SAPS - Student Sheet 12 - Fast tissue culture This method of tissue culture uses seedlings as the explants. A newer method of demonstrating tissue culture in the classroom is with cauliflowers, which can be seen here.
Plant tissue culture refers to the growth of individual cells, tissues or, as in this case, organs on an artificial medium. The aseptic techniques which are usually associated with tissue culture experiments are not necessary here. This is because the explants will continue to grow on agar alone. Serious bacterial or fungal contamination is unlikely because the medium contains no sugar and the time scale is so short.
Seedlings of Sinapis alba and rapid-cycling Brassica rapa grow very quickly. Under ideal conditions (20 - 26oC under a light bank), the seedlings sown in step 1 could be ready for use in step 2 within 2 - 3 days. New leaves should appear on explants within 2 - 3 days and even new roots or flower buds within 7 - 10 days.
Notes on the questions
- The agar provides water and support for the seedling explants.
- It would be difficult to 'plant' the explants in the agar at the bottom of longer tubes.
- Maintain humidity/prevent moisture loss; prevent contamination; allow light in, etc.
- Safety: Despite the low risk of contamination, tubes should remain closed throughout in case any potentially dangerous micro-organisms start growing on the agar. All tubes should be disposed of safely at the end of the experiment, preferably by placing in an autoclave at 120°C and 15 p.s.i. for 15 minutes.
- The time taken for each explant to start growing a new leaf, new roots, or develop flower buds, can be recorded. A new leaf/root can be defined by size/length (e.g. record a new leaf only when it reaches 3 mm long). The percentage of explants showing a response (there is usually a high degree of variability) can be recorded. Large sample sizes should enable students to show statistically significant differences between various treatments.
- The cotyledons are photosynthetic organs and contain their own stores of nutrients. Only one cotyledon attached to the shoot apex is sufficient to ensure continued growth without adding nutrients to the medium.
- Perhaps some indication of the extent to which development of the explants is dependent on the presence of apex, hypocotyl or cotyledons.
Ideas for investigation: e.g. for Sc1 investigations at GCSE, or A-level.
Vary the growth conditions e.g. light/dark, different wavelengths of light, different temperatures. Compare material isolated from different species.
Remove one or both cotyledons and compare growth with that of complete explants. Try growing isolated cotyledons only.
Explant response is enhanced by the addition of nutrients to the agar. Ready-prepared mixes, e.g. Murashige and Skoog, are available from Philip Harris, Sigma etc. Ask for 'MS salts - without added sugar'. The plasticity of explants grown on a range of media throws light on plant development and plant repair mechanisms.
Acknowledgements:
Mark Hanley-Browne, Charterhouse School, Dr Mick Fuller, University of Plymouth and Fran Fuller, South Devon College. The work on the protocol described here was carried out with the help of Dr David Hanke of the Dept. of Plant Sciences, University of Cambridge.