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Plant Biotechnology IMETHODOLOGY
Ivo Frébort
Centre of the Region Haná for Biotechnological and Agricultural ResearchOlomouc, Czech Republic
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Summary of the plant biotechnology lectures
Plant Biotechnology I - Methodology
• Crop breeding by humans
• Genetic transformation of plants
• Gene editing technology
Plant Biotechnology II - Applications
• Production of GM plants worldwide
• Herbicide tolerance, pest and pathogen resistance
• Nutrition improvement
• Risk assesment and GM control in the EU
• Examples from CRH
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Present crops resulted from the process of changing the genetic information of plants that has started more than 10,000 years ago
Crop breeding by humans
Triticum urartu(2n = 2x = 14; AA)
Aegilops speltoides2n = 2x = 14;BB
Aegilops squorrosa2n = 2x = 14; DD
+ + =
Hordeum spontaneum2n = 2x = 14
Hordeum chilense2n = 2x = 14
+=
Barley
Triticum aestivum(2n = 6x = 42; AABBDD)
Bread wheat
Hordeum vulgare2n = 2x = 14
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Goals of recent crop breeding
Goals of the breeders (traits to be incorporated):
• Increased quality and yield of the crop
• Increased stress tolerance (salinity, temperature, drought)
• Resistance to viruses, fungi and bacteria
• Increased tolerance to insect pests
• Increased tolerance to herbicides (weed control)
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Growing human population, soildegradation, climate changes …
High demand for sustainable growth of agricultural production
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Global food production sustainability issue
• Demand for food crops expected to grow by 70-80% between 2000 and 2050
• In next 50 years, humans must produce as much food as has been produced in the entire history of humankind
• Green revolution – Norman Borlaug, 1940-1960‘s, 5-fold increase in yield• 2nd Green Revolution is needed!
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1. Selection (crossing and screening, diversity)- Interbreeding of related (crossable) species
2. Hybridization (heterosis and hybrid vigor)- Back-crossing of progenies with parental lines- Hybrids made from distant lines
3. Polyploidy (chemical induction/colchicine – increased variation)
4. Induced mutation (chemicals, radiation)
Conventional breeding methodsChanges introduced to the plant genome are usually not known to the breeder or consumer
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Dimethylsulfate (DMS)Ethyl metanesulfonate (EMS)
Introduced after WWII
• Chemical mutagenesis– carcinogenes, chemical warfare
• Produce many mutants• Test those that survive• Select usable ones
Mutation breeding
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Radiation: gamma rays (60Co), X-rays
Atomic gardening
Institute of Radiation Breeding, Hitachiohmiya, Japan
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Nowadays crops are often radiation mutants
Most of these mutations have never been mapped
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• Somatoclonal variation
- use of callus derived lines
• Marker assisted selection
- DNA markers and fingerprinting
• Genetic modification- introduction of new genes- silencing of gene expression
Molecular plant breeding
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Methods for genetic modification of plants
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Plants that have been genetically transformed
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Gene gun – particle bombardment
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Tranformation by Agrobacterium tumefaciens
Ti plasmid
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Chemistry of Agrobacterium invasion
Produced by wounded plant,sensed by Agrobacterium
Opines, sources of energy Hormones - auxins and cytokinins; induce crown gall formation
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Vir proteins provideinsertion of T-DNA intoplant chromosome
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Arabidopsis thaliana
Sites of T-DNA insertion
Insertion produces a heterozygote – insertion in one of the paired chromosomes
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Exploitation of Agrobacterium genetics
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Composition of gene insertion cassette
PromoterConstitutive: 35S (CaMV), ubiquitin, actin
Tissue specific: 2A11 (tomato) – fruit specific; Lhcb3 (A. thaliana) leaf specific, light regulated; alkaline phosphatase – root specific, phosphate induced, etc.
Inducible: In2-2 (benzensulfonamide herbicide Safener), tet (tetracycline), pOp(dexamethasone)
Transcription terminator - Nos (nopaline synthase TT)
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Selection of transformants – selection markers
Antibiotics: kanamycin A, hygromycin B (aminotriglycosides, inhibit protein synthesis)
Marker gene: amidoglycoside phosphotransferase (nptIII, hpt)
Herbicides: glyphosate - N-(phosphonomethyl)glycine (inhibits synthesis of aromatic amino acids (5-enolpyruvylshikimate-3-phosphate synthase, EPSPS)
Marker gene: Agrobacterium strain CP4 EPSPS (resistant to glyphosate), glyphosateoxidoreductase (gox, Ochrobactrum anthropi)
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Selection of transformants – selection markers II
Sugars: sucrose in the medium is needed for young plants grown in vitro; if xylose and mannose are given instead of sucrose, hexokinase forms mannose-6-phosphate that cannot proceed to glycolysis and accumulates causing toxicity
Marker gene: mannose-6-phosphate isomerase (mpi) – converts mannose-6 phosphate to
fructose-6-phosphate that enters glycolysis
Hormones: cytokinins are needed to regenerate shoots from calli
Marker gene: isopentenyl transferase (ipt), produces excess of cytokinins
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Removal of selection markers – recombinase systems
Cre recombinase• from P1 bacteriophage• acts on 34 bp lox excise sites
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Gene silencing by interfering RNAsmiRNA (21-24 nt), piRNA (26-31 nt), siRNA (20-25 nt)
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CRISPR/Cas – prokaryotic immune systemClustered Regularly Interspaced Short Palindromic Repeats
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CRISPR/Cas technology allows precise genome editing
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AtCKX2-GFP: ER → secreted
AtCKX3-GFP: vacuolesGFP (control): cytosol and nucleus
Localization of plant proteins with GFP
Aequorea victoria
Gene for green fluorescent protein (GFP) from jellyfish,fluorescence induced by blue light (440-480 nm)
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Marker and reporter genes used in plants
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Floral dipping – perfect for Arabidopsis
(a) Plants for floral dipping(b) Dipping in Agrobacterium cell
suspension for 10 s(c) Wrapping with plastic films to
maintain high humidity for 16–24 h
(d) Growing for 1 month(e) Harvesting of seeds(f) Selection of transformants
• 20% efficiency• improved by vacuum infiltration• surfactant Silwet L-77• works also for alfalfa and petunia
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Transformation using leaf discs - dicots
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Regeneration of transformed tobacco
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• Monocots much more difficult to transform than dicots• A. tumefaciens (supervirulent strain) applied on wounded immature embryo• Acetosyringone added to stimulate the infection• Difficult plant regeneration from calli, fine tuning of cytokinin and auxin needed
Mrízová et al., submitted manuscript
Transformation of monocots
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Advantages:• transgene not transmitted by pollen• specific sites for homologous recombination• high expression• suitable for bacterial genes• no gene silencing
Disadvantages• biolistic method, difficult to shoot through the chloroplast membrane• very low efficiency
Plastid transformation – insertion into chloroplast DNA
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Further reading
Molecular Biotechnology: Principles and Applications of Recombinant DNA, 4th Edition
Bernard R. Glick, Jack J. Pasternak, Cheryl L. PattenISBN: 978-1-55581-498-4