Propagation tests trials and experiments.

[Tsuchibuta]

I'm about to begin doing a number of test trials and experiments in propagation starting this weekend. If you have a method you would like to see documented let me know and maybe I can try it out as well. Please keep in mind these tests are going to be geared to the home hobbiest. Also I want to try alternative methods that are more excess-able to those of us that don't live where normal propagation supplies can be had.

Currently my tests will include:
Media:
- Phyto p723
- Phyto p658
- Phyto p748
- C.D. Moulton's corn starch media
- OSP1

Vessels:
- Mason jars
- Polypro deli style culture vessels
- Plain plastic soda bottles
- recycled jars spaghetti sauce and such (sorry do not have kids or close to anyone that does so baby food jars or out - besides they are well tested)
- Plastic Ziplock bags

Sterlization methods:
- Pressure canner
- Microwave
- Roby's chlorine gas method
- aerosol disinfectant vs bleach water mist vs disinfectant wipes (this test is because some people are very sensitive to bleach)


Tests will be preformed using homemade glove box made from clear plastic tote that is easily obtainable.

I will try to post and take as many pictures as possible. Also if there is any specific data you would like to see let me know so I can track that as well.

Currently I plan to track: Contamination, Germination, Growth rate, cost.

[Gage]

I love it! Can't wait to hear of your results!

[Discus]

How many replicates will you be doing for each treatment? And what species are you trying it with?

You can also try vented vs non-vented containers, or perhaps different potential vents.

If you haven't seen it, the OSP's media book (Orchid Seed Germination Media) is pretty epic.

There was one recipe in there that seemed pretty easy to make with common "household" ingredients - if you want it, let me know.

You can also try things like with/without potato cubes etc. etc. etc. There are hundreds of variations you could do...

You can also mess about with temperatures and lighting periods.

If you want to be able to make any actual conclusions though, experimental design is quite important.

[Tsuchibuta]

Originally Posted by Discus

How many replicates will you be doing for each treatment? And what species are you trying it with?

I am going to try to do as many as possible with the quantity of seed that I have and can acquire throughout the trials. Currently I have Trias nasuta, Dendrobium hymenianthum, and thanks to King of Orchids Eulophia andamanensis. Also I will be doing some replate tests with some of the slower growing plantlets in my Max x Zygo cross and Gongora hybrid. I will be adding more seed as I acquire it.

Originally Posted by Discus

You can also try vented vs non-vented containers, or perhaps different potential vents.

I had thought of this but forgot to include it above.

Originally Posted by Discus

If you haven't seen it, the OSP's media book (Orchid Seed Germination Media) is pretty epic.


There was one recipe in there that seemed pretty easy to make with common "household" ingredients - if you want it, let me know.

I would love to see the recipe. At one time I was going to order the book but then decided that it was more space conservative to have a company flask for me. Now that my situation is reversed I am back to wanted to learn more.

Originally Posted by Discus

You can also try things like with/without potato cubes etc. etc. etc. There are hundreds of variations you could do...

You can also mess about with temperatures and lighting periods.

If you want to be able to make any actual conclusions though, experimental design is quite important.

I would be definitely interested in additives, but I may have to add that study once I have acquired more available seed. Lighting will be easy to do, and can definitely look to that - however initially I will keep temperature constant among the test cultures so that data will not be affected. Then once a baseline is established I can go further.

More than anything I want photo/video documentation of the findings because many people are visual learners, or just plain skeptical and though there are some really phenomenal books on the subject, few have detailed photos of the process completely through.

[orchidsarefun]

....its not easy.
I also tried a couple of different things. What worked in terms of non-contamination of the vessels was to put them in a plastic storage box and then cover the entire box with plastic wrap. So I did not cover each vessel with plastic wrap.
In fact 5/8 of the vessels individually covered with plastic wrap became contaminated with a fungus.

[Tsuchibuta]

Interesting, I will keep that in mind.

[Discus]

Paranoia is usually key to asepsis... My introduction to this was tissue culture of coelacanth skin cells on a ship. It was not a fun place to start, but I did at least have a laminar flow cabinet and fairly comprehensive instructions.

re: those media, there are actually a whole bunch of slightly different formulations of fertiliser/sugar/agar/(other things) where (other things) are banana pulp, fruit juice, coconut milk and that sort of thing. These are rather easier than the commercial ones to make up, or the more complex ones requiring accurate balances (or messing around with stock solutions to get milligrams of stuff). I've avoided all the ones that need specific chemicals or things like "peptone" that may be hard to come by. The penultimate one is quite interesting in that it doesn't use agar.

Recipes are generally made up to 1l unless otherwise stated.

