micropropagation studies in dracaena and cordyline - ETD ...

3.2.2 Quality of Chemicals
All chemicals used in the experiments were of analytical grade.
3.3 Preparation of culture media
The stock solutions were mixed in the required proportion along with growth
regulators and sucrose (3%). Then the volume was adjusted by adding double distilled water.
The pH of the medium was adjusted 5.6 to 5.8 using digital pH meter by adding either 0.1N
HCl or NaOH drop wise while stirring with the help magnetic stirrer. Volume was finally made
up to required level and agar (0.7%) was added to the medium. Agar in the medium was
made to melt by gentle heating and the medium was poured into the baby jar bottles having
300 ml capacity.
3.4 Sterilization procedures
3.4.1 Media
Baby jars or culture tubes containing the media were autoclaved at a temperature of
121ºC at 15 lb per square inch pressure for 20 minutes.
3.4.2 Instruments
All the instruments used for sterile handling and transfer of cultures were sterilized by
autoclaving at a temperature of 121ºC at 15 lb per square inch pressure for 20 minutes.
3.4.3 Sterile technique in laminar air flow cabinets
All the sterile transfer work was carried out in a laminar air flow cabinet. Before
starting any sterile operation, the inner surface of the cabinet was swabbed with 70% ethyl
alcohol. The UV lamp provided within the cabinet was then switched on for 15 to 20 minutes
before use, followed by the airflow. During the course of transfer, between each transfer of
explants to culture bottles or tubes, the instruments were dipped in alcohol and flame
sterilized.
3.5 Culture establishments
3.5.1 Inoculation
Before inoculation of explants, the ultra violet light of laminar air flow was switched on
for 20 minutes for sterilization of the area. Inoculation was carried out under aseptic condition
under laminar air flow.
3.5.2 Culture condition
The culture tube or jams bottles were incubated in culture room having control over
temperature and light. The temperature of culture room was maintained at 25 ± 2ºC with light
intensity of 16 hours light and 8 hours dark.
3.5.3 Subculture
Microshoots formed in the bottles were taken out and were separated by dissecting
them in the sterile environment of laminar airflow cabinet with sterile dissecting needle/scalpel
and forceps. Then they were placed back in the test tubes/ bottles containing the fresh media.