Hi guys
Anyone here sow there own paph (or Phrag) seeds or make there own crosses? Anyone have any tips? Successes/ failures?

I'm new to flasking (since september), and would appreciate any advice! Does someone have some approximate germination times for paphs?

I sowed some bellatulum and exul seeds last week. I also sowed villosum, but both flasks are contaminated. I used Western 3 media with 10% coconut water. Any thoughts on good media for paphs? Or sterilizing seed.

Kyle

I do paph flasking.

Success tip 1: Green Pod Sowing
Success tip 2: Replate early, Replate often.
Success tip 3: Western Medias are good to use.
Success tip 4: Know what coconut water actually is. This is mostly orchid voodoo, but it has reasons for helping. There are much better things than coconut and banana.
Success tip 5: Sterilize seed with 1-3% bleach solution, for 15 minutes, shake vigerously, use a surfactant with it.

Thanks steve.

About the replate early/often tip. How many times do you ahve to replate paphs? I figures once would be enough. Do you have some approximate germination times? I have bellatulum and exul in flask right now (two weeks).

My understanding is that coconut has a bunch of cytokinins in its 'water' (germoplast, I think is the technical term). I don't know much about banana except that it is a big no-no for paphs, but I assume aside from lots of sugar/carbohydrates it has some level of hormones. Can you recommend some alternatives.

Thanks
Steve

Regarding replates. The longer the media goes without replating, the more likely you will get contamination. It isn't that our sterile culture actually kills or removes the possibility of infection, but that it greatly deters it. Agar itself is used because bacteria cannot digest it. PPM is used as a biostat. You just have to find out what timing works well for you. Germination can happen very quickly, like a week, or very slow, like a couple months. It just depends on if the seed feels like it. I have heard some outrageous rumor that some sort of paph seed wouldn't germinate in flask until 7 years later. Like anyone would keep a dud flask that long...

Coconut water has natural cytokinins and auxins, and the food in it is endosperm, because supposedly the seed has none. Seed sure does last a long time for not having endosperm tho... curious. I am in doubt of this.

If you know what is in coconut water (above,) and why people cling to it (because they don't have the slightest idea of hormone ratios, levels, activity, phytochemistry) you can move beyond the obvious limitations of those who use it. Yes, there are much better alternatives. Consider, even Knudson didn't develop these medias for orchids, and everything after him was just a modification of the wrong foundation for the wrong house.

Also, nobody wants to share what is in their secret agar formula. This is some folks life work. Watch out that you don't trash it by just telling everyone everything. Just for example, when I first started learning about the needs of orchid seed and media, I myself postulated on the use of coconut water within just a few minutes where it took others apparently decades. Why? All you need is to educate yourself about these subjects.

If you're looking for the quick and dirty way to get good success, the W2.5 or W3 with coconut water will work great for you. Best of luck!

I'm happy to hear that you endorse Western Three with coconut.

I’m not sure that I agree with you regarding constant replating equaling less contamination. I use a glove box, which greatly reduces my contamination rates, but If I can get a flask to go ten days without a problem I am, >90% of the time, in the clear. Every time I open a flask I am allowing potential pathogen inside. I am very new at this, and you have years of experience, but until I run into a problem I am only going to replate when seedlings are crowded. Do you ever come across the problem that seedlings use up all the nutrients in the media before they are ready to come out of the flask?

I agree that when possible it is better to use synthetic coconut water, but since we don't know the exact composition or the relationship/synergy of the components, we are left with have no choice but to use it. The first coconut I opened to use the water had spoiled (cloudy water/bad smell). The variability of quality of coconut water or banana mash makes me nervous. I know Phytotech sells both coconut water and banana powder, but shipping to Canada is outrageous!

Another question for you, what pH are you sowing the seeds at? I used 5.8.

Kyle

I have used a flow hood and a glove box. I much prefer the flow hood, but the glovebox certainly has its uses.

The reason I say to replate it that there are problems that you will come upon. Not just contamination. For one, after a certain time, the plants may appear to be at a standstill in their growth. The reason may be that the plants sucked up the nutrient, which modified the pH of the media, and made the rest of the nutrients unavailable in that range. Time to replate. 5.8 should be okay, maybe lower it a little depending on the plants.

Hi Steve,

Replating becasue the pH has changed makes a lot of sense. Thanks for explaining it clearly.

I would love to use a flow hood, but I don't see this hobby developing to the point of justifying the expense for a few years now. Until then I look for cheap ones on E-bay...

