lecture 12 Flashcards
agrobacterium-mediated plant transformation continued non-bacterial ways of transforming plants - why bother with other methods? - Guns - biolistic transformation - organelle transformation - what are plastids are why transform them? - plastid transformation — the problem of heteroplasmy - biotechnological applications — Bt toxin - viruses as a gene delivery system Using examples, give two advantages of expressing a foreign protein in the plastid over the nucleus? Describe ho
What determines whether a gall is rooty or shooty or elsewise?
- Tms and Tmr
- mutation in cytokinin gene (Tmr) gives a rooty phenotype (i.e. only capable of producing auxin)
- reversing the situation gives a shooty phenotype
What other plant pathogens cause galls by secreting plant hormones?
- witches broom on birch (cytokinin secretion)
- olive branch with a gall caused by Pseudomonas savastanoi (auxin secretion)
What are the good features of Agrobacterium?
- transfers DNA to plant cells
- selection system for transformed cells
- organised structures (leaves, roots) can form
What are the bad features of Agrobacterium?
- opines
- gall formation
- Ti plasmid is too big for recombinant DNA work
What is the binary vector system?
- creating two plasmids
- T-DNA and Vir genes are on separate plasmids
- T-DNA plasmid is small and is stable in both E. coli and Agrobacterium
- Select transformed plant cells using an antibiotic resistance gene
- just have to have the left and right border
- get rid of the bad aspects of agrobacterium (‘Disarmed’ Ti plasmid)
What is the basic protocol for Agrobacterium transformation?
- prepare explants (leaf pieces, hypocotyl segments, etc)
- dip in Agrobacterium solution containing vector
- place infected explants on agar plates containing cytokinin and auxin
- culture with Agrobacterium
- remove Agrobacterium infection with antibiotics
- tissue culture to induce root/shoot formation
- transfer transgenic plants to soil
Why bother with other methods?
- some plant species are highly resistant to Agrobacterium infection or cannot be regenerated from susceptible tissues
What did John Sanford do?
- wanted to insert genes into pollen grains through holes drilled into the surface
- didn’t work
- thought a ‘bullet’ might work better
- the gene gun
- bought a cheap air gun and some onions and fiddled with the gas blast until he found conditions that were “less than catastrophic”
- later showed this could be used to introduce DNA into cells
- sold idea to Dupont, who produced a range of ‘biolistic’ devices (including hand-held guns)
- used routinely for transient as well as stable plant transformation
What is the difference between transient and stable transformation?
Transient transformation:
- introduced DNA is expressed but not integrated into host cell chromosomes
- because introduced DNA isn’t copied during mitosis, expression will decline over time
Stable transformation:
- introduced DNA is stably integrated into host cell DNA and passed on to daughter nuclei after mitosis
- transgenic plants can only be regenerated from a stably transformed cell
