Biology Reference
In-Depth Information
4. Rotary shaker.
5. Sterile scalpels, forceps, and Petri dishes.
6. Filtration equipment (Buchner funnel and fl ask for fi ltration
under reduced pressure).
1. Ethanol solution: 70 % (v/v) in water.
2. Calcium hypochlorite solution: 7 % (w/v) in water.
3. Tween 20.
4. Sterile distilled water.
2.3 Sterilizing
Solutions
1. Murashige and Skoog (MS) basal salt mixture (Duchefa or
Sigma) [
29
].
2. Gamborg B
5
basal salt mixture (Duchefa or Sigma) [
30
].
3. Kinetin (Sigma) 200 mg/L. Dissolve 0.02 g kinetin in 90 mL
water. Add, while stirring, drop by drop, 1 M NaOH. Add
water to a volume of 100 mL. Mix and store at 4 °C.
4. 1-Naphthaleneacetic acid (NAA) (Sigma) 200 mg/L. Dissolve
0.02 g NAA in 90 mL water. Add, while stirring, drop by drop,
1 M NaOH. Add water to a volume of 100 mL. Mix and store
at 4 °C.
5. Morel vitamins. Prepare 100 mL of a 1,000× stock of Morel
vitamins [
31
] in water. While stirring, add 10 mg calcium
pantothenate, 1 mg of biotin, 10 mg of nicotinic acid, 10 mg
of thiamine, 10 mg of pyridoxine, and 10 g of myoinositol.
Store at 4 °C.
6. Acid (1 N HCl) and alkali (1 M NaOH or KOH) solutions to
adjust the pH of the media.
7. Plant Agar
®
(Duchefa).
8. Induction medium. Add 4.3 g MS basal salt mixture, 30 g
sucrose, and 0.25 g casein hydrolysate to 1 L distillated water
(
see
Note 1
). Stir and then add 1 mL 200 mg/L kinetin, 0.5 mL
200 mg/L NAA, and 1 mL of 1,000× Morel vitamins. While
stirring, adjust the medium to pH 6.0 using HCl, NaOH, or
KOH. For solid media, add 7.0 g of agar per liter of media and
autoclave for 20 min at 121 °C (
see
Note 2
). After that, distribute
the medium in sterilized Petri dish (
see
Note 3
).
2.4 Plant
Culture Medium
9. Growth medium. Add 3.05 g Gamborg B
5
basal salt mixture,
20 g sucrose, and 0.25 g casein hydrolysate to 1 L distillated
water (
see
Note 1
). Stir and then add 1 mL 200 mg/L kinetin,
0.5 mL 100 mg/L NAA, and 1 mL of Morel vitamins [
31
].
While stirring, adjust the medium to pH 6.0 using HCl,
NaOH, or KOH and then distribute the medium in 250 mL
Erlenmeyer fl asks which contains 100 mL of Gamborg B
5
. For
solid media, add 0.7 g of agar in each, and autoclave them for
20 min (
see
Note 2
) at 121 °C.
