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TRANSGENIC TECHNOLOGY. 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.
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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
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’
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
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
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
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) • 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
Agrobacterium 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 • 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
Ti plasmid of A. tumefaciens 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!
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
Agrobacterium and genetic engineering: • Engineering the Ti plasmid
Co-integrative and binary vectors LB RB Co-integrative Binary vector
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
Factor determining the success • Species • Genotypes • Explant • Agrobacterium strains • Plasmid
Direct gene transfer Introducing gene directly to the target cell
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
Microprojectile bombardment 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.
Cell’s DNA DNA coated golden particles Gene gun Plant cell A plant cell with the new gene Cell division Transgenic plant “Gene Gun” Technique
Electroporation • 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%
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
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%
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
Competent cells 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 regenerable cells Transformed cells Cells containing new DNA that are able to regenerate into a new plant
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
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 screening agent like antibiotic resistance. The NptII gene encoding Neomycin phospho-transferase II phosphorylates kanamycin group antibiotics and is commonly used.
Screening Investigating based on a large number of organism for the presence of a particular property as in screening for a mutation for antibiotic resistance • Screen at the level of the intact plant • Screen 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
Selection methods • The most common procedure was mass-selection which in turn was subdivided into negative and positive • 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 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
Selection Strategies • Positive • Negative • Visual
Positive selection • 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
Negative selection • 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. • All cells growing on selective agent will die leaving only non-growing cells to now grow. • Useful for selecting auxotrophs.
Visual selection • Only useful for colored or fluorescent compounds • Plate out at about 50,000 cells per plate. • Pick off colored / fluorescent compounds • Possible to screen about 1,000,000 cells in a days work.
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
Regeneration Plant tissue culture uses growth regulators and nutrients to regenerate plants in vitro Regeneration of shoots from leaf protoplasts in Arabidopsis thaliana
Cloning Vectors Plasmids that can be modified to carry new genes • Plasmids useful as cloning vectors must have • a replicator (origin of replication) • a selectable marker (antibiotic resistance gene) • a cloning site (site where insertion of foreign DNA will not disrupt replication or inactivate essential markers
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 • Ends of the plasmid/fragment are closed to form a "recombinant plasmid" • Plasmid can replicate when placed in a suitable bacterial host
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 • Construct foreign DNA with same two restriction enzymes • Foreign DNA can only be inserted in one direction
