1. TRANSGENIC TECHNOLOGY

2. Traits that plant breeders would like in plants High primary productivity High primary productivity High crop yield High crop yield High nutritional quality High nutritional quality Adaptation to inter- cropping Adaptation to inter- cropping Nitrogen Fixation Nitrogen Fixation Drought resistance Drought resistance Pest resistance Pest resistance Adaptation to mechanised farming Adaptation to mechanised farming Insensitivity to photo-period Insensitivity to photo-period Elimination of toxic compounds Elimination of toxic compounds

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 - Microprojectile bombardment - Electroporation - Polyethylene glycol (PEG) - Glass-beads - Silicon carbide whiskers Method depends on plant type, cost, application

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

7. Agrobacterium 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. Agrobacterium tumefaciens Characteristics Characteristics 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 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

9. Agrobacterium tumefaciens Lives in intercellular spaces of the plant Lives in intercellular spaces of the plant Plasmid contains genes responsible for the disease Plasmid 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 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

10. Ti plasmid of A. tumefaciens

11. 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! Ti plasmid of A. tumefaciens

12. 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

13. Agrobacterium and genetic engineering: Engineering the Ti plasmid

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

15. 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

17. Factor determining the success Species Species Genotypes Genotypes Explant Explant Agrobacterium strains Agrobacterium strains Plasmid Plasmid

18. Direct gene transfer Introducing gene directly to the target cell

19. 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

20. 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. Microprojectile bombardment

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

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

24. Explants: cells and protoplasts Most direct way to introduce foreign DNA into the nucleus Achieved by electromechanically operated devices Labour intensive and slow Transformation frequency is very high, typically up to ca. 30% Electroporation

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

26. 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

27. 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

28. Not all cells take up DNA & not all cells can regenerate Need an efficient regeneration system and transformation system i.e. lots of cells take up DNA and lots of cells regenerate into a plant to maximize chance of both happening Transformed cellsregenerable cells Cells containing new DNA that are able to regenerate into a new plant Competent cells

29. 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

30. 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 screening agent like antibiotic resistance. The NptII gene encoding Neomycin phospho-transferase II phosphorylates kanamycin group antibiotics and is commonly used. Usual to use a screening agent like antibiotic resistance. The NptII gene encoding Neomycin phospho-transferase II phosphorylates kanamycin group antibiotics and is commonly used.

31. Screening Screen at the level of the intact plant Screen at the level of the intact plant Screen in culture Screen 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 Investigating based on a large number of organism for the presence of a particular property as in screening for a mutation for antibiotic resistance

32. Selection methods The most common procedure was mass-selection which in turn was subdivided into negative and positive The most common procedure was mass-selection which in turn was subdivided into negative and positive Negative selection 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 The remaining best individuals are propagated as much as necessary Positive selection 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

33. Selection Strategies Positive Positive Negative Negative Visual Visual

34. 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

35. 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.

36. Visual selection Only useful for colored or fluorescent compounds Only useful for colored or fluorescent compounds Plate out at about 50,000 cells per plate. Plate out at about 50,000 cells per plate. Pick off colored / fluorescent compounds Pick off colored / 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.

37. Positive and Visual Selection

38. 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

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

40. Somatic embryogenesis in peanut

41. Organogenesis

42. Gene construct

43. Cloning Vectors Plasmids that can be modified to carry new genes Plasmids useful as cloning vectors must have Plasmids useful as cloning vectors must have a replicator (origin of replication)a replicator (origin of replication) a selectable marker (antibiotic resistance gene)a selectable marker (antibiotic resistance gene) a cloning site (site where insertion of foreign DNA will not disrupt replication or inactivate essential markersa cloning site (site where insertion of foreign DNA will not disrupt replication or inactivate essential markers

46. A typical plasmid vector with a polylinker

47. Chimeric Plasmids Named for mythological beasts with body parts from several creatures After cleavage of a plasmid with a restriction enzyme, a foreign DNA fragment can be inserted After cleavage of a plasmid with a restriction enzyme, a foreign DNA fragment can be inserted Ends of the plasmid/fragment are closed to form a "recombinant plasmid" Ends of the plasmid/fragment are closed to form a "recombinant plasmid" Plasmid can replicate when placed in a suitable bacterial host Plasmid can replicate when placed in a suitable bacterial host

50. Directional Cloning Often one desires to insert foreign DNA in a particular orientation This can be done by making two cleavages with two different restriction enzymes This can be done by making two cleavages with two different restriction enzymes Construct foreign DNA with same two restriction enzymes Construct foreign DNA with same two restriction enzymes Foreign DNA can only be inserted in one direction Foreign DNA can only be inserted in one direction

52. 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

53. 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)

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

55. 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.

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

57. 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.

58. Inducible expression

59. 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 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.

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

61. 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

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

63. 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

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