Tissue culture is applied in plant research for such purposes as the growing of new plants, which in some cases undergo genetic alterations. The plant of interest is taken through the tissue culture process and grown in a controlled environment. This process involves the use of small pieces of a given plant tissue (plant of interest). Once the tissue is obtained, it is then cultured in the appropriate medium under sterile conditions so as to prevent various types of microorganisms from affecting the process.
• Healthy and disease-free explants (Sprouts) of potato (Solanum tuberosum L.) are collected from tubers.
• One-week old healthy sprouts are taken.
• Then wash in running tap water for at least 40-50 minutes.
• Then the sprouts are taken to the laminar airflow for further sterilization to avoid any type of pathogenic contamination.
• At first the sprouts are washed with 70% ethanol for only 40-50 seconds. [Use autoclaved glass vessels]
• Then rinse with autoclaved distilled water for one time for 3 minutes.
• After washing sprouts are treated with 0.1% HgCl2 for 7 minutes and 2-3 drops of Tween-20 for 7 minutes.
• Then again sprouts are washed with autoclaved distill water for 3 times (each time for 5 minutes).
• Now sprouts are ready to transfer into medium.
Three to four days old MS media, is used for new sprout inoculation. Now sterilize sprouts are transferred into media containing test tube/ glass vessels (one sprout per test tube/glass vessel) by using autoclaved scalpel and forceps.
After 20-21 days of inoculation potato shots are selected for subculture. In this case under laminar air flow, nodal segment of shoot is cut, transferred into the MS media, and kept it in the growth room.
The plantlets produced in this manner can be effected with virus. Only meristem culture can ensure virus-free potato plantlet. So, to ensure whether the plantlets are virus free or not, ELISA Test is conducted for individual lots. In this way if a lot is virus positive then the whole lot is to be immediately discarded.
Research Assistant, ASRBC