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Flashcards in lecture 12 Deck (9)
1

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

2

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)

3

What are the good features of Agrobacterium?

- transfers DNA to plant cells
- selection system for transformed cells
- organised structures (leaves, roots) can form

4

What are the bad features of Agrobacterium?

- opines
- gall formation
- Ti plasmid is too big for recombinant DNA work

5

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)

6

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

7

Why bother with other methods?

- some plant species are highly resistant to Agrobacterium infection or cannot be regenerated from susceptible tissues

8

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

9

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