1. Materials required
For media preparation An electronic balance
 pH meter
 Refrigerator
 Water demineralizer
 An Autoclave
 Shaker , gravity with platform for suspension culture.
 Glass bottles or containers
 Beakers
For transferring & culturing Laminar air flow
 Petri dishes
 Scalpel
 Forceps
Method
Media preparation
First the bottles were well washed and cleaned with detergents (T-pol). They were
autoclaved at 121 ˚C for 105 kPa for 20 minutes. Meanwhile the media preparation was
done.
 For Anthurium- Anthurium establishment media called ½ Ms media was
prepared.
 For Orchids- Kundson:C media was prepared for Orchid seed plantation.
( Vacin and Went media for meristem plantation.)
½ Ms media- (for 4mL)
reagent
Amount
Stock A
Stock B
Stock C
Glycine
Pyridonine
Nicotinic acid
Thiamin
Coco. water
BAP
200 ml
10 ml
20 ml
10 ml
10 ml
10 ml
10 ml
600 ml
40 ml

2. 2,4D
Distilled water
Myo inositol
Sugar
Agar
pH = 5.8-5.6
4 ml
3086.0 ml
0.4 g
120 g
32 g
Stock A- (500ml)
Compound
Concentration /(mg/l)
NH4NO3
1650
Need amount to prepare
500ml (g)
8.24g
KNO3
1900
9.50
MgSO4.7H2O
370
1.85
KH2PO4
170
0.83
CaCl2.2H2O
440
2.2
Compound
Concentration (mg/l)
Need amount to prepare
250ml (g)
H3BO3
MnSO4.7H2O
ZnSO4.7H2O
KI
Na2MoO4.2H2O
CuSO4.5H2O
CoCl2.6H2O
6.3
22.3
8.6
0.83
0.25
0.025
0.025
0.15
0.55
0.125
0.02
0.005
0.006
0.006
Stock B- (250ml)
Stock C- (250ml)
Compound
Concentration (mg/l)
Na-EDTA
FeSO4.5H2O
37.34
27.8
Need amount to prepare
250ml (g)
0.09
0.07

3. Knudson:C media
Reagent
Amount
Stock A
Stock B
10 ml
10 ml
StockC
Fe Stock
Coconut water
Distilled water
Banana
Sugar
Agar
10 ml
10 ml
250 ml
210 ml
500 ml
20 g
8g
pH = 4.8-5.2
Stock preparation –
Stock
Compound
Need amount to prepare
250ml (g)
Stock A
Ca(NO3)2
25.0
Stock B
(NH4)2SO4
MgSO4.7H2O
MnSO4.4 H2O
KH2PO4
12.5
6.25
0.00018
6.25
FeSO4.7 H2O
Na-EDTA
0.695
0.932
Stock C
Fe Stock
The prepared media was poured into the bottles autoclaved. Bottles containing
media solution were sealed with foil papers, polythene and rubber bands. They were
again autoclaved at 121˚C, 105kPa, for 20 minutes.

4. Preparation of tissues for culturing- (Anthurium baby leaves)
1. Strerilization
The whole leaf was washed consecutively with Lysol, Tpol, and distilled water.
Then it was dipped in a fungicide Kaptan for 20 minutes. It was removed from the
fungicide and cleaned with distilled water to wash off the fungicide completely.
Then the leaf was cut into 3 or 4 pieces and the pieces were dipped in a distilled
water beaker. This whole procedure was done in the preparation room.
The beaker was carried onto the laminar flow hood in transferring room. Under
the pure air flow first the leaf pieces were wash with 70% ethanol dipping then in a 70%
ethanol beaker. They were cleaned with distilled water twice.
Then they were dipped in a 5% Chlorox + T20 solution. It should be shaken for 5
minutes and then the pieces were cleaned with distilled water twice.
Finally they were washed by 5% Chlorox solution and washed off by distilled
water. They were again dipped in a distilled water beaker.
Culturing
All the pieces in distilled water beaker were again cut into 2cm × 2cm small
pieces. Those small pieces were lay up on the solidified agar medium (prepared and
autoclaved) as the lower side down to touch the medium. 1 or 2 pieces were placed on
one bottle.
Then the cultured bottles were carried to in to the culture room to incubate them
under room temperature supplying other suitable conditions.
Transferring
The grown plantlets (here Anthurium and Orchid) in cultured media bottles were
transferred into another media bottles for further growing.
The plantlets were uprooted from medium and they were grouped according to the
size and growth rate. So the plants in same size and age were again implanted in new
medium.
Then the bottles were sealed with foil papers, polythene, and rubber bands. And
then the bottles were again transfer to the culture room for futher growing.
This whole transferring procedure was done on the laminar flow hood in transfer
room.