2. TRANSGENIC TECHNOLOGY

3. getting DNA into a cell getting it stably integrated getting a plant back from the cell Plant transformation

4. 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

5. 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

6. Electrical method 1.Electroporation (electropermeabilization) 2.Cells or protoplast are subjected to short electrical pulse Physical Methods 1.Particle bombardment 2.Microinjection 3.Silicon Carbide whiskers

7. Agrobacterium-mediated transformation A natural genetic engineer A natural genetic engineer 2 species 2 species A.tumefaciens (produces a gall)A.tumefaciens (produces a gall) A. rhizogenes (produces roots)A. rhizogenes (produces roots) Oncogenes (for auxin and cytokinin synthesis) + Opines Oncogenes (for auxin and cytokinin synthesis) + Opines In the presence of exudates (e.g. acetosyringone) from wounded plants, Virulence (Vir) genes are activated and cause the t-DNA to be transferred to plants. Everything between the left and right border is transferred. In the presence of exudates (e.g. acetosyringone) from wounded plants, Virulence (Vir) genes are activated and cause the t-DNA to be transferred to plants. Everything between the left and right border is transferred.

8. BACTERIAL GALL DISEASES Galls: Galls: overgrowth or proliferation of tissue, primarily due to increased cell division (hyperplasia) and increased cell size (hypertrophy). Bacterial Galls: Bacterial Galls: induced by bacteria in 3 different genera. AgrobacteriumAgrobacterium PseudomonasPseudomonas ClavibacterClavibacter Genes for plant hormone production found on bacterial plasmids! Genes for plant hormone production found on bacterial plasmids!

9. Crown Gall Disease: Agrobacterium tumefaciens Gram - Gram - Dicots Dicots Worldwide Worldwide

10. Disease Cycle

11. Agrobacterium tumefaciens Characteristics Characteristics Plant parasite that causes Crown Gall DiseasePlant parasite that causes Crown Gall Disease Encodes a large (~250kbp) plasmid called Tumor-inducing (Ti) plasmidEncodes 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 )

12. Agrobacterium tumefaciens 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 Then integrated into plant genome Then integrated into plant genome 2 x 23bp direct repeats play an important role in the excision and integration process 2 x 23bp direct repeats play an important role in the excision and integration process

13. 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

14. 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

15. Ti plasmid of A. tumefaciens

17. 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!

18. Agrobacterium tumefaciens How is T-DNA modified to allow genes of interest to be inserted? How is T-DNA modified to allow genes of interest to be inserted? In vitro modification of Ti plasmidIn vitro modification of Ti plasmid T-DNA tumor causing genes are deleted and replaced with desirable genes (under proper regulatory control) T-DNA tumor causing genes are deleted and replaced with desirable genes (under proper regulatory control) Insertion genes are retained (vir genes) Insertion genes are retained (vir genes) Selectable marker gene added to track plant cells successfully rendered transgenic [antibiotic resistance gene  geneticin (G418) or hygromycin] Selectable marker gene added to track plant cells successfully rendered transgenic [antibiotic resistance gene  geneticin (G418) or hygromycin] Ti plasmid is reintroduced into A. tumefaciens Ti plasmid is reintroduced into A. tumefaciens A. tumefaciens is co-cultured with plant leaf disks under hormone conditions favoring callus development (undifferentiated) A. tumefaciens is co-cultured with plant leaf disks under hormone conditions favoring callus development (undifferentiated) Antibacterial agents (e.g. chloramphenicol) added to kill A. tumefaciens Antibacterial agents (e.g. chloramphenicol) added to kill A. tumefaciens G418 or hygromycin added to kill non-transgenic plant cells G418 or hygromycin added to kill non-transgenic plant cells Surviving cells = transgenic plant cells Surviving cells = transgenic plant cells

19. Agrobacterium and genetic engineering: Engineering the Ti plasmid

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

21. cause ‘Crown gall’ disease Agrobacterium tumefaciens Agrobacterium-mediated transformation Agrobacterium is a ‘natural genetic engineer’ i.e. it transfers some of its DNA to plants

22. Electroporate T- DNA vector into Agrobacterium and select for tet r Expose wounded plant cells to transformed agro strain Induce plant regeneration and select for Kan r cell growth

25. Explants: cells and protoplasts Most direct way to introduce foreign DNA into the nucleus Electroporation

26. Diagram of one technique

27. 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

28. 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.

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

31. 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

32. Silicon carbide forms long, needle like crystals Cells are vortex mixed in the present of whiskers and DNA DNA can be introduced in the cells following penetration by the whiskers Silicon Carbide Whiskers

33. Gene construct

34. Vectors Promoter/terminator reporter genes selectable marker genes ‘genes of interest’.

35. Vectors 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

36. Promoter 1.A nucleotide sequence within an operon 2.Lying in front of the structural gene or genes 3.Serves as a recognition site and point of attachment for the RNA polymerase 4.It is starting point for transcription of the structural genes 5.It contains many elements which are involved in producing specific pattern and level of expression 6.It can be derived from pathogen, virus, plants themselves, artificial promoter

37. Types of Promoter Promoter always expressed in most tissue (constitutive) 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 Tissue specific promoter -. Haesa promoter -. Agl12 promoter Inducible promoter Inducible promoter -. Aux promoter Artificial promoter Artificial promoter -. Mac promoter (Mas and 35 s promoter)

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

39. 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.

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

41. 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.

42. Inducible expression

43. 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.

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

45. 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

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

47. Gene of interest Sequence of DNA which will be inserted to the host cell and its product will be studied or beneficial for mankind Origin of gene interest: 1.Non plant genes 2.Plant genes

48. Exogenous genes (non-plant genes) pathogen-derived genes bacterial genes any other organism Endogenous genes (Plant genes ) Enzymes in biochemical pathway Natural resistance genes

49. 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

50. 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.

51. 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

52. Selection Strategies Positive Positive Negative Negative Visual Visual

53. 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

54. Negative selection Add in an agent that kills dividing cells e.g. chlorate / BUdR. Add in an agent that kills dividing cells e.g. chlorate / BUdR. Plate out leave for a suitable time, wash out agent then put on growth medium. 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. All cells growing on selective agent will die leaving only non-growing cells to now grow. Useful for selecting auxotrophs. Useful for selecting auxotrophs.

55. Visual selection Only useful for coloured or fluorescent compounds Only useful for coloured or fluorescent compounds Plate out at about 50,000 cells per plate. Plate out at about 50,000 cells per plate. Pick off coloured / fluorescent compounds Pick off coloured / fluorescent compounds Possible to screen about 1,000,000 cells in a days work. Possible to screen about 1,000,000 cells in a days work.

56. Positive and Visual Selection

57. 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

58. Regeneration Regeneration of shoots from leaf protoplasts in Arabidopsis thaliana Plant tissue culture uses growth regulators and nutrients to regenerate plants in vitro

59. Somatic embryogenesis in peanut