Sophrolaelia Dry Seed Flasking - first attempt

[czayta]

Hi everyone This weekend I attempted my first try at flasking. I was given some Sophrolaelia 'Cheerio' seeds to try flasking from an orchid grower up the road from me.

I started by rinsing all the flasks in bleach water. I had a majority of baby food jars with Magenta B caps and some large Mason jars for my mother flasks. I am not convinced on the Magenta B caps because I feel like they don’t seal 100%. The caps just lightly pop onto the rim of the baby food jar. We will see with time if any contamination grows.

I purchased my media from the OSP.


I used P669 modified germination media and mixed it with 1 L of boiled, distilled water.


I’m not sure if I mixed it long enough because some of my flasks didn't set/gel/solidify and some separated to a layer of clear on top and black at the bottom.


I poured the media into all of the jars and positioned them in my pressure cooker.


I had autoclave tape that I put on some of them as well. The tape turned black which happens after exposure to temperatures commonly used in sterilization processes, typically 121°C in a steam autoclave, or in this case a pressure cooker. I pressure cooked them for 15minutes at 15psi.


I took the media out of the pressure cooker and let it cool down on the counter with a towel over it. When it was cooled I sealed them and put parafilm around the edges of each flask.


The next day I went to the lab to sterilize and flask the seeds. I did it two ways: syringe with a filter disk and in a test tube. I read online about a way to sterilizing in a test tube with a bleach solution and then centrifuging the seeds, aspirating the bleach, and rinsing with sterile water.

The seed pod had been cracked open for over a month so he warned me that the seeds may not be viable.


I looked at the seeds under a microscope and this is what I saw. I don’t think they are viable. But I flasked them anyway for practice.


This is an image I found online of viable orchid seeds.


I did the syringe method by dropping a little bit of the seeds into the syringe and then inserting the plunger. I pulled up some 5% bleach water (with a drop of dish soap) and vortexed the seeds for 1 minute. I let them sit for 10 minutes after that before rinsing with sterile water twice. I used a Nalgene Syringe Filter to push all the bleach water through the filter. Then I rinsed it with sterile water a few times.


For the test tube method I scraped a bunch of seeds into the bottom of a 15mL conical tube, added 5% bleach water, vortexed for 1 minute, and then centrifuged the seeds at 3500 rpm for 4 minutes.


A pellet was formed at the bottom of the test tube and I aspirated the bleach water solution off of the cell pellet. I then rinsed with sterile water, vortexed, and centrifuged again. Here they are suspended in the bleach solution:


I like this method better than the filter paper method because I feel like i lost a lot of cells in the filter apparatus that I used and that the cells weren't adequately cleaned of the bleach solution.

Once the cells were sterilized I set up the laminar flow hood. I turned the blower on for 20 minutes and sterilized the work surface with 70% isopropyl alcohol. I wiped it down again and cleaned everything off as I transfer it into the hood. I was finally ready to inoculate the flasks. Using a sterile pipette I aspirated some of the clean seeds into each flask and sealed them back up right away.

I wrapped each flask with parafilm after and took them all home.


I have them under a T5HO four foot long fixture with two bulbs on a 12hr daylight cycle at 74°F during the day and 65°F at night.


Some seeds splashed up on the sides during transportation:


It was a very fun experiment, but I have found myself sighing and thinking that it might all be for naught because the seeds don't appear to be viable. Right now I am just hoping for no contamination. It was good practice and I discovered my aseptic technique is very rusty lol. If anyone has any advice on how I could improve this process it would be appreciated. In August my Phal NoID pod will be ready for green pod harvesting so I am going to give that another go. Very excited. Sowing orchid seeds and flasking is fun!

[orchidsarefun]

Good Luck, I tried something similar and it didn't work out. I must admit that it wasn't as sophisticated an operation as yours ! If anyone can do it, you evidently can !

[Cntry]

Wow I hope this works for you. It was really cool to see all the pictures you did of each step. Thanks for sharing the pictures and all the information with us all here.

[WhiteRabbit]

Very cool!

I'm hoping your efforts were not in vain! Keep us updated, please?

[czayta]

Of course! As of today nothing (of any sort) is growing. To be honest, I'm not expecting much from this batch given how the seeds looked under the microscope. :P

I found a really thorough link with information about preparing media, dry seed sowing, and green seed sowing.

Media prep & Dry Seed sowing:
PAPUA NEW GUINEA ORCHID NEWS

Green seed sowing:
PAPUA NEW GUINEA ORCHID NEWS

[lotis146]

Best of luck to you! I look forward to your updates. And at least like you said this will have been good practice for your next attempts, even if this isn't successful.

[czayta]

Time for a quick update. One week later and no signs of germination, contamination or growth of any sort in the six flasks that I sowed Sl. Cheerio seeds into.



I had a control flask that I purposely exposed to air and wanted to become contaminated, and a little white colony of something has started growing in that one.



I think its good that contamination is growing in the control flask but nothing in the main flasks so far though. Does anyone know when I should expect germination to occur for these seeds?

I read this: "If the seeds are viable and the conditions are correct, they will germinate in several days or weeks (sometimes months)" so I'm going to give it three months before I clean the flasks out and try again with other seeds I think.

[czayta]

Quick update.

Contamination in two of the flasks. They were both the flasks that came from 'Test Tube 2' so maybe those seeds didn't get sterilized properly. I have disposed of those flasks. Of the remaining flasks there isn't any growth in any of them yet. Its been a little over 3 weeks.

[Cntry]

I still have my fingers crossed that you get something out of there other then contamination.

[epiphyte78]

Really excellent documentation! I'm quite impressed with how professional your first attempt was.

I'm also impressed that you went ahead and flasked the seeds even though you established that they didn't appear to be viable.

But I'm wondering whether the seeds really weren't viable because they were too old. What would the "viability over time" curve look like?

Day 1: 90% viability
Day 2: ...
Day 3: ...
Day 30: 0% viability

100% viability loss after 30 days strikes me as a bit off. You'd figure that most Cattleya alliance would have been naturally selected for seeds that had a greater margin of error. Because...what if the pod ripened and released its seeds 2 or 3 weeks before the start of the rainy season? Only the seeds with the greatest longevity would survive. Or perhaps orchids that failed to accurately predict the onset of the rainy season took themselves out of the gene pool long ago?

Personally I'm trying to germinate orchid seeds symbiotically. This year I've been pretty good at consistently pollinating my orchids. Right now I probably have 30+ pods in various stages of development.

The next time you plan on flasking...feel free to PM me if you're interested in additional seeds. Also, I could send you some fresh Cattleya alliance seed if you'd like to graph its viability over time. My Cattleya Penny Kuroda has 6 fat seed pods on it. Most of the pollen came from Mexican Laelias. I'd be interested to know if the seeds are viable and if so, what their longevity is.