Steve, when its not possible to use green pod and you are sterilizing dry seed, what concentration of bleach are you suing and how long are leaving the seeds in the solution. I was very disapointed to lose all my godfroya seeds due to improper sterilizing. My bleach solution is as follows, 5ml bleach in 95ml of distilled water. Any advice in the dry seed area?

Kyle

I haven't flasked in a few years (lack of time, not lack of interest), but I had a fair amount of success with sigma P6668. Added 1 four ounce jar of banana baby food per liter for replates. And some other stuff, but I'd have to find my notebooks to recall what that was. Are there better media? Sure... That is just what I used. Worked well on maudiae type paphs and novelty crosses, and I did a few phrags too. Steve is right, nobody wants to share their secret formulas. As a scientist, that bothers me quite a bit, but that is the way the market works. Seems like there should be some sort of 'open source' flasking information so that everybody could play.

As to some of the comments that Steve had... Everybody learns how to flask differently, and everybody has a slightly different interpretation of why some things work the way they do. That might also help to explain why there are so many widely disparate flasking media out there for paphs, they were derived independently. I learned to do tissue culture in graduate school, and spent literally 8 hours (or more) a day in a tissue culture hood doing mammalian cell culture. Plant culture is different in the reagents used, but not the technique.

1. If your medium becomes contaminated over time, it isn't because of lack of replates. Either it wasn't completely sterile to begin with (easy with a commercial autoclave, hard on the stovetop), you are using less than ideal technique, or there is unfiltered air getting into the flask somehow. If you find that you are playing tag with contamination and that replating helps you keep ahead of the game, I'd suggest there might be a technique or sterilization issue to blame.

2. I'm sure some bacteria can eat agar... Generally that isn't the limitation, the bacteria and fungi are after the sugars, which we can't eliminate from our flasks. I always found that the choice of gelling agent was really a matter of convenience. Agar is cheap. Some gelling agents work better at the pH range we are working at than others, agar is pretty flexible there.

3. I like the observation that replating more frequently can overcome changes in the medium (pH, nutrient removal, etc), and can induce 'stuck' plants to grow again. I'd bet most people never figure that out... I personally suspect a lot of the problem is due to a change in nutrient concentration due to dehydration of the medium. Paphs stay in flask a long time, and the medium dries out over time.

Other random things: I always used dry seed. I'm not sure that was good... 5% bleach, a drop of detergent, and about 10 minutes was what I used for disinfecting the seed. Seemed to work more often than not, but there were batches that were hopelessly contaminated. Some people have recommended calcium hypochlorite, some recommend acidified bleach. Do a google search on Aaron Hicks, he has a lot of information on his website about flasking and decontaminating seed. Don't give up on the seed germinating, I found some crosses took almost a year to germinate. As long as the flask isn't contaminated, it doesn't hurt to leave it on the shelf.

Seed sterilization is tricky. Some folks will suggest 1% bleach for 5 to 10 minutes. Others will say 10% for 30 minutes. The truth is somewhere in between.

For example, I had tons of the chinese paph seed I purchased, but we got huge amounts of contam with strict protocol. The seed was just too contaminated.

Something to help you, however, is to prime the seed. By this, I mean to soak the seed in a sugar solution for 48 hours prior. This will help to get the bacteria and fungus to start coming out of their dormancy and protection. At this point is when you nuke them. They are out of their shell and exposed to what they were hoping was going to be a good environment.

Regarding the hoods and replating. I replate more often then not, simply because the more time the flask spends in an unsterile room, the greater the chances of contam are. Even in the hood, so air flow could possibly be swept back in. I would much rather replate before contamination, than after. Not only that, but it helps the plants.

Steve
What do you use to check the pH of your in flask medium? Since its mostly solid, and not very deep in the flask I would think a pH probe would have problems with this situation.

Rick,
To test the pH of the media I use a 'Hanna Checker 1' that I calibrate before each use. I check the pH before I disperse the media into each jar, prior to sterilizing it.

Steve, I have been using a 24 hour soak in sugar water. I assume that my godfroyea seed was heavily contaminatied to begin with and the the contamination was of no fault of mine. I thought that there might be a small chance that I was no sterilizing long enough or with a high enough concentration of bleach.

Kyle

I figured just about any probe is fine for checking the media while it is still liquid and out of the flask. I just wondered how would you check the pH of the old media (in a flask full of embryos) to determine if the pH has changed? i.e spent media

I don't particularly like using it, but litmus paper would work.