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melkasewwandi

The document outlines materials and methods for media preparation and plant tissue culture of Anthurium and orchids. Key steps include: 1) Autoclaving bottles and preparing 1/2 MS media for Anthurium and Knudson:C media for orchids. Media components and preparation are detailed. 2) Sterilizing and cutting Anthurium baby leaves into small pieces which are placed on solidified agar medium in bottles. 3) Incubating cultured bottles in culture room and transferring grown plantlets to new media bottles for further growth. The transferring is done on a laminar flow hood.

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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
2,4D 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
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.
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.

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Method

  • 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.