Regeneration Prac

Question 1

regeneration

Answer 1

• remaking of an entire organ(ism) from a single/small group of cells
• differentiated -> de-differentiated -> differentiated reprogramming
• e.g. cutting
• needs totipotency and certain hormonal conditions

Question 2

totipotency

Answer 2

demonstrated in carrots in the ’50s

Question 3

regeneration expts:

Answer 3

• tissue culture experiments w explants in agar

Question 4

when is regeneration used?

Answer 4

1) asexual reproduction
2) herbivory recovery
3) clonal propagation in biotechnology

Question 5

Questions:

Answer 5

1) How do plant hormones affect regeneration in tobacco?
2) How does Agrobacterium infection affect regeneration in carrots

Question 6

Treatments for expt1

Answer 6

different auxin:cytokinin ratios

Question 7

Treatments for expt2

Answer 7

different strains: tumefaciens, rhizogenes (hairy roots)

Question 8

Agrobacterium

Answer 8

• tumourigenic
• modifies growth and metabolism
• GE bacteria (hijacks developmental programming)
• Ti plasmids (excision, transfer and nuclear integration for expression)

Question 9

callus

Answer 9

undifferentiated wound tissue that precedes organogenesis

Question 10

aseptic techniques

Answer 10

• during tissue manipulation to avoid microbial contamination
• sterile airflow cabinets (far inside) and sterile surfaces such as Petri dishes
• do not lean on/touch/breathe over

Question 11

Phytohormone equipment

Answer 11

• sterile leaf material from 6-8 week old tobacco seedlings
• 3x tissue culture media of different phytohormone ratios (1 minimal culture medium, no hormone; 1 10x cytokinin, 1 4x auxin)

Question 12

Phytohormone protocol

Answer 12

1) using sterile forceps and scalpel, cut 1cm^2 explants from leaf tissue (avoid major veins and damage)
2) plate 4-5 evenly distributed explants and firmly press for good contact
3) seal to prevent drying
4) culture for 7 weeks at 22degrees in sunlight

Question 13

tumours

Answer 13

row on minimum culture media because Ti can synthesise cytokinin and auxin; transforming effect

Question 14

Agrobacterium protocol

Answer 14

1) cut 6-10cm carrot pieces and note root and shoot poles
2) remove epidermis and blemishes w sterile scalpel
3) shake in 20% bleach for 20 minutes at intervals the discard
4) shake in sterile water x3
5) transfer to sterile tile in airflow cabinet
6) top and tail using sterile scalpel
7) cut 5-8mm transverse sections using forceps and scalpel
8) put 3-5 pieces on MSO agar
9) inoculate, leave control
10) seal w parafilm

Question 14

calli

Answer 14

• oxidation of tissue
• brown/grey/black
• form at the cutting borders on the meristem cells that release the hormone

Question 15

what happens to the explants?q

Answer 15

• swelling and discolouration
• the parts that have lifted are lighter (less nutrients, less calli)

Question 16

exogenous auxin and cytokinin are necessary for

Answer 16

regeneration from tobacco leaf explants

Question 17

relative concentrations of auxin and cytokinin determine

Answer 17

developmental identity (shoot versus root)

Question 18

infection with different Agrobacterium species differentially affects

Answer 18

the identity of resultant regenerate organs

Question 19

different Agrobacterium species may determine different regenerant organ identities by

Answer 19

influencing the relative concentration of auxin and cytokinin

Question 20

Minimal culture medium result for tobacco

Answer 20

no regeneration

Question 21

10x cytokinin result

Answer 21

low auxin/cytokinin ratio promotes shoot regeneration

Question 22

4x auxin result

Answer 22

high auxin/cytokinin ratio promotes root regeneration

Question 23

Agrobacterium control

Answer 23

no change

Question 24

Agrobacterium tumefaciens

Answer 24

tumour formation

Question 25

Agrobacterium rhizogenes

Answer 25

root regeneration

Question 26

different Agrobacterium species promote different

Answer 26

developmental fates.

Question 27

Compare the experiments

Answer 27

A. rhizogenes looks like high auxin/cytokinin

Question 28

Why is important wrt crown gall disease?

Answer 28

tumefaciens causes crown gall because of its tumourigenesis. Find a way to modulate its cytokinin/auxin ratio, and maybe the result would be different?

Question 29

What would happen if you had insensitivity?

Answer 29

you would need a higher/lower ratio

Question 30

What does auxin perception do?

Answer 30

regulates auxin dose, therefore the ratio received and the regeneration response