Flasking 101 - A how-to guide

[King_of_orchid_growing:)]

Seeds from the genus Disa from species such as Disa uniflora, Disa tripetaloides, Disa aurata, and Disa cardinalis have been sown using an ex-vitro method of boiling some sphagnum moss. There are details about this all over the web.

[Royal]

Thanks for the info, King. But since this is "flasking - 101" we should probably stick to in vitro methods. (just to keep things strait for the intended newbie audience)

[Zozzl]

A few mistakes I made in my first attempts:

1) Using containers that were not able to be adequately tightened/sealed. Hoping and praying does not work
2) Homemade media does not work as well and the laboratory stuff.
3) Probably picking up the containers a lot after the seeds are sown is not the best idea.

I did notice that laboratory media does give much better growth than the first homemade formula I used.

Pat

[Royal]

That's a good point, Pat. There are countless ways that it can be done, just as King points out, but certain ways to eliminate variables and increase the likely hood of success. There was a similar discussion on a different thread concerning homemade media. If you have limited resources, you can make do with many different materials and techniques. But why just "make do" if the real stuff is only a few bucks for a liter. That's like saying you don't need a telephone if you have a few coconuts and a string lying around. Just go buy the ten dollar phone!

Certain steps, like autoclave sterilization, proven media formulas, and chlorine as a seed sterilant are the standard. These are the techniques derived from decades of research and currently in use by most large scale laboratories. They are, by no means, the only way, but they a good starting point and can eliminate much of the trial and error for the beginner. This is why I thought this thread was such a good idea. Get the basics out there so the newbie doesn't have to sort though all sorts of specialized info for someone else's preferred technique just to find the facts.

[Royal]

Ok, it has been a week and no other responses. I'm moving on to sowing.

So, we have good seed, we chose our media, we have some small jars with lids, and we know how to sterilize them with the media already poured. We also have a glove-box or or similar "clean" workspace. Here's where the rubber hits the road - putting seed inside the sterile jars on the germination media. There are quite a few ways to get this done, but a common factor is that anything that goes in a flask will have to be sterile or we'll grow mold instead of orchids. Two basic methods are used: sowing dry seed, and sowing from a green immature seed capsule.

The "green pod" method is popular due to the fact that seed inside a capsule that has not yet split is still sterile, it has never been exposed to any contaminants. The outside of the capsule is cleaned rigorously(usually with soap, bleach, and/or peroxide), then sliced open with a sterile tool (in your sterile workspace). Seed can just be scraped from the inside of the pod and tapped into the flask. A few drops of sterile water can help disperse the clumps about the surface of the medium but is not necessary. The trick here is to time the development of the seed so that they are mature enough to be viable, but the pod itself is still sealed (not yet splitting). Too early and seeds might not germinate, too late and the pod can split.

I normally harvest and sow dry seed. I like the ability to store some for later or share it with OB members. The catch is that we must assume that it is contaminated with airborne particles of "bad stuff" so the seed itself must be disinfected. Traditionally (back to Knudson), chlorine has been used as the seed sterilant. Growers used to use calcium hypoclorite, but chlorine is chlorine so most of us just use household bleach (6% sodium hypochorite). A 1:10 solution is a good place to start (0.06% available chlorine), but may need to be adjusted to the variety. Contact time of about 10 minutes is a good starting point, but you can experiment with shorter times or even lower concentrations.

Dry seed can be tricky to work with. It can carry a static charge that makes it stick to everything. Sometimes it will want to float and is tough to get saturated with the bleach solution. Vacuum pressure and ultrasonic waves are a few proven methods of fully infiltrating the seed testa and getting them to sink. Another negative is that the bleach needs to be rinsed from the seed. I skipped this step, only diluting the bleach before just dumping it all. I got mixed results, but I'm not satisfied - I'm sure it's my failure to rinse enough. Some decant as best as they can, some use filters. I'm leaning toward the filters but I'm still exploring my options. The syringe method sounds better and better.

[Zozzl]

Royal, rinsing the seed is the most difficult part for me also. I wish they was an easier way to do it. I have heard about the filters but I am not actually sure how to use them. Anyone?

Pat
p.s. Great post!

[King_of_orchid_growing:)]

You mean coffee filters?

It's a pain to collect the seeds and put them into the flasks.

Sometimes I end up collecting the filter fibers along with the seeds. The problem with this is that there may be contaminants in them too even if you sterilize the seeds using the filters. I believe my contamination problems come from the bits of filter fibers that get scraped up with the seeds. Those always seem to be the sites of contamination.

Just my . At this point it's just speculation.

[Royal]

Pat, Some people make little packets of seed sealed in a filter paper. The whole thing is sanitized and rinsed as one, then cut open and the seeds scraped out. Is this what you are talking about King?

AJ Hicks sells the Whatman glassine filters, they shouldn't disintegrate in the bleach like the coffee filters. He also sells autoclaveable wash bottles and filter funnels. If using these filters, there is no need for agar in the germination media and all you'd need is a thin layer on which to float the filter. I've never used filters, so I'm speculating now too.

[King_of_orchid_growing:)]

No. I pour the seeds into a coffee filter and put the whole thing in a bowl of isopropyl alcohol for about 5 to 10 minutes, then take that out and put the coffee filters with seeds in another bowl with 3% hydrogen peroxide for the rest of the time. While it is in the hydrogen peroxide I use a replating fork to scoop up the seeds and put them into the flasks.

I actually find my method to be time consuming. I'd like to find faster, better, more efficient methods. There are times when I won't have the time to be fooling around with orchid seeds all day.

I want them disinfected and in the flippin' jars asap.

[mnoor]

Being a complete novice to this orchids matters and out of curiosity, I hand pollinate 2 dendrobiums on 8 Jan 2009. Today, I have a seed pod that has split opened and there are many tiny orchids seeds dropped on some of the leaves. Upon realising it, I save the pod and kept in an envelope and store it in a refrigerator.

Accordingly, dendrobium seed pods will mature around 12 months. I am puzzled as to why my seed pod has opened up and released the seeds just within 7 months!

Allow me to describe the environment which my orchids are growing:-

1) I am staying in an apartment that is 10 storeys high.

2) Day temperature fluctuate between 28 - 32 degrees centigrade

3) There isn't much temperature fluctuation during the night as all my orchids plants are kept at my apartment's balcony, though it is opened in the night, the plants do not get any dew drops.

4) Humidity is very high at my place, 80% is common and if it rained, it can be 100%.

5) Watering is done once or twice a week depending on the days, if more dry days I will water it twice and will not water until the medium is really touch dry.

6) The seed pod is dry but the color is still green with some visible yellowish color indicating going to reach maturity.

My question is:-

1) Is the seed pod ready for flasking?

2) Can it be a hiccup in its maturity process that it is now split opened before maturity rendering it a useless seed pod?

3) Can it be because of the strong wind at my apartment that has caused the leaves frictionised the seed pod that cause it to split opened prematurely?

Any input is indeed, much appreciated. Sorry for this lengthy message.