Introduction
The
callus culture is one of the ways to grow a tissue culture. The culture is grown on a solid gel medium, which has had a number of ingredients added to it to aid growth. These include macro and micro nutrients, nitrogen, potassium, phosphorus and growth regulators. The callus can also be made to differentiate into certain tissues that would be found on a new plant by addition of hormones or enzymes. This is known as
totipotency.
In relation callus cultures made from root explants (which is what i'll be doing), the culture grown is an undifferentiated callus. Eventually embryoids will appear and these can be transferred to a gel medium that will sustain them.
The experiment was carried out over 4 weeks.Week 1 was to set up and the rest were for observation of the cultures.
The experiment.
Materials/equipment - Tap root of a carrot (Daucus carota)
- Scapel
- Sterile 50ml screw-capped tube
- 10% v/v sodium hypoclorite solution
- Waste beaker
- Petri dish
- Agar medium containing 2,4-D
Procedure - Cut a piece of tap root 5cm long (discard both ends) and remove epidermis. Keep track of which end was the root pole. Then sterilise.
- Put the tap root in the screw-capped tube and cover it with the 10% sodium hypoclorite solution. Shake for 5sec every 5min for 20min.
- Pour out the solution into a waste beaker
- Wash the root 4 times by putting sterile water in the tube and shaking
- Put root on sterile petri dish and cut 1cm from each end. Discard this.
- Cut 3-5 transverse sections, 1-3mm thick, across the tap root. Transfer these (shoot-pole uppermost) to a fresh sterile petri dish. All this should be done in a laminar flow cabinet.
- Cut even smaller sections from those above. They should be approx a 5mm square.
- Put these into petri dishes containing agar medium and the hormone 2,4-D.
- Seal and label them, and incubate them in dark at 25 degrees Celsius.
- Observe over a 4 week period and record any change.
- After 4 weeks the callus should be getting developed enough so that they can be sub-cultured into different petri-dishes. Any unhealthy tissue should be removed.
Results Carrot callus cells appear as whitish areas on the surface of the carrot disc. Within 5 weeks the culture can be up to 5 times the size of the original carrot disc. At this point it is time to move the callus onto a more suitable medium to maintain its growth.
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