From: "Mellard, David" <dam7@cdc.gov>

For those not familiar, here's Malmgren's amino acid-based media for
micropropagating terrestrial orchids.

calcium phosphate (Ca)3(PO4)2   50 - 100 mg
magnesium sulfate MgSO4 7H20    50 - 100 mg
potassium phosphate KH2PO4      50 - 100 mg
sugar                           10 g in sowing medium,
                                15 - 20 grams in growing medium
agar-agar                      apporximately 6 grams
pineapple juice                 10 - 25 ml
Vamin (amino acid)              5 ml ( approx 300 mg aa)
Soluvit (vitamins)                     0.1 - 0.3 amp
tap water                              1 liter
charcoal                               approximately 0.5 gm
(kinetine 2 - 5 mg for certain orchids)

Vamin and Soluvit are amino acid and vitamin solutions, respectively,
and are sold only in certain European countries, even though Vamin is
made right here in the US (North Carolina to be specific).  So close,
yet so far away, just like the song.

Carson Whitlow passed on a recommendation from someone at RBG Kew
(sorry, I forget her name) about American substitues for Vamin and
Soluvit.  They are Sigma's RPMI 1640  amino acid  solution (50X) (Sigma
# R7131, cell culture catalog) and Sigma's Nitsch and Nitsch Vitamin
Powder (1,000X) (Sigma # N0390, Sigma plant culture catalog).  It was
fun figuring out that I had to look in two different catalogs to find
them.

In addition to replacing the Vamin and the Soluvit, I also decided to
replace the agar with phytagel.  That turned out to be a small headache
because the first batch I made using 2.0 gm/L of phytagel didn't gel.  I
tried several things next, increasing the concentration to 2.5 gm/L,
then 3.5 gm/L then 4.5 gm/L, then distilled water instead of tap water.
Remembering that too high a concentration of cation will affect gelling
I decreased the concentration, then went back to 75 mg/L.  Finally, it
gelled at 10 gm/L.  Something was wrong.

That was last weekend and I have since talked with Sigma who said that I
should increase the cation (Ca and Mg) levels then I can go back to 2.5
gm/L phytagel.  Last night I tested 150 mg/L calcium phosphate and 150
mg/L magnesium sulfate.  It gelled but still had too much liquid.
Tonight, I will try 200 mg/L of each. I'm pretty sure that will solve
the problem.

Here's the germination formula as it stands today.

10 gm           sucrose
2.5 gm          phytagel
0.5 gm          charcoal
0.1 gm          Nitsch & Nitsch vitamin power, 1000X (Sigma N-0390)
5 ml            RPMI 1640 amino acid solution, 50X (Sigma R-7131),
yields
                   approximately 300 mg amino acids if my math is
correct.
200 mg          (Ca)3(PO4)2
200 mg          MgSO4 7H20
200 mg          KH2PO4
20 ml           pineapple juice
1 L             tap water

The amino acids and the vitamins are not an exact match for what
Malmgren used.  My feeling is that it probably doesn't matter.  The more
I read about media components and sterilization/dormancy techniques, the
more I realize that this is just as much an art form as it is a science.
Science helps but unless you are able to exactly duplicate someone
else's methods, or rather if you change one small technique that puts
you back to figuring out what works for you.

I went into details for several reasons. First, the hope that someone
will (gently) add to what I've said, or describe what they do.  For
instance, coconut water or potato cubes can be used instead of pineapple
juice.  What's not clear (at least to me) is which one is better overall
or better for certain species.

Now I haven't even started on sterilization and breaking dormancy and
whether NaOCl or CaOCl is better.  That's another email.  I will let
people know that I started my first set of cyp seed last week (acaule,
pubescens, parviflorum, plus Paph micranthrum) but it's far too early to
determine success.

David Mellard