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TRANSGENIC TECHNOLOGY

TRANSGENIC TECHNOLOGY. 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

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TRANSGENIC TECHNOLOGY

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  1. TRANSGENIC TECHNOLOGY

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

  3. 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’

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

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

  6. A natural genetic engineer 2 species A.tumefaciens (produces a gall) A. rhizogenes (produces roots) 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. Agrobacterium-mediated transformation

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

  8. Crown Gall Disease: Agrobacterium tumefaciens • Gram - • Dicots • Worldwide

  9. Disease Cycle

  10. Agrobacterium tumefaciens • Characteristics • Plant parasite that causes Crown Gall Disease • 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)

  11. Agrobacterium tumefaciens • 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 • 2 x 23bp direct repeats play an important role in the excision and integration process

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

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

  14. Ti plasmid of A. tumefaciens

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

  16. Agrobacterium tumefaciens • How is T-DNA modified to allow genes of interest to be inserted? • In vitro modification of Ti plasmid • T-DNA tumor causing genes are deleted and replaced with desirable genes (under proper regulatory control) • Insertion genes are retained (vir genes) • 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 • 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 • G418 or hygromycin added to kill non-transgenic plant cells • Surviving cells = transgenic plant cells

  17. Agrobacterium and genetic engineering: • Engineering the Ti plasmid

  18. Co-integrative and binary vectors LB RB Co-integrative Binary vector

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

  20. Expose wounded plant cells to transformed agro strain Electroporate T-DNA vector into Agrobacterium and select for tetr Agrobacterium Mediated Transfer Induce plant regeneration and select for Kanr cell growth

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

  22. Diagram of one technique

  23. 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

  24. 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.

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

  26. 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%

  27. Silicon Carbide Whiskers • 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

  28. Gene construct

  29. Gene construct Vectors Promoter/terminator reporter genes selectable marker genes ‘genes of interest’.

  30. 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

  31. 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

  32. 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)

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

  34. GUS 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) 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.

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

  36. 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.

  37. Inducible expression

  38. GFP (Green Fluorescent Protein) • Fluoresces green under UV illumination • Problems with a cryptic intron now resolved. • Has been used for selection on its own. 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

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

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

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

  42. 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: • Non plant genes • Plant genes

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

  44. Screening technique 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

  45. 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. • 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.

  46. 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

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