TRANSGENIC TECHNOLOGY

1. TRANSGENIC TECHNOLOGY

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

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

4. 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’

5. Transformation methods DNA must be introduced into plant cells Indirect- Agrobacterium tumefaciens Direct- microprojectile bombardment - electroporation - Polyethylene glycol (PEG)-mediated - glass-beads - silicon carbide whiskers Method depends on plant type, cost, application

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

16. 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!

17. 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

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

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

20. 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

21. 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

24. 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

25. 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.

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

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

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

30. Somatic embryogenesis in peanut

31. Screening Technique

32. Not all cells take up DNA & not all cells can regenerate so 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 regenerable cells Transformed cells Cells containing new DNA that are able to regenerate into a new plant

33. 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

34. Selection • 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.

35. Reportergenes - easy to visualise or assay most common - ß-glucuronidase (GUS) (E.coli) - green fluorescent protein (GFP) (jellyfish) - luciferase (firefly, bacterial, jellyfish etc)

36. 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.

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

38. 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.

39. 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

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

41. Selectable marker genes - let you kill cells that haven’t taken up DNA- usually genes that confer resistance to a phytotoxic substance Most common: antibiotic resistance - e.g. kanamycin, hygromycin [ only phytotoxic antibiotics can be used] herbicide resistance - e.g. phosphinothricin (PPT); glyphosate

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

43. Pathogen resistance Herbicide resistance Bioreactors/molecular farming Delivery systems Plant improvement Gene silencing/ downregulation APPLICATIONS transfer of exogenous genes manipulation of endogenous genes

44. Gene silencing/ downregulation of endogenous genes Antisense RNA – delayed ripening; FlavR SavR tomatoes - modified flower colour (paler flowers) Post-transcriptional gene silencing induces cytoplasmic RNA degradation system induced by dsRNA highly sequence specific

45. Applications of Plant Biotechnology • Crop Improvement • The following traits are potentially useful to plant genetic engineering: controlling insects, manipulating petal color, production of industrially important compounds, and plant growth in harsh conditions. • Genetically Engineered Traits: The Big Six. • Herbicide Resistance • Herbicides are a huge industry, with herbicide use quadrupling between 1966 and 1991, so plants that resist chemicals that kill them are a growing need. • Critics claim that genetically engineered plants will lead to more chemical use, and possible development of weeds resistant to the chemicals.

46. Applications of Plant Biotechnology • Glyphosate Resistance • Marketed under the name Roundup, glyphosate inhibits the enzyme EPSPS, makes aromatic amino acids. • The gene encoding EPSPS has been transferred from glyphosate-resistant E. coli into plants, allowing plants to be resistant. • Glufosinate Resistance • Glufosinate (the active ingredient being phosphinothricin) mimics the structure of the amino acid glutamine, which blocks the enzyme glutamate synthase. • Plants receive a gene from the bacterium Streptomyces that produce a protein that inactivates the herbicide.

47. Applications of Plant Biotechnology • Bromoxynil Resistance • A gene encoding the enzyme bromoxynil nitrilase (BXN) is transferred from Klebsiella pneumoniae bacteria to plants. • Nitrilase inactivates the Bromoxynil before it kills the plant. • Sulfonylurea. • Kills plants by blocking an enzyme needed for synthesis of the amino acids valine, leucine, and isoleucine. • Resistance generated by mutating a gene in tobacco plants, and transferring the mutated gene into crop plants.

48. Applications of Plant Biotechnology • Insect Resistance • The Bt toxin isolated from Bacillus thuringiensis has been used in plants. The gene has been placed in corn, cotton, and potato, and has been marketed. • Plant protease inhibitors have been explored since the 1990s: • Naturally produced by plants, are produced in response to wounding. • They inhibit insect digestive enzymes after insects ingest them, causing starvation. • Tobacco, potato, and peas have been engineered to resist insects such as weevils that damage crops while they are in storage • Results have not been as promising as with Bt toxin, because it is believed that insects evolved resistance to protease inhibitors.

49. Applications of Plant Biotechnology • Virus Resistance • Chemicals are used to control the insect vectors of viruses, but controlling the disease itself is difficult because the disease spreads quickly. • Plants may be engineered with genes for resistance to viruses, bacteria, and fungi. • Virus-resistant plants have a viral protein coat gene that is overproduced, preventing the virus from reproducing in the host cell, because the plant shuts off the virus’ protein coat gene in response to the overproduction. • Coat protein genes are involved in resistance to diseases such as cucumber mosaic virus, tobacco rattle virus, and potato virus X.

50. Applications of Plant Biotechnology • Resistance genes for diseases such as fungal rust disease and tobacco mosaic virus have been isolated from plants and may be transferred to crop plants. • Yellow Squash and Zucchini • Seeds are available that are resistant to watermelon mottle virus, zucchini yellow mosaic virus, and cucumber mosaic virus. • Potato. • Monsanto developed potatoes resistant to potato leaf roll virus and potato virus X, which also contained a Bt toxin gene as a pesticide. • hain restaurants do not use genetically engineered potatoes due to public pressures. • Papaya. • Varieties resistant to papaya ring spot virus have been developed.