Transgenic Development (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

THE SCIENCE OF GENETIC ENGINEERING

Isolate desired gene for a new trait from any organism Isolate plasmid DNA Gene inserted into plasmid.

Introduce modified plasmid into bacterium for replication.

Grow in culture to replicate

Plant transformation

 getting DNA into a cell  getting it stably integrated  getting a plant back from the cell

Requirement

1. a suitable transformation method 2. a means of screening for transformants 3. an efficient regeneration system 4. genes/constructs v ectors reporter genes ‘genes of interest’ Promoter/terminator selectable marker genes

Transformation technique

   Biological.

• Agrobacterium mediated transformation.

Mechanical.

• Particle bombardment.

• Electroporation.

• Microinjection.

Chemical.

• Polyethylene glycol.

Transformation methods

DNA must be introduced into plant cells

Indirect

Agrobacterium tumefaciens

Direct

1. Microprojectile bombardment 2. Electroporation 3. 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

Agrobacterium tumefaciens

Agrobacterium Genomic DNA Genomic DNA (carries the gene of interest ) Plant cell Ti plasmid Restriction enzyme A

Restriction enzyme A

Empty plasmid

+

Gene of interest Ti plasmid with the gene of interest

Agrobacterium tumefaciens

Ti plasmid with

the new gene +

cell’s DNA Transformation Agrobacterium Plant cell The new gene Transgenic plant Cell division

T-DNA

binary vector

A. tumefaciens

Success Factor

   Species Genotypes  Explant Agrobacterium strains  Plasmid

Direct gene transfer

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

Electroporation

 Explants: cells and protoplasts  Most direct way to introduce foreign DNA into the nucleus  Achieved by electromechanically operated devices  Transformation frequency is high

Electroporation Technique

Plant cell Power supply

Duracell

Protoplast DNA containing

the gene of interest

DNA inside the plant cell The plant cell with

the new gene

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%

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

 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.

“Gene Gun” Technique

DNA coated golden particles Cell’s DNA Plant cell Gene gun A plant cell with

the new gene

Transgenic plant Cell division

Model from BioRad: Biorad's Helios Gene Gun

♣ ♣ ♣

In Planta Transformation

Meristem transformation Floral dip method Pollen transformation

Screening technique

Technique which is exploited to screen the transformation product (transformant Cell) Reason: 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

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

Selection Strategies

 Positive  Negative  Visual Selectable marker gene Selectable marker gene Reporter gene

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      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

  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     Add in an agent that kills dividing cells 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.

Positive and Visual Selection

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

Transformation series of events

Callus formation Transform individual cells Auxins Remove from sterile conditions Cytokinins

Gene construct

Bam

HI

LB

T

35S

nptII

P

35S

P SAG12 ipt

T nos T

35S

gus-intron

P

35S

RB

Gene construct

Vectors Promoter/terminator Reporter genes Selectable marker genes ‘Genes of interest

’.

Vectors

A vehicle such as plasmid or virus for carrying recombinant DNA into a living cell    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

Ti plasmid

The binary Ti plasmid system

Binary vector system

Binary vector system

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, artificial promoter

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)

Reporter gene

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

GUS

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.

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)

5 -



- glucuronidase Genes

  very stable enzyme cleaves  -D glucuronide linkage  simple biochemical reaction • It must take care to stay in linear range  detection sensitivity depends on substrate used in enzymatic assay (fast) • colorimetric and fluorescent substrates available

  

5 -



-glucuronidase Genes

Advantages • low background • can require little equipment (spectrophotometer) • stable enzyme at 37ºC Disadvantages • sensitive assays require expensive substrates or considerable equipment • stability of the enzyme makes it a poor choice for reporter in transient transfections (high background = low dynamic range) Primary applications • typically used in transgenic plants with X-gus colorimetric reporter

β Glucorodinase gene

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 non destructive assay so the same cells can be monitored all the way through   It fluoresces green under UV illumination It has been used for selection on its own

Green fluorescent protein (GFP)

    Source is bioluminescent jellyfish Aequora victoria  GFP is an intermediate in the bioluminescent reaction Absorbs UV (~360 nm) and emits visible light.

 has been engineered to produce many different colors (green, blue, yellow, red)  These are useful in fluorescent resonance energy transfer experiments Simply express in target cells and detect with fluorometer or fluorescence microscope Sensitivity is low  GFP is non catalytic, 1  M concentration in cells is required to exceed auto-fluorescence

Green fluorescent protein (GFP

)  Advantages • can detect in living cells • inexpensive (no substrate)  Disadvantages • low sensitivity and dynamic range • equipment requirements  Primary applications • lineage tracer and reporter in transgenic embryos

GFP

protoplast colony derived from protoplast mass of callus regenerated plant

Luciferase

   luc gene encodes an enzyme that is responsible for bioluminescence in the firefly. This is one of the few examples of a bioluminescent reaction that only requires enzyme, substrate and ATP. Rapid and simple biochemical assay. Read in minutes Two phases to the reaction, flash and glow. These can be used to design different types of assays.