Bridgen Medium
30ml sucrose (table sugar)
237ml tap water
118ml of nutrient soln*
1/2 250mg inositol tablet
1/4 vitamin tablet with thiamine
30ml agar flakes
Combine in flask; boil while stirring.
*1/4tsp 10-10-10 fertiliser in 4 litres of water

Fernandez Media
100g Banana Puree
12g agar
15g sucrose
300mg vitamin B tablet
900ml distilled water
suggested mods include addition 250g puree mature tomato; filtered or 200mL pineapple juice.

Another mod is
20g peters 20-20-20
100g banana puree
12g agar
15g sucrose
0.25ml superthrive

Fertiliser based medium (epiphytes):
10g honey
10g sucrose
2ml Wuxalk 12-4-6 fert
6g agar

Fournier
6g fertiliser
38-43g chinese food store agar
"add sugar, super ripe bananas and other components as required" makes 4l
ferts can be: G&B 20-10-20 cattleyas; Peters 12-36-14 for ghost (leafless) orchids; 10-60-10 bloom plus for phals; replate cattleyas & ghost miracle gro roses 18-24-16.

Hyponex (fertiliser) media
Several are listed; the simplest is
3g hyponex
35g sucrose
15g agar
(for cattleyas)

Seeing as you're trying a Den, they suggest for dens:
3g hypones
35g sucrose
10-20% apple juice by volume
15g agar

Macdonald
In a large pot - 3/4oz agar from chinese food store; add 6 cups water set heat on high. Blend 4 bananas that have been frozen and thawed together with 1/2 tsp peters 20-20-20 with minor elements, 1/2 tsp superthrive. Add to pot. Stir to avoid burning. Add 2tsp powdered activated charcoal (crush fishtank charcoal in dry blender). Stir to be homogenous; portion out into 24 mason jars. Autoclave at 15psi for 15 minutes. Mason jars should have 1/8th inch vent hole, suitably capped.
If desired, medium may be improved with B vitamins; add .5 to 10ml of a vitamin B1 or B-50 tablet dissolved in 120ml water.

Modified Kyoto
3g hyponex
20g sucrose
8g agar.

OSP1
2 level tsp sucrose
1/2 level tsp miracle gro 15-30-15
150ml banana baby food (no added sugar version)
Pineapple juice can be used instead of banana.
Mix well.
This is then placed into small culture vessels with a sheet of Whatman #4 filter paper or other suitable absorbent, non-tearing paper at the base. Sufficient liquid added to saturate paper.

Banana may be omitted or reduced for germination. For replate, banana is used and an inert medium (perlite) used to support seedlings. Nutrient solution added to just below level of perlite in vessel; Medium is autoclaved, seedlings replated onto surface.

Stoutemeyer and Cooke
2g water soluble fertiliser (2 level tsp)
20-30g sucrose (5-7 level tsp)
10-15g agar (12 level tsp)
For replate, add 100g banana; reduce agar to 8 level tsp
Recommended ferts are peters houseplant or orchid, miracle-gro or stern's miracid.

Remember to check/adjust pH to suitable value in all.

These recipes are from Orchid Seed Germination Media - A compendium of Formulations by Aaron J. Hicks, Edited by Katy Lynn. This is available from the OSP; I gave the link in an earlier post in this thread. Well worth acquiring for its in depth discussion of this topic and its complexities.

It might be of interest to trial a fert-based one with Ray's new seaweed enhanced MSU formula.(http://www.orchidboard.com/community...stimulant.html)

[Tsuchibuta]

Very interesting, I'm really curious about the non agar based media as well (hence the corn starch media I am hoping to try) as I find that acquiring agar is often the most difficult part for the hobbyist if they don't know or have access to good sources.

[Discus]

If you're struggling to find agar (a lot of oriental and health food shops stock it, the latter as a vegetarian friendly alternative to gelatin) perhaps you could try gelatin? You may even be able to get jello to work, if you account for the sugar and its osmolarity!
Certainly, if you've gotten hold of the P series of nutrient bases, you should be able to get agar from the same supplier!
You could also try alginate, although I think that requires quite a bit of calcium ions to set. Even carrageenan or gellan might work.

[Tsuchibuta]

I have the P series because I ordered them when I visited family back in the States. Shipping from Phytotech to the island is outrageous. If I was back in my hometown in the US I would have no problem locating it, because I lived in a big city. However now not only do I live on an island it's is a sparsely populated one at that. Hence what sparked my interest in non traditional methods. Worse comes to worse I can order agar and have it shipped to my family in the states then forwarded to me. (which is actually cheaper than the international shipping from phyto) - I really don't want to do this though as it defeats the purpose of my trials - because if I have access to preformulated then I would/ could just use that and there would be no need to for the homemade method.

As of yet I haven't located it sold on the island, but I will check the couple of Asian supermarkets when I get a chance. I'm not getting my hopes up though.