lecture 12

Question 1

What determines whether a gall is rooty or shooty or elsewise?

Answer 1

• 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

Question 2

What other plant pathogens cause galls by secreting plant hormones?

Answer 2

• witches broom on birch (cytokinin secretion)
• olive branch with a gall caused by Pseudomonas savastanoi (auxin secretion)

Question 3

What are the good features of Agrobacterium?

Answer 3

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

Question 4

What are the bad features of Agrobacterium?

Answer 4

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

Question 5

What is the binary vector system?

Answer 5

• 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)

Question 6

What is the basic protocol for Agrobacterium transformation?

Answer 6

• 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

Question 7

Why bother with other methods?

Answer 7

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

Question 8

What did John Sanford do?

Answer 8

• 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

Question 9

What is the difference between transient and stable transformation?

Answer 9

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