1. Plant Genetic Engineering

2. 1.a suitable transformation method 2.a means of screening for transformants 3.an efficient regeneration system 4.genes/constructs Vectors Promoter/terminator reporter genes selectable marker genes ‘genes of interest’ Requirement

3. Transformation methods DNA must be introduced into plant cells Indirect - Agrobacterium tumefaciens Direct - Chemical method - Electrical method - Physical methods Chemical Method 1.Use of PEG (Polyethylene glycol (PEG)-mediated ) 2.Protoplasts are incubated with a solution of DNA and PEG

4. Electrical method Electroporation (electropermeabilization) Physical Methods 1.Particle bombardment 2.Microinjection 3.Silicon Carbide whiskers

5. Agrobacterium tumefaciens Plant parasite that causes Crown Gall Disease Plant parasite that causes Crown Gall Disease Encodes a large (~250kbp) plasmid called Tumor-inducing (Ti) plasmid Encodes a large (~250kbp) plasmid called Tumor-inducing (Ti) plasmid Portion of the Ti plasmid is transferred between bacterial cells and plant cells  T-DNA (Tumor DNA ) Portion of the Ti plasmid is transferred between bacterial cells and plant cells  T-DNA (Tumor DNA ) T-DNA integrates stably into plant genome T-DNA integrates stably into plant genome Single stranded T-DNA fragment is converted to dsDNA fragment by plant cell Single stranded T-DNA fragment is converted to dsDNA fragment by plant cell 1. Then integrated into plant genome 2. 2 x 23bp direct repeats play an important role in the excision and integration process the excision and integration process

6. Agrobacterium tumefaciens Tumor formation = hyperplasia Tumor formation = hyperplasia Hormone imbalance Hormone imbalance Caused by A. tumefaciens Caused by A. tumefaciens Lives in intercellular spaces of the plantLives in intercellular spaces of the plant Plasmid contains genes responsible for the diseasePlasmid contains genes responsible for the disease Part of plasmid is inserted into plant DNA Part of plasmid is inserted into plant DNA Wound = entry point  10-14 days later, tumor forms Wound = entry point  10-14 days later, tumor forms

7. Agrobacterium tumefaciens What is naturally encoded in T-DNA? What is naturally encoded in T-DNA? Enzymes for auxin and cytokinin synthesisEnzymes for auxin and cytokinin synthesis Causing hormone imbalance  tumor formation/undifferentiated callus Causing hormone imbalance  tumor formation/undifferentiated callus Mutants in enzymes have been characterized Mutants in enzymes have been characterized Opine synthesis genes (e.g. octopine or nopaline)Opine synthesis genes (e.g. octopine or nopaline) Carbon and nitrogen source for A. tumefaciens growth Carbon and nitrogen source for A. tumefaciens growth Insertion genes Insertion genes Virulence (vir) genesVirulence (vir) genes Allow excision and integration into plant genomeAllow excision and integration into plant genome

8. 1.Auxin, cytokinin, opine synthetic genes transferred to plant 2.Plant makes all 3 compounds 3.Auxins and cytokines cause gall formation 4.Opines provide unique carbon/nitrogen source only A. tumefaciens can use!

9. Agrobacterium and genetic engineering: Engineering the Ti plasmid

10. Co-integrative and binary vectors Binary vector LBRB Co-integrative

12. Explants: cells and protoplasts Most direct way to introduce foreign DNA into the nucleus Achieved by electromechanically operated devices that control the insertion of fine glass needles into the nuclei of individuals cells, culture induced embryo, protoplast Labour intensive and slow Transformation frequency is very high, typically up to ca. 30% Electroporation

14. Microprojectile bombardment uses a ‘gene gun’ DNA is coated onto gold (or tungsten) particles (inert) gold is propelled by helium into plant cells if DNA goes into the nucleus it can be integrated into the plant chromosomes cells can be regenerated to whole plants

15. Pressure gauge Disk with DNA-coated particles Stop plate Sample goes here Vacuum line Gas line Rupture disk Vacuum chamber

16. In the "biolistic" (a cross between biology and ballistics )or "gene gun" method, microscopic gold beads are coated with the gene of interest and shot into the plant cell with a pulse of helium. In the "biolistic" (a cross between biology and ballistics )or "gene gun" method, microscopic gold beads are coated with the gene of interest and shot into the plant cell with a pulse of helium. Once inside the cell, the gene comes off the bead and integrates into the cell's genome. Once inside the cell, the gene comes off the bead and integrates into the cell's genome.

