Protocol of Callus Culture
Callus tissue can be induced from different plant parts of may plant species, however, carrot is a highly standarize material. The callus culture from exercise tap root of carrot is described below:
1. A fresh tap root of carrot is taken and washed thoroughly under running tap water to remove all surface dirts.
2. The tap root is then dipped into 5% “Teepol” for 10 minutes and then the root is washed. The carrot root, sterilized forceps, scalpels, other instruments, autoclaved nutrient medium Petri dishes are then transferred to laminar air flow or inoculation chamber. Throughout the manipulation sequences forceps, scalpels must be kept in 95% ethanol and flamed thoroughly before use.
3. The tap root surface sterilized by immersing in 70% v/v ethanol for 60 seconds, followed by 20-25 minutes in sodium hypochlorite ( 0.8% available chlorine).
4. The root is washed three times with sterilized distilled water to remove completely hypochlorite.
5. The carrot is then transferred to a sterilized petri dish containing a filter paper. A series of transverse slice 1mm in thickness is cut from the tap root using a sharp scalpel.
6. Each piece is transfer to another sterile petri dish. Each piece contains a whitish circular ring of cambium around the pith. An area of 4mm2 across the cambium is cut from each piece so that each piece contains part of phloem. Cambium and xylem size and thickness of explant should be uniform.
7. Always the lid of petri dish is replaced after each manipulation.
8. The closure from a culture tube is removed and flamed the uppermost 20mm of the open end. While holding the tube at an angle of 45 0, an explant is transferred using forceps onto surface of the surface of the agarified nutrient medium. Nutrient medium is Gamborg’s B5 or Ms medium supplemented with 0.5 mg/, 2,4-D.
9. The closure is immediately placed on the open mouth of each tube, Date, medium and name of the plant are written on the culture tube by a glass marking pen or pencil.
10. Culture tubes after inoculation are taken to the culture room where they are placed in the racks. Cultures are incubated in dark at 25 0C.
11. Usually, after 4 weeks in culture the explants incubated on medium with 2,4-D will form a substantial callus. The whole callus mass is taken out aseptically on a sterile petri dish and should be divided into two or three pieces.
12. Each piece of callus tissue is transferred to a tube containing fresh same medium.
13. Prolonged culture of carrot tissue products large calluses.