Plant Biotechnology I METHODOLOGY. Centre of the Region Haná for Biotechnological and Agricultural Research Olomouc, Czech Republic.

Transcription

1 Plant Biotechnology I METHODOLOGY Centre of the Region Haná for Biotechnological and Agricultural Research Olomouc, Czech Republic Ivo Frébort

2 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

3 Crop breeding by humans Present crops resulted from the process of changing the genetic information of plants that has started more than 10,000 years ago Bread wheat + + = Triticum urartu (2n = 2x = 14; AA) Aegilops speltoides 2n = 2x = 14;BB Aegilops squorrosa 2n = 2x = 14; DD Triticum aestivum (2n = 6x = 42; AABBDD) + = Barley Hordeum chilense 2n = 2x = 14 Hordeum spontaneum 2n = 2x = 14 Hordeum vulgare 2n = 2x = 14

4 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)

5 High demand for sustainable growth of agricultural production Growing human population, soil degradation, climate changes

6 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, s, 5-fold increase in yield 2 nd Green Revolution is needed!

7 Conventional breeding methods Changes introduced to the plant genome are usually not known to the breeder or consumer 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)

8 Mutation breeding Introduced after WWII Chemical mutagenesis carcinogenes, chemical warfare Produce many mutants Test those that survive Select usable ones Ethyl metanesulfonate (EMS) Dimethylsulfate (DMS)

9 Atomic gardening Radiation: gamma rays ( 60 Co), X-rays Institute of Radiation Breeding, Hitachiohmiya, Japan

10 Nowadays crops are often radiation mutants Most of these mutations have never been mapped

11 Molecular plant breeding Somatoclonal variation - use of callus derived lines Marker assisted selection - DNA markers and fingerprinting Genetic modification - introduction of new genes - silencing of gene expression

12 Methods for genetic modification of plants

13 Plants that have been genetically transformed

14 Gene gun particle bombardment

15 Ti plasmid Tranformation by Agrobacterium tumefaciens

16 Chemistry of Agrobacterium invasion Produced by wounded plant, sensed by Agrobacterium Opines, sources of energy Hormones - auxins and cytokinins; induce crown gall formation

17 Vir proteins provide insertion of T-DNA into plant chromosome

18 Sites of T-DNA insertion Insertion produces a heterozygote insertion in one of the paired chromosomes Arabidopsis thaliana

19 Exploitation of Agrobacterium genetics

20 Composition of gene insertion cassette Promoter Constitutive: 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)

21 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), glyphosate oxidoreductase (gox, Ochrobactrum anthropi)

22 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

23 Removal of selection markers recombinase systems Cre recombinase from P1 bacteriophage acts on 34 bp lox excise sites

24 Gene silencing by interfering RNAs mirna (21-24 nt), pirna (26-31 nt), sirna (20-25 nt)

25 CRISPR/Cas prokaryotic immune system Clustered Regularly Interspaced Short Palindromic Repeats

26 CRISPR/Cas technology allows precise genome editing

27 Localization of plant proteins with GFP Gene for green fluorescent protein (GFP) from jellyfish, fluorescence induced by blue light ( nm) Aequorea victoria AtCKX2-GFP: ER secreted GFP (control): cytosol and nucleus AtCKX3-GFP: vacuoles

28 Marker and reporter genes used in plants

29 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 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

30 Transformation using leaf discs - dicots

31 Regeneration of transformed tobacco

32 Transformation of monocots 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

33 Plastid transformation insertion into chloroplast DNA 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

34 Further reading Molecular Biotechnology: Principles and Applications of Recombinant DNA, 4th Edition Bernard R. Glick, Jack J. Pasternak, Cheryl L. Patten ISBN: