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!