PRODUCTION OF TRUE TO TYPE PLANTLETS
RAPID MULTIPLICATION INDEPENDENT OF SEASONAL CONSTRAINTS
MAINTAINING AND IMPROVING THE PRODUCTIVITY OF OUTSTANDING VARIETIES IN THE FIELD
PRODUCTION OF DISEASE FREE PLANTING MATERIAL FROM APICAL MERISTEM
TISSUE CULTURE TECHNIQUE
The tissue culture technique has the following important components:
1) Taking of spindle explants/Apical meristem from moist hot air treated seed
cane crop of six to eight months age.
2) Media preparation
3) Inoculation
4) Multiplication of cultures
5) In vitro rooting
6) Pre hardening
7) Transfer of plantlet from laboratory to Polybags
8) Hardening
9) Transplanting in the field
TAKING SPINDLE EXPLANTS
The spindle explants are excised from cane tops of field grown moist hot air treated seed crop of 6-8 months age, after removing outer leaf sheaths which are washed with Tepol/Savlon to remove dust particles and contaminants, if any. Subsequent operations are done in the inoculation chamber of the laboratory. Two types of media are used, solid and liquid. The explants are established on solid medium and the established cultures are multiplied on shoot proliferation liquid media with shoot growth promoting hormones.
MEDIA PREPARATION
There are a number of media formulations for culturing of plants, but most widely used media for tissue culture is MS (1962) Medium, named after the scientists Murashige and Skoog (1962). There are 5 stocks named MS I,II,III,IV and V having major salts, minor salts, vitamins and amino acids. Agar, Sucrose and growth hormones are added, later on, to the media. Stocks are stored in the refrigerator after use to avoid precipitation and contamination. After preparing the media by adding different stocks, sucrose and growth regulators, final volume is made by adding distilled purified water. Tap water is not used because it may result in precipitation of salts. Media pH is adjusted between 5.5 to 5.8, by pH meter. Media is poured in glass bottles. After tightening the lids, media is transferred into Autoclave by loading on carriage system for sterilization from any contamination. It is autoclaved at 121 degrees Centigrade, 15 lb. Pressure for 15-20 minutes. Glass vessels, test tubes, petri plates are also autoclaved/sterilized. Autoclaved media is then brought out and stored for 24 hours so that if any contamination is still there, it could be detected. This medium is then used for inoculation.