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SEED TECHNOLOGY TISSUE CULTURE The tissue culture technique in sugarcane can be used for rapid multiplication of newly developed high yielding, high sugar, disease resistant varieties and rejuvenation of outstanding varieties under cultivation. The vegetative propagation of sugarcane through seed cane cuttings is cumbersome requiring larger quantities of vegetative seed material and seed multiplication rate is too low, needing about ten years for a new variety to be released for cultivation and cover larger areas in the field subsequently. With tissue culture technique, it is possible to release a variety within five years and propagate it quickly in the field. The productivity of the important varieties can also be maintained and improved for their longer life span in the field. The micro propagation technique used in this technology has the following advantages: 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: Taking of spindle explants/Apical meristem from moist hot air treated seed cane crop of six to eight months age. Media preparation Inoculation Multiplication of cultures In vitro rooting Pre hardening Transfer of plantlet from laboratory to Polybags Hardening 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. INOCULATION It has the laminar Air Flow cabinet, which generates clean air, with HEPA filter of pore size 0.2 mm. Bacteria and fungal spores, higher than 0.2 mm gets killed and cannot pass through these filters. Excised explants are surface sterilized with 0.1% Mercuric chloride solution for 10 minutes, after three washings with autoclaved distilled water. The outer leaves are aseptically removed to obtain suitable spindle explants (0.5 – 1.0 cm). Individual explants are inoculated/cultured on MS (1962) establishment media. MULTIPLICATION The established cultures are then multiplied on multiplication media by re culturing and sub culturing these cultures after cleaning on fresh liquid media. These are sub cultured by changing the media after every 15 days. INCUBATION After marking the culture bottles for different varieties, cultures are incubated in the incubation room with a specified temperature between 25 ± 2 Degrees Centigrade which is maintained with the help of air conditioners, as at lower temperature, growth slows down and higher temperature (³ 30 Deg. Centigrade) results in the death of the plants. Light is another important factor; cultures are incubated on the culture racks fitted with fluorescent tube light rods to meet requirements of high intensity light (5000 Lux) for the growth of cultures. Duration of light exposure is kept between 16 hours light and 8 hours dark. High humidity (60-70%) is maintained in the growth room to avoid media desiccation. IN VITRO ROOTING The multiplied cultures are rooted in the laboratory on rooting media. Shoot clumps of 10-15 shoots are transferred to liquid media with elevated sucrose concentration and supplemented with root promoting hormones. Fairly good rooting occurs after two weeks of culturing. PRE-HARDENING The well rooted plants are removed from the culture vessels, washed thoroughly with running tap water; placed in a bucket full of water and left for one or two hours. Plantlets are individually separated making them single. These plantlets are again washed and kept in a tray with cotton soaked with water or water filled bottles. These are kept 3 to 4 days in high light and high humidity conditions in growth room. HARDENING After slight root elongation, plantlets are transferred to small soil filled polythene bags in the green house for hardening. Before transferring the plantlets to poly bags, rooted and laboratory pre-hardened plantlets are given fungicidal treatment to avoid fungal growth in poly bags. Watering of the plantlets is done 3 to 4 times a day and nutrient solution added to meet the requirements of major and micronutrients. After one and a half month, the plantlets from green house are shifted to a shade house for further hardening to avoid field mortality.
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