SAPS - Student Sheet 13 - Fast tissue culture part2 This is more demanding than the protocol outlined in Student Sheet 12 and is more suitable for year 12-13 students. Some previous knowledge of aseptic technique would be an advantage.
The aim is to examine the growth of root explants over a 7 day period. The addition of sugar to the medium increases the risk of fungal and bacterial contamination unless you take precautions to keep the explants and apparatus sterile. The methods outlined will give adequate sterility if the instructions are followed carefully.
Practical points for setting up
1. All media and containers must be sterile. For sterilisation of media, water and glassware, we recommend autoclaving for 15 mins at 121°C and 15psi. However sufficiently sterile water can be obtained by boiling distilled water in a beaker covered with foil for 15 min and leaving it to cool.
2. To prepare the germination pots, add appropriate amounts of 1% water agar and then autoclave. Baby food jars are excellent for this. Reusable transparent caps or closures for them can be bought from Sigma Chemicals. Alternatively, sterile clear pots can be bought, and sterile agar can be poured into them.
3. Use four sterile media which contain 0%, 0.5%, 1.0% and 3% sucrose to investigate the effects of sucrose on root growth. Sterile Petri dishes may be bought from the manufacturers. NB 1% sucrose agar can be made by dissolving 1g sucrose in 100 ml of water, adding 1g of agar and autoclaving as above.
Potentially this protocol will generate a great deal of quantative information for students to handle, and could form the basis of highly original projects. For example, students could measure the increase in root length each day to see if the increase is linear or exponential. They could count the number of lateral roots produced by mid root explants over time, correlating this to the length of the explant.
To simplify this protocol, each student could use and collect data from a single explant type on different media, or look at the two explants on a single medium. Combining the results would give a comprehensive set of class data.
Safety
All plates must be sealed after Step 5 and not re-opened. Dispose of all plates by autoclaving as above.
Make sure that the ethanol container is kept away from naked flames, and have a glass lid readily accessible to cover the container in case of accidental fires.
Acknowledgements to:
Mark Hanley-Browne (Charterhouse School), Mick Fuller (University of Plymouth, and Fran Fuller (South Devon College). Artwork by Linda Gray.
All plates must be sealed after Step 5 and not re-opened. Dispose of all plates by autoclaving as above.