Lab1

EstablishmentandMaintenanceofCarrotCallus

Objective:Studentswillsterilizecarrottissueandestablishitintissueculture

Protocol:

1. In the lab, rinse a carrot root under running tap water to remove any surface soil.
Make sure the root is undamaged; particularly that it doesnt have any large
cracks. Cut off the top and bottom on the carrot and peel off the epidermis with a
peeler. Trim the carrot into 6 - 10mm sections and place them into a beaker in the
flow hood. The carrot has already be sliced up for you.
2. You will be given carrot root slices that have been soaked in 2.5mg/L 2,4-D for 24
hr. Treat them as follows, the same as the untreated slices, but keep them
separate.
3. Cover the carrot sections with about 150ml 70% EtOH and sitr for 5 minutes.
Pour off the EtOH into a waste beaker.
4. Immediately cover the carrot sections with about 150ml of a 20% bleach solution
+ a drop of Tween 20 (a surfactant) for approximately 20 min, then decant off the
bleach solution.
5. Rinse the carrots three times with sterile water (100 150ml), covering the
sections and stirring for 1-2 min each time.
6. Transfer a section into a sterile dish. Using scalpel and forceps, cut small (5mm
square) pieces of root, making sure to include the cambium (the innermost ring
of the root). Sterilize Instruments often.
7. Place 4 explants in each of the provided Petri plates. You will be given two types
of plates - containing MS medium and MS medium + 2,4-D (prepared on 1/5).
Prepare one of each media type for each root treatment.
8. Place 4 of the slices into a Petri plate with sterile filter paper which has been
moistened with sterile distilled water.
9. Seal each dish with parafilm, label and incubate in the dark.
10. After 4 weeks, subculture the callus onto the same medium, discarding necrotic
tissue.
11. After another 4 weeks, transfer all tissue to hormone-free medium (MS media
without 2,4-D).