Protocol for Organogenesis in Tobacco Callus
This is an experiment in which mature tobacco stem is initiated to give rise to callus tissue. Under appropriate hormonal condition callus is induced to form either root or shoot primordia. The protocol is given below:
1. The upper part of the stem of 3-4 ft tall tobacco plants are harvested and cut into 2 cm long internodes segment.
2. Surface sterilization of tissue is done by immersing the stem pieces in 70% v/v ethanol for 30 seconds, followed by 15 minutes incubation in sodium hypochlorite. Then tissue is washed in several changes of sterile distilled water.
3. The stem explants are taken in a sterilized petri dish and cut longitudinally in two equal pieces and inoculated onto Murashige and Skoog’s solid medium supplemented with 2 mg indole acetic acid (IAA) and 0.2 mg kinetin. The cultures are then incubated at 25 0C with an illumination of about 2000 lux.
4. Organogenesis in callus culture can be stimulated by transferring tobacco callus onto MS medium with different auxin or cytokinins rations. Shoot primordia develop within three weeks of transfer of callus to MS medium with IAA at 0.02 mg and kinetin at 1 mg/ lambada. Root formation occurs within 2-3 weeks of transfer of callus to MS medium supplemented with 0.2 mg / lambada.
5. Callus tissue which is white or yellow in colour, begins to form in two weeks and after six weeks it should be sub cultured to fresh medium.
6. After 6 weeks, rootless shoots can be excised and placed onto the root inducing medium with 0.2 mg/lambada, IAA and 0.02 mg/ lambada kinetin.
7. It is possible to transplant tobacco plantlet to soil. It should be noted that aseptic procedure is not required for the transplantation of plantlets. The plantlets are removed from the culture vessels and are care should be taken not to damage root or shoot system. The plantlets are carefully washed with tap water to remove the residual agar medium. Individual plantlets are separated out and transplanted into pots containing seedling compost. The soil is watered. The pot is covered with small inverted polythene bag. This will reduce the amount of water lost by plantlets due to transpiration. After 7 days several small holes are made in polythene bags and gradually enlarged during next 2-3 weeks. At this stage, tobacco plantlets should be sufficiently “hardened off” to allow the complete removal of plastic bag. They can be grown to maturity in green house.