• Addition of substrates and ATP causes a flash of light that decays after a few seconds when [ATP] drops • after the flash, a stable, less intense “glow” reaction continues for many hours - AMP is responsible for this

Luciferase

 flash reaction is ~20x more sensitive than glow  glow reaction is more stable • allows use of scintillation counter • no injection of substrates required • potential for simple automation in microplate format

Luciferase

 Advantages • large dynamic range up to 7 decades, depending on instrument and chemistry • rapid, suitable for automation • instability of luciferase at 37 ° C (1/2 life of <1hr) • inexpensive • widely used  disadvantages • Equipment requirement • luminometer (very big differences between models) • liquid scintillation counter (photon counter)

Selectable Marker Gene

Gene which confer tolerance to a phytotoxic substance Most common: 1. antibiotic resistance kanamycin (geneticin), hygromycin Kanamycin arrest bacterial cell growth by blocking various steps in protein synthesis 2. herbicide resistance phosphinothricin (bialapos); glyphosate

Effect of Selectable Marker Non-transgenic =

Lacks Kan or Bar Gene

Plant dies in presence of selective compound X Transgenic =

Has Kan or Bar Gene

Plant grows in presence of selective compound

Kanamycin

  Targets 30s ribosomal subunit, causing a frameshift in every translation Bacteriostatic: bacterium is unable to produce any proteins correctly, leading to a halt in growth and eventually cell death

Kanamycin use/resistance

   Over-use of kanamycin has led to many wild bacteria possessing resistance plasmids As a result of this (as well as a lot of side effects in humans), kanamycin is widely used for genetic purposes rather than medicinal purposes, especially in transgenic plants Resistance is often to a family of related antibiotics, and can include antibiotic-degrading enzymes or proteins protecting the 30s subunit

G418-Gentamycin

 source: aminoglycoside antibiotic related to gentamycin  activity: broad action against prokaryotic and eukaryotic cells • inhibits protein synthesis by blocking initiation  resistance - bacterial neo gene (neomycin phosphotransferase, encoded by Tn5 encodes resistance to kanamycin, neomycin, G418 • but also cross protects against bleomycin and relatives.

G418 - Gentamycin

 Stability: • 6 months frozen  selection conditions: • E. coli: 5  g/ml • Eukaryotic cells:  300-1000  g/ml. G418 requires careful optimization for cell types and lot to lot variations  Kill curves required  It requires at least seven days to obtain resistant colonies, two weeks is more typical

G418 - Gentamycin

Increasing dose ->  use and availability: • perhaps the most widely used selection in mammalian cells • vectors very widely available

Hygromycin

 source: aminoglycoside antibiotic from Streptomyces hygroscopicus.  Activity: kills bacteria, fungi and higher eukaryotic cells by inhibiting protein synthesis • interferes with translocation causing misreading of mRNA  resistance: conferred by the bacterial gene hph • no cross resistance with other selective antibiotics

Hygromycin

 stability: • one year at 4 ºC, 1 month at 37 ºC  selection conditions: • E. coli: 50  g/ml • Eukaryotic cell lines:  50 - 1000  g/ml (must be optimized)  10 days- 3 weeks required to generate effect  use and availability: • vectors containing hygromycin resistance gene are widely available • in use for many years

Glyphosate resistance

 Glyphosate = “Roundup”, “Tumbleweed” = Systemic herbicide  Glyphosate inhibits EPSP synthase (S enolpyruvlshikimate-3 phosphate – involved in chloroplast amino acid synthesis)  Escherichia coli EPSP synthase = mutant form  sensitive to glyphosate less  Cloned via Ti plasmid into soybeans, tobacco, petunias • Increased crop yields of crops treated with herbicides

RoundUp Sensitive Plants Shikimic acid + Phosphoenol pyruvate + Glyphosate

Plant

X 3-Enolpyruvyl shikimic acid-5-phosphate X Without amino acids, plant dies X Aromatic X

RoundUp Resistant Plants Shikimic acid + Phosphoenol pyruvate + Glyphosate

Bacterial EPSP synthase

RoundUp has no effect; enzyme is resistant to herbicide 3-enolpyruvyl shikimic acid-5-phosphate (EPSP) With amino acids, plant lives Aromatic amino acids

Bialaphos

    Glufosinate – active substance of a broad-spectrum herbicide = synthetical copy of the aminoacid phosphinothricin produced by Streptomyces

viridochomogenes

Inhibit glutamine-synthetase (important enzyme in nitrogen-cycle of plants) caused plant dies Herbicide-tolerance is reached by gene-transfer from the bacterium to the plant The transfered gene encodes for the enzyme phophinothricin-acetyl-transferase degrade glufosinate

Bialaphos

* Bialaphos (Phosphinothricin-alanyl-alanine) is an herbicide that inhibits a key enzyme in the nitrogen assimilation pathway, glutamine synthetase, leading to accumulation of toxic levels of ammonia in both bacteria and plant cells

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