Equipment for In Vitro Asymbiotic Orchid Seed Germination (Flasking)
By Mano Fonooni

All Phases of orchid seed sowing done in absolutely 100% sterile and germ free environment.

To sow orchid seeds a sterile space to work on is necessary.
So you need a Glove box or make a more preferable laminar flow clean air station.
The laminar flow is a lot more expensive, but gives one lots of freedom of movement as well as efficiency.
 


laminar flow clean air station
Glove box

 

 

To grow your  orchid seeds in a sterile condition then you need
wide mouth seed sowing flasks or similar containers.

      

 

In order to provide water and nutrition to your orchid seeds in the flask,
a special blend of nutrition and chemical with specific ph level needed.
You can buy premixed powder or mixed them yourself !

This is a sample of chemical that needed to make 1000 ml. for final volume.

 
K 4003
K 4128
P 0931
P 1056
P 6668
P 6793
M 5524 		 
Knudson C modified orchid medium
Knudson C orchid medium, morel modification
Phytamax orchid maintenance medium without charcoal
Phytamax orchid medium with charcoal and banana powder
Phytamax orchid maintenance medium
Phytamax orchid multiplication medium
Murashige and Skoog basal salt mixture (MS)
  (listed for purposes of comparison only)
 
Component (mg/L) K 4003 K 4128 P 0931 P 1056 P 6668 P 6793 M 5524*
Aluminum chloride • 6H2O              
Ammonium nitrate   500.0 825.0 825.0 825.0 825.0 1650.0
Ammonium phosphate monobasic              
Ammonium sulfate 500.0 500.0          
Boric acid 0.056   3.1 3.1 3.1 3.1 6.2
Calcium chloride anhydrous     166.0 166.0 166.0 166.0 332.2
Calcium nitrate 694.4 347.2          
Calcium phosphate tribasic              
Cobalt chloride • 6H2O     0.0125 0.0125 0.0125 0.0125 0.025
Cupric sulfate • 5H2O 0.0624   0.0125 0.0125 0.0125 0.0125 0.025
Na2-EDTA     37.24 37.24 37.24 37.24 37.26
ZnNa2-EDTA              
Ferric chloride • 6H2O              
Ferric citrate              
Ferric sulfate              
Ferric tartrate • 2H2O              
Ferrous sulfate • 7H2O 25.0 25.0 27.85 27.85 27.85 27.85 27.8
Magnesium sulfate 122.125 122.125 90.35 90.35 90.35 90.35 180.7
Manganese chloride • 4H2O              
Manganese sulfate • H2O 5.682 5.682 8.45 8.45 8.45 8.45 16.9
Molybdenum trioxide 0.016            
Molybdic acid (sodium salt) • 2H2O     0.125 0.125 0.125 0.125 0.25
Nickel chloride • 6H2O              
Nickel sulfate • 6H2O              
Potassium chloride   250.0          
Potassium iodide     0.415 0.415 0.415 0.415 0.83
Potassium nitrate     950.0 950.0 950.0 950.0 1900.0
Potassium phosphate monobasic 250.0 250.0 85.0 85.0 85.0 85.0 170.0
Potassium sulfate              
Sodium nitrate              
Sodium phosphate monobasic              
Sodium sulfate              
Zinc nitrate • 6H2O              
Zinc sulfate • 7H2O 0.331   5.3 5.3 5.3 5.3 8.6
Organics (mg/L) K 4003 K 4128 P 0931 P 1056 P 6668 P 6793 M 5524*
Activated charcoal       2000.0 2000.0    
Adenine hemisulfate              
Agar              
Banana powder (50% malto-dextrin)       30,000      
6-Benzylaminopurine (BA)           2.0  
D-Biotin              
6gamma-gamma-
Dimethylallylaminopurine		              
Glycine (free base)              
Indole-3-acetic acid              
Indole-3-butyric acid              
Kinetin              
MES (free acid)     1000.0 1000.0 1000.0 1000.0  
myo-Inositol     100.0 100.0 100.0 100.0  
alpha-Naphthaleneacetic acid           0.5  
Nictotinic acid (free acid)     1.0 1.0 1.0 0.5  
Peptone     2000.0 2000.0 2000.0 2000.0  
Pyridoxine • HCl     1.0 1.0 1.0 0.5  
Sucrose 20,000 20,000 20,000 20,000 20,000 20,000  
Thiamine • HCl     10.0 10.0 10.0 1.0  
  K 4003 K 4128 P 0931 P 1056 P 6668 P 6793 M 5524*
Grams of powder to prepare 1 L 21.6 22.0 25.3 57.3 27.3 25.3 4.3
pH ± 0.5 at room temperature 4.7 4.8 5.0 5.2 5.4 5.0 3.9

*Composition for M 5524, Murashige and Skoog Basal Salt Mixture, listed for purposes of
  comparison only.

THIS PRODUCT IS INTENDED FOR LABORATORY USE ONLY.
NOT FOR DRUG, HOUSEHOLD, OR OTHER USES.
PREPARATION: Preparing the medium in a concentrated form is not recommended as some salt complexes may precipitate. Supplements may be added prior to sterilization or added aseptically to a sterile medium. Certain supplements (i.e. heat labile) may require filter sterilization and may affect the shelf life of the medium. The basic steps for preparing the culture medium are the following:
1. Using a container twice the size of the desired final volume, measure out approximately 90% of the final required volume of tissue culture grade water (e.g. Sigma Product No. W-3500) . Example: 900 ml for a final volume of 1000 ml.

To prepare Flasks with above chemical and nutrition you need an Autoclave equipment to sterilize them.

     
       Autoclave            

After you sterilized chemical with correct ph in the flasks using Autoclave. Then you take all sterilized equipment and seed pods under  laminar flow clean air or inside the glove box. You have to sterilize outside of flasks and the air inside of  the glove box too . Finally you sow the seeds and close the flasks top under 100% sterile condition.

Once you have successfully made your seed sowing flasks, you must find a warm (70 to 75 degree f.), and well lighted place to grow them.  Therefore you need Hybridizing Incubators.


Hybridizing Incubator

 

Other equipment:, Ph meter., Replete forks., Scalpel., Alcohol.
Clorox., Marker pen., Gloves., and Distill water. If all Phases of orchid seed sowing  have not completed in absolutely 100% sterile and germ free environment,
then you will see molds in your flasks in about one week.
That means you have to do all over again, if you have another seed pod !
  All research laboratories use many high tech equipment to achieve plant production through tissue culture, it is important to note that many home gardeners and hobbyists could substitute the high tech equipment and instruments with ordinary house hold items.



Good luck !
 By Mano Fonnoni

LINK TO :  ORCHID SEED SOWING MANUAL

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