Plant Genetic Engineering

Genetic Engineering The process of manipulating and transferring instructions carried by genes from one cell to another Why do scientists want to change gene instructions? to produce needed chemicals to carry out useful processes to give an organism desired characteristics

DNA IS EVERYWHERE

Plant Genetic Engineering Process Cell Extracted DNA Cell division Transgenic plant A single gene Transformation Plant cell

Why Plants? Plants are also very flexible and can produce a wide variety of proteins. Plants are also very flexible and can produce a wide variety of proteins. Crop plants can synthesize a wide variety of proteins that are free of mammalian toxins and pathogens. Crop plants can synthesize a wide variety of proteins that are free of mammalian toxins and pathogens. Crop plants produce large amounts of biomass at low cost and require limited facilities. Crop plants produce large amounts of biomass at low cost and require limited facilities. Crops are therefore well suited for the production of safe low-cost proteins Crops are therefore well suited for the production of safe low-cost proteins

getting DNA into a cell getting it stably integrated getting a plant back from the cell Plant transformation Requirement 1.a suitable transformation method 2.a means of screening for transformants 3.an efficient regeneration system

Transformation methods DNA must be introduced into plant cells Indirect - Agrobacterium tumefaciens Direct Microprojectile bombardment Electroporation Microinjection Method depends on plant type, cost, application

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

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

Agrobacterium tumefaciens Ti plasmid Agrobacterium Genomic DNA Plant cell the gene of interest Genomic DNA (carries the gene of interest) + Ti plasmid with the gene of interest Gene of interest Empty plasmid A Restriction enzyme A

Agrobacterium Ti plasmid with the new gene Plant cell cell’s DNA Transgenic plant Cell division The new gene + Transformation Agrobacterium tumefaciens

A. tumefaciens binary vector T-DNA

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

Direct gene transfer Introducing gene directly to the target cell 1.Electroporation 2.Microinjection 3.Particle Bombardment

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

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

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

Pressure gauge Disk with DNA-coated particles Stop plate Sample goes here Vacuum line Gas line Rupture disk Vacuum chamber

“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

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.

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

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

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

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

Selection Strategies Positive Positive Negative Negative Visual Visual

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

Positive and Visual Selection

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

Transformation series of events Transform individual cells Callus formation Auxins CytokininsRemove from sterile conditions

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

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.

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

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.

GFP protoplast colony derived from protoplast mass of callus regenerated plant

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

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