Media for growing Triphysaria in vitro

¼ Hoagland’s solution (0.25 X) (1 liter)

900 ml deionized H2O:

1.25 ml 1 M Ca(NO3)2

1.25 ml 1 M KNO3

0.5 ml 1M MgSO4

0.25 ml 1M KH2PO4

0.25 ml Iron-EDTA solution

0.25 ml 1000X micronutrients

7.5 gm sucrose

pH to 6.1 with KOH

Add H2O to 1L

For square plates, add 10 g (1%) Phytagar. For germination plates and multiwell trays, add 6 g (0.6%).

Autoclave 50 min on liquid cycle and cool in 65ºC water bath.  One litre of media will make approximately 20 plates.  Carefully fill each plate approximately half full with media and dry in flow hood with covers on.  Once the agar has dried, plates can be stored at 4ºC, inverted, in sterile plastic sleeves (original packaging) or individually sealed with parafilm.  Before plating seeds, dry and warm plates in the flow hood for about 15 minutes.   

 Micronutrient solution (1000X)

Prepare 100 ml of micronutrient solution using 1 M stock solutions of each component

70.0 mM H3BO3

14.0 mM MnCl2

0.5 mM CuSO4

1.0 mM ZnSO4

0.2 mM NaMoO4

10.0 mM NaCl

0.01   mM CoCl2

Filter sterilize

Iron-EDTA Solution (1000X)

To 900 ml H2O add

2.5 gm FeSO4-7H2O

3.36 gm NaEDTA

Bring to boil, then stir 30 on magnetic stirrer while cooling

Make up to 1 L with H2O