Plant transformation Flashcards
(20 cards)
What is the aim of plant transformation?
To generate whole plants that carry the foreign gene in every cell, that are transmitted to the next generation VIA SEED.
What are the main steps of plant transformation?
- Introduce a construct with desired gene
- Screen transformed cells (e.g. by using antibiotic / herbicide resistance genes)
- Regeneration of whole plant
What are some tissue culture variables?
- Culture media (macro and micro nutrients)
- Hormone type and concentration
- Light conditions, temp etc.
What is regeneration dependent on?
Supply of phytohormones (balance of auxin and cytokinin)
- Intermediate ratio of auxin: cytokinin to induce proliferation of cells
- High cytokinin levels to induce shoot formation
- High auxin levels to induce root formation
How are transformed cells selected?
Selection genes confer resistance to antibiotics and herbicides
Non transformed cells will be killed
Selection gene must be on the same gene of interest
e.g. Glyphosate in Agrobacterium
What does vector mediated transformation rely on?
Relies on Agrobacterium tumefaciens
What is Agrobacterium tumefaciens?
- Bacteria found in the rhizosphere
- Causes crown gall disease in plant cells
- Virulence is caused by the Ti plasmid
- Transfers T-DNA into nuclear DNA
- Disarmed plasmid is used in biotech
How is Agrobacterium tumefaciens used in biotech?
Tumour genes are deleted from T-DNA and replaced by selectable markers
Ti plasmid is too large to bioengineer (2000kbp)
So Ti plasmid virulence genes and T-DNA is cloned into separate plasmids (Binary vector)
T-DNA plasmid given E.coli and Agrobacterium replication origin
T-DNA cloning done in E.coli before shuttling into Agrobacterium
What does binary vector mean?
Uses 2 plasmids:
- T-DNA vector (carries gene construct) ~20 kbp
- pVIR plasmid (contains vir genes, retained in Agrobacterium), shuttle vector from E.coli to Agrobacterium ~170 kbp
What is a typical T-DNA construct?
- Promoter (most commonly CaMV 35S, has a high level of expression in multple cell types)
- DNA to be expressed
- Terminator (polyA addition sequence from CaMV 35S or nos gene, prevents read through into next gene)
- Selectable marker gene
- Right and left T-DNA borders
What is direct transformation?
Using a gene gun: tissue bombardment / biolistics
(Key to cereal transformation)
How can direct transformation lead to gene silencing?
Greater frequency of transgene rearrangement and transgene copy number than Agrobacterium mediated transformation, can lead to gene silencing
What is biolistics?
Tiny metal beads coated in foreign DNA shot at plant cells
What are biolistics beads made of?
Tungsten or gold
~ 1 micron diameter
What genetic material is required for direct transformation?
No T-DNA required, can be linear
But selectable marker is required
How does DNA incorporate in direct transformation?
DNA diffuses off of beads, and is often transiently expressed (temporarily)
If DNA enters the nucleus, there is a chance it becomes part of the hosts DNA permanently. Likely through host’s DNA repair mechanism.
Less commonly, DNA can enter other organelles (chloroplast / mitochondria)
What are the major differences between the DNA construct for direct vs. vector mediated transformation?
- Direct DNA fragment can be linear
- Does not T-DNA borders
Why is chloroplast transformation beneficial?
- Maternally inherited, DNA not transferred in pollen so reduced risk of escape and GM contamination
- Can utilise homologous recombination
- Multiple chloroplasts, meaning multiple genomes so high expression
Chloroplasts are bacterial in origin, what does this mean for transformation?
- Promoters must be recognised by a plastic RNA pol
- 5’ UTR requires ribosome binding site (Shine Dalgarno)
- Selection gene must be specific to chloroplast aadA gene
- Multiple genomes means it is likely that few will be translated, meaning several rounds of selection needed for homoplasy
- Can stack multiple genes in operons
What are the possible options for chloroplast transformation?
- Homologous recombination to knock out a specific chloroplast gene
- Site-directed mutagenesis, inserting a mutant version of a chloroplast gene
- Co-transformation, 2 constructs targeted to separate parts of the chloroplast genome
