo (nucleus), stem cells, fertilized egg
 can target several genes at once
Application of transgenic animals
 disease-resistant livestock
 in vivo immunization - overexpress genes encoding monoclonal antibodies
 eliminate production of host cell components interacting with infectious agent
 improving milk quality
 increase casein contents let to increase cheese production
 decrease lactose content by overexpress lactase
 abolish lacto globulin expression (for milk allergic consumer)
 improving animal production traits
 transgenic fish - enhanced growth 3-5 times (growth hormone)
 transgenic pig - production of omega-3-fatty acids (roundworm gene)
 transgenic poultry - lower cholesterol and fat in eggs
 biopharming
Biopharming
 use of plants or animals for the production of useful molecules
 industrial products
 proteins (enzymes)
 fats and oils
 polymers and waxes
 pharmaceuticals
 recombinant human proteins
 therapeutic proteins and pharmaceuticals
 vaccines and antibodies
Biopharming
 industrial products from plants
 cheap and easy to produce
 free of animal viruses
 risk of food supply contamination
 environmental contamination
 EXAMPLES (transgenic corn, Sigma):
 trypsin
o traditionally isolated from bovine pancreas
o first large scale transgenic plant product
o worldwide market = US$120 million
 avidin
o medical diagnostics
 β-glucuronidase
o visual marker in research labs
Biopharming
 edible vaccines from plants
 no purification required
 no hazards associated with injections
 may be grown locally where needed
 no transportation costs
 no need for refrigeration or special storage
 EXAMPLES:
 HIV-suppressing protein in spinach
 rabies virus G protein in tomato
 vaccine for rotavirus or hepatitis 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
 plant-made pharmaceuticals
 therapeutic proteins and intermediates
 EXAMPLES: proteins to treat cystic fibrosis, HIV, hypertension
Biopharming
 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-antitrypsin
 GOAT: human antithrombin III (FDA approved),
tissue plasminogen activator, malaria antigen
 production of materials in milk
 BioSteel from spider silk (Nexia Biotech)
GMO benefits
 crops
 increased stress tolerance
 improved resistance to disease, pests and herbicides
 increased nutrients, yields, enhanced taste and quality
 animals
 improved animal health, resistance, productivity and feed efficiency
 better yields of meat, eggs, and milk
 environment
 more efficient processing
 conservation of soil, water, and energy
 better natural waste management
 society
 increased food security for growing populations
 climate change induced drought
GMO controversies
 safety
 human health – toxicity, allergens, antibiotic resistance, unknown effects
 environment - unintended transfer through cross-pollination,
unknown effects on other organisms, loss of biodiversity
 ethics
 tampering with nature by mixing genes among species / cloning
 violation of natural organism´s intrinsic values
 stress for animals
 access and intellectual property
 domination of world food production by few companies
 increasing dependence on industrialized nations by developing countries
GMO future
 GMO crop first commercialized in 1996
 17.3 million farmers grew biotech crops on 170 million hectares
 90% of new users are small resource-poor farmers in developing countries
 EU research on risk of GMOs over the past two decades unable to detect
any risks that have not yet been known from conventional agriculture*
* EU Commission (2012): A Decade of EU-funded GMO Impacts Research
Questions
Reading
 U . S . A g e n c y fo r I n t e r n a t i o n a l D e v e l o p m e n t , A g r i c u l t u ra l
B i o t e c h n o l o g y S u p p o r t P r o j e c t I I , a n d t h e P r o g r a m fo r
B i o s a fe t y S y s t e m s
 H o w a r e B i o t e c h C r o p s & F o o d s A s s e s s e d f o r S a f e t y ? ,
D e v e l o p i n g a B i o s a f e t y S y s te m ( B R I E F # 5 a n d # 6 )