This is a blog for an experiment i carried out for my Botany course. The experiment was to prepare Sinapis alba (white mustard) organ cultures:
Plant tissue culture is the the growth of individual cells, tissues or, as in this case, organs on an artificial medium .
The extreme aseptic techniques that are carried out with tissue culture experiments are not needed here. This is because the explants will continue to grow on agar alone. Serious bacterial/fungal contamination will probably not arise because the medium contains no sugar .The agar only provides water and support for the seedling explants as the cotyledons contain stored carbohydrates, proteins and lipids. They are photosynthetic once they are above the ground to produce their own food.
What was used in this experiment:
- Parafilm
- Sinapis alba (White mustard) seedlings
( The seeds were sown on damp tissue 3-4 days before the experiment and incubated at 20°c in a light place).
- McCartney bottles containing 1% w/v agar
The McCartney bottles were sterilized and the agar was made up (1g per 100ml of distilled water) and then it was autoclaved. After the agar had cooled slightly, using sterile techniques, the agar medium was poured into the McCartney bottles until they were just slight less than half filled. They were then left for the agar to solidify and were then sealed with parafilm.
- Scissors
How it was done:
- The S alba seeds were germinated in a warm light place for 2-3 days until the cotyledons had just started to unfold.
- The tops of the seedlings were cut just below the shoot apex. These are the explants.
- One explants was put into each bottle of containing McCartney agar. The cut end of the shoot was pushed gently into the agar, making sure that the cotyledons didn't touch the surface of the agar.
- The bottles were covered with parafilm and incubated at 18-20°c.
- The bottles were not opened for 4 weeks to ensure that the humidity was maintained and to reduce the risk of contamination by microbes.
- After the 4 weeks the cultures were opened and observed for any changes and any signs of root initiation.
One of my classmates has good pictures of her seedling explants in agar.
http://botanisegalway.blogspot.com/
what happened after five weeks?
Well not much happened. None of the seedling explants grew any roots. They did all grow a little in size and one had a turned a rather sad pale yellow colour.
Why did this happened:
I believe there was a couple of reasons behind my seedling explants not growing roots:
- My inexperience. I think i need to build up my technique and experience as i was not as careful as i had hoped to be. When pushing the shoot into the agar i don't think i pushed them in properly as some of them fell over .
- I think that the seedlings may have been a little to young and that they should have been a little older so that they were stronger and easier to put into the agar.
