Methods continued...
Preparing the agar:
The agar was prepared using about 2.5 g of gelrite, 2.2g of MS media and 10g
of sucrose for every one liter of agar prepared. The agar also contained the
differing molar concentrations of salt (25mmol/L, 50mmol/L, 75 mmol/L) that
were added to the agar; unless it was one of the controls, in which case no
salt was added. After measuring out each of the substances and adding about
1000 ml of water, the solutions were mixed using a magnetic stirrer. The pH
of the solution was also measured. The ideal pH for all the agars is a pH
around 5.8. If the solution was not at its optimum pH then a base (usually
potassium hydroxide) or an acid (hydrochloric acid) was added to bring the
pH close to 5.8. These solutions were then autoclaved for an hour in order
to kill all the bacteria in the agar. Then, the hot liquid was quickly poured
into Petri dishes because the agar usually solidifies really fast if its not
poured immediately.
Cleaning the seeds:
First, the seeds were counted out and the number was determined based on the
number of seeds needed for planting. The A. thaliana seeds were first rinsed
using 70% ethanol and then followed by 30% bleach to remove the ethanol. This
was then cleaned three times with double distilled water. In each of the solutions,
the seeds were spun around in a centrifuge for about a minute. The seeds were
then soaked in sterile water for two days at 0ˇăC. The seeds of each of the
plant mutants were cleaned this way. The plant hormone mutants that were utilized
in this experiment were Wild type - Columbia (Col) (3), Ethylene insensitive
- ein 2-1 (28), Ethylene overproducer - eto-2 (4), ABA deficient
- aba 1-5 (15), Auxin resistant - axr 2-1 (10), BR insensitive
- bri 1-201(1) and BR overproducer - BRI - GF(9).
Planting the seeds:
Using tongs, the seeds were sown in rows in the agar, with about ten seeds
per plate. This was performed in a sterile environment. These were wrapped
and placed in growth chambers, where the plants were allowed to grow for two
weeks.