Plant Biotechnology IMETHODOLOGY

Ivo Frébort

Centre of the Region Haná for Biotechnological and Agricultural ResearchOlomouc, Czech Republic

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

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

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)

Growing human population, soildegradation, climate changes …

High demand for sustainable growth of agricultural production

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!

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

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

Radiation: gamma rays (60Co), X-rays

Atomic gardening

Institute of Radiation Breeding, Hitachiohmiya, Japan

Nowadays crops are often radiation mutants

Most of these mutations have never been mapped

• 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

Methods for genetic modification of plants

Plants that have been genetically transformed

Gene gun – particle bombardment

Tranformation by Agrobacterium tumefaciens

Ti plasmid

Chemistry of Agrobacterium invasion

Produced by wounded plant,sensed by Agrobacterium

Opines, sources of energy Hormones - auxins and cytokinins; induce crown gall formation

Vir proteins provideinsertion of T-DNA intoplant chromosome

Arabidopsis thaliana

Sites of T-DNA insertion

Insertion produces a heterozygote – insertion in one of the paired chromosomes

Exploitation of Agrobacterium genetics

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)

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)

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

Removal of selection markers – recombinase systems

Cre recombinase• from P1 bacteriophage• acts on 34 bp lox excise sites

Gene silencing by interfering RNAsmiRNA (21-24 nt), piRNA (26-31 nt), siRNA (20-25 nt)

CRISPR/Cas – prokaryotic immune systemClustered Regularly Interspaced Short Palindromic Repeats

CRISPR/Cas technology allows precise genome editing

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)

Marker and reporter genes used in plants

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

Transformation using leaf discs - dicots

Regeneration of transformed tobacco

• 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

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

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