Transgenic Development (Plant Genetic Engineering)

1. Transgenic Development (Plant Genetic Engineering)

2. Why do scientists want to change gene instructions? • to produce needed chemicals • to carry out useful processes • to give an organism desired characteristics Genetic Engineering The process of manipulating and transferring instructions carried by genes from one cell to another

3. THE SCIENCE OF GENETIC ENGINEERING Isolate desired gene for a new trait from any organism Isolate plasmid DNA Gene inserted into plasmid. Introduce modified plasmid into bacterium for replication. Grow in culture to replicate

5. Plant transformation • getting DNA into a cell • getting it stably integrated • getting a plant back from the cell Requirement • a suitable transformation method • a means of screening for transformants • an efficient regeneration system • genes/constructs • vectors Promoter/terminator • reporter genes selectable marker genes • ‘genes of interest’

6. Transformation technique • Biological. • Agrobacterium mediated transformation. • Mechanical. • Particle bombardment. • Electroporation. • Microinjection. • Chemical. • Polyethylene glycol.

7. Transformation methods DNA must be introduced into plant cells IndirectAgrobacterium tumefaciens Direct1.Microprojectile bombardment 2. Electroporation 3. Microinjection Method depends on plant type, cost, application

8. Transformation by the help of agrobacterium Agrobacterium-mediated transformation Agrobacterium is a ‘natural genetic engineer’ i.e. it transfers some of its DNA to plants

9. Agrobacterium Plant cell Genomic DNA (carries the gene of interest) Genomic DNA Restriction enzyme A Restriction enzyme A Ti plasmid + Gene of interest Empty plasmid Ti plasmid with the gene of interest Agrobacterium tumefaciens

10. Ti plasmid with the new gene cell’s DNA + Transformation The new gene Agrobacterium Plant cell Transgenic plant Cell division Agrobacterium tumefaciens

11. T-DNA binary vector A. tumefaciens

12. Success Factor • Species • Genotypes • Explant • Agrobacterium strains • Plasmid

13. Direct gene transfer Introducing gene directly to the target cell • Electroporation • Microinjection • Particle Bombardment

14. Electroporation • Explants: cells and protoplasts • Most direct way to introduce foreign DNA into the nucleus • Achieved by electromechanically operated devices • Transformation frequency is high

16. Plant cell Duracell Protoplast The plant cell with the new gene DNA inside the plant cell DNA containing the gene of interest Electroporation Technique Power supply

17. Microinjection • 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%

18. 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

19. 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.

20. Cell’s DNA DNA coated golden particles Gene gun Plant cell A plant cell with the new gene Transgenic plant Cell division “Gene Gun” Technique

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

23. In Planta Transformation ♣ Meristem transformation ♣ Floral dip method ♣ Pollen transformation

24. Screening technique Technique which is exploited to screen the transformation product (transformant Cell) Reason: 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

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

26. Selection Strategies • Positive Selectable marker gene • Negative Selectable marker gene • Visual Reporter gene

27. Positive selection • Only individuals with characters satisfying the breeders are selected from population to be used as parents of the next generation • Seed from selected individuals are mixed, then progenies are grown together • 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 • Plate out and pick off growing colonies. • Possible to select one colony from millions of plated cells in a days work. • Need a strong selection pressure - get escapes

28. Negative selection • The most primitive and least widely used method which can lead to improvement only in exceptional cases • It implies culling out of all poorly developed and less productive individuals in a population whose productivity is to be genetically improved • Add in an agent that kills dividing cells • Plate out leave for a suitable time, wash out agent then put on growth medium. • All cells growing on selective agent will die leaving only non-growing cells to now grow. • Useful for selecting auxotrophs.

29. Positive and Visual Selection

30. Regeneration System 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

31. Transformation series of events Callus formation Transform individual cells Auxins Remove from sterile conditions Cytokinins

32. Gene construct

33. Gene construct Vectors Promoter/terminator Reporter genes Selectable marker genes ‘Genes of interest’.

34. Vectors A vehicle such as plasmid or virus for carrying recombinant DNA into a living cell • Ti-plasmid based vector • a. Co-integrative plasmid • b. Binary plasmid • Coli-plasmid based vector • a. Cloning vector • b. Chimeric Plasmid • Viral vector a. It is normally not stably integrated into the plant cell b. It may be intolerant of changes to the organization of its genome c. Genome may show instability

35. Ti plasmid

36. The binary Ti plasmid system

37. Binary vector system

38. Binary vector system

39. Promoter • A nucleotide sequence within an operon • Lying in front of the structural gene or genes • Serves as a recognition site and point of attachment for the RNA polymerase • It is starting point for transcription of the structural genes • It contains many elements which are involved in producing specific pattern and level of expression • It can be derived from pathogen, virus, plants themselves, artificial promoter

40. Types of Promoter • Promoter always expressed in most tissue (constitutive) -. 35 s promoter from CaMV Virus -. Nos, Ocs and Mas Promoter from bacteria -. Actin promoter from monocot -. Ubiquitin promoter from monocot -. Adh1 promoter from monocot -. pEMU promoter from monocot • Tissue specific promoter -. Haesa promoter -. Agl12 promoter • Inducible promoter -. Aux promoter • Artificial promoter -. Mac promoter (Mas and 35 s promoter)

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

42. GUS 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. Cells that are transformed with GUS will form a blue precipitate when tissue is soaked in the GUS substrate and incubated at 37oC this is a destructive assay (cells die)

43. 5 -- glucuronidase Genes • very stable enzyme • cleaves -D glucuronide linkage • simple biochemical reaction • It must take care to stay in linear range • detection sensitivity depends on substrate used in enzymatic assay (fast) • colorimetric and fluorescent substrates available

44. 5 - -glucuronidase Genes • Advantages • low background • can require little equipment (spectrophotometer) • stable enzyme at 37ºC • Disadvantages • sensitive assays require expensive substrates or considerable equipment • stability of the enzyme makes it a poor choice for reporter in transient transfections (high background = low dynamic range) • Primary applications • typically used in transgenic plants with X-gus colorimetric reporter

45. β-Glucorodinase gene Bombardment of GUS gene - transient expression Stable expression of GUS in moss Phloem-limited expression of GUS

46. GFP (Green Fluorescent Protein) GFP glows bright green when irradiated by blue or UV light This is a non destructive assay so the same cells can be monitored all the way through • It fluoresces green under UV illumination • It has been used for selection on its own

47. Green fluorescent protein (GFP) • Source is bioluminescent jellyfish Aequoravictoria • GFP is an intermediate in the bioluminescent reaction • Absorbs UV (~360 nm) and emits visible light. • has been engineered to produce many different colors (green, blue, yellow, red) • These are useful in fluorescent resonance energy transfer experiments • Simply express in target cells and detect with fluorometer or fluorescence microscope • Sensitivity is low • GFP is non catalytic, 1 M concentration in cells is required to exceed auto-fluorescence

48. Green fluorescent protein (GFP) • Advantages • can detect in living cells • inexpensive (no substrate) • Disadvantages • low sensitivity and dynamic range • equipment requirements • Primary applications • lineage tracer and reporter in transgenic embryos

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

50. Luciferase • luc gene encodes an enzyme that is responsible for bioluminescence in the firefly. This is one of the few examples of a bioluminescent reaction that only requires enzyme, substrate and ATP. • Rapid and simple biochemical assay. Read in minutes • Two phases to the reaction, flash and glow. These can be used to design different types of assays. • Addition of substrates and ATP causes a flash of light that decays after a few seconds when [ATP] drops • after the flash, a stable, less intense “glow” reaction continues for many hours - AMP is responsible for this