18. Model from BioRad: Biorad's Helios Gene Gun Model from BioRad: Biorad's Helios Gene Gun

19. Most direct way to introduce foreign DNA into the nucleus Achieved by electromechanically operated devices that control the insertion of fine glass needles into the nuclei of individuals cells, culture induced embryo, protoplast Labour intensive and slow Transformation frequency is very high, typically up to ca. 30% Microinjection

22. There are many thousands of cells in a leaf disc or callus clump - only a proportion of these will have taken up the DNA therefore can get hundreds of plants back - maybe only 1% will be transformed How do we know which plants have taken up the DNA? Could test each plant - slow, costly Or use reporter genes & selectable marker genes Screening technique

23. Screening Transformation frequency is low (Max 3% of all cells) and unless there is a selective advantage for transformed cells, these will be overgrown by non- transformed. Transformation frequency is low (Max 3% of all cells) and unless there is a selective advantage for transformed cells, these will be overgrown by non- transformed. Usual to use a positive selective agent like antibiotic resistance. The NptII gene encoding Neomycin phospho-transferase II phosphorylates kanamycin group antibiotics and is commonly used. Usual to use a positive selective agent like antibiotic resistance. The NptII gene encoding Neomycin phospho-transferase II phosphorylates kanamycin group antibiotics and is commonly used.

24. Screening (selection) Select at the level of the intact plant Select at the level of the intact plant Select in culture Select in culture single cell is selection unitsingle cell is selection unit possible to plate up to 1,000,000 cells on a Petri-dish.possible to plate up to 1,000,000 cells on a Petri-dish. Progressive selection over a number of phasesProgressive selection over a number of phases

25. Selection Strategies Positive Positive Negative Negative Visual Visual

26. Positive selection Add into medium a toxic compound e.g. antibiotic, herbicide Add into medium a toxic compound e.g. antibiotic, herbicide Only those cells able to grow in the presence of the selective agent give colonies Only those cells able to grow in the presence of the selective agent give colonies Plate out and pick off growing colonies. Plate out and pick off growing colonies. Possible to select one colony from millions of plated cells in a days work. Possible to select one colony from millions of plated cells in a days work. Need a strong selection pressure - get escapes Need a strong selection pressure - get escapes

27. Positive and Visual Selection

28. How do we get plants back from cells? We use tissue culture techniques to regenerate whole plants from single cells getting a plant back from a single cell is important so that every cell has the new DNA Regeneration System

29. Transformation series of events Transform individual cells Callus formation Auxins CytokininsRemove from sterile conditions

30. easy to visualise or assay - ß-glucuronidase (GUS) (E.coli) -green fluorescent protein (GFP) (jellyfish) - luciferase (firefly) Reporter gene

31. GUS Cells that are transformed with GUS will form a blue precipitate when tissue is soaked in the GUS substrate and incubated at 37 o C this is a destructive assay (cells die) The UidA gene encoding activity is commonly used. Gives a blue colour from a colourless substrate (X-glu) for a qualitative assay. Also causes fluorescence from Methyl Umbelliferyl Glucuronide (MUG) for a quantitative assay.

32. GUS Bombardment of GUS gene - transient expression Stable expression of GUS in moss Phloem-limited expression of GUS

33. HAESA gene encodes a receptor protein kinase that controls floral organ abscission. (A) transgenic plant expressing a HAESA::GUS fusion. It is expressed in the floral abscission zone at the base of an Arabidopsis flower. Transgenic plants that harbor the AGL12::GUS fusions show root- specific expression.

34. Inducible expression

35. GFP (Green Fluorescent Protein) GFP glows bright green when irradiated by blue or UV light This is a nondestructive assay so the same cells can be monitored all the way through Fluoresces green under UV illumination Fluoresces green under UV illumination Problems with a cryptic intron now resolved. Problems with a cryptic intron now resolved. Has been used for selection on its own. Has been used for selection on its own.

36. GFP protoplast colony derived from protoplast mass of callus regenerated plant

37. let you kill cells that haven’t taken up DNA- usually genes that confer resistance to a phytotoxic substance Most common: 1.antibiotic resistance kanamycin, hygromycin 2. herbicide resistance phosphinothricin (bialapos); glyphosate Selectable Marker Gene

38. Only those cells that have taken up the DNA can grow on media containing the selection agent

40. A. tumefaciens binary vector T-DNA