Adventures in Flasking

[Dorchid]

I figured I’d take a stab at flasking some seed as I grow mushrooms and have the gear and experience working with invitro culture and aseptic technique. The Odcdm. was selfed on March 18 and took 8 months for the capsule to mature. Seed extraction and flasking were all carried out in front of a large laminar flow "hood". Unfortunately, I didn't catch the capsule before it dehisced and didn't have any media on hand, so stored the capsule in an envelope in my still air box for a few days while waiting on the media to arrive. I cleaned up the seed in a H2O2 with a dab of soap as a surfactant (forgot to order Tween20). This was carried out in a syringe for 10 min (I followed the method on the herebutnot video). Not super pumped on this technique but we'll see what happends. I made up some flasks with pint mason jars and plastic lids modified with a hole and polyfill to serve as a gas exchange filter and PhytoTech P723 Orchid Seed Sowing Media. The media was supplemented with 5% coconut water per PhytoTech, though I just used store-bought VitaCoco. I didn't adjust pH or treat the media in any other way. Media was pre-poured and sterilized for 40 min in flask. Each flask was sown with 3 drops of the H2O2 solution containing seeds and will be stored in my tent with roughly 12/12 light cycle, ~75F and 75-80% RH. Hopefully, germination will occur in the next month or so...I'll keep this post updated as things progress or fail. Please leave any questions or comments.

[Mr.Fakename]

Fingers crossed for germination, and nicely explained!

I think it would be safer to rinse the seeds in sterile water, rather than keeping them in hydrogen peroxide. Even though it becomes water under light, long exposure time can damage seeds.

Second comment, 40 minutes of autoclaving seems overkill for the amount of medium in each flask. Chemicals contained in it are (mostly) heat sensitive.
If the lids are microwave-resistant, sterilising using a good ol' kitchen microwave saves a lot of time and trouble.

[Dorchid]

Thanks Mr. F! Hopefully the components weren’t denatured with the extra time. I usually run 30-40 min on media sterilization which is indeed overkill even considering they aren’t for plant propagation. That said, I’d never use the microwave to sterilize media. I have another pod to harvest and will sterilize the media for 20 min. Yeah, I wasn’t too enthused with the H2O2 bath and would rather do a dilute bleach bath and sterile water rinse...I was lacking some things to carry out that procedure. The video I referenced purported good results so we’ll see what happens. Appreciate the input!

[Dorchid]

I sterilized a new batch of media per phytotech at 15psi for 25 min. which includes ramp up (Thanks Mr. F for jogging my senses!). This round I used half pint jars which should make for easier replating should protocorns form. Additionally, this was a “green pod” which I decontaminated with a 30 minute soak in bleach, a quick scrub with toothbrush, rinse in sterile DI water, spray with isopropyl and flamed! With flamed scalpel, I cut off the end and then down 2 seams, pulled up slightly at the cut end and it popped right open. While holding with forceps, I lightly tapped the capsule with the scalpel while holding over the flasks and the seeds snowed down onto the media. This was a much easier process than washing the seeds of a dehisced capsule.

[Dorchid]

The odcdm. project didn’t turn out. I didn’t get any germination from the seed as I think the ph of the medium was too low. I’m starting again with some noid phal seeds from a green capsule. The cap was washed in 50% Clorox solution for 25 min and rinsed multiple times with sterilized ro water. The contents were extremely fluffy and transferred to around 40ml of sterilized ro water and aspirated into a sterilized syringe. P723 was again used and I made 3 different batches. Two were ph 5.8, one with coconut water and the other without and the third was ph 5.6 with coconut water. PPM was used in all. 27 plates were poured and around 5 drops of seed solution applied to each. After distributing around the plate and allowing the seed to settle, the excess water was poured off and the plates wrapped in parafilm. They are currently in a tent with 16/8 moderate light (25W COB) with a temp range of around 65F-73F. All work was done in front of the hood.

Pollen parent


Cap parent



Cap at 6 months


Excess water poured off




Possible germination after 15 days


Homemade laminar flow station

[Dorchid]

Just found my loop and got a good look at the seeds. Those on the 5.6 ph formulation are swollen and green; Much more so than those on the 5.8.

[orchidman77]

Love the detail and pictures! Great job with germination and good luck - I'm excited to see updates.

David

[Dusty Ol' Man]

Th8s is so far over my head, but it amazes me the a .2 difference in pH makes that much of a difference.

[Roberta]

Originally Posted by Dusty Ol' Man

Th8s is so far over my head, but it amazes me the a .2 difference in pH makes that much of a difference.

Remember, the pH scale is exponential.

[tmoney]

Originally Posted by Roberta

Remember, the pH scale is exponential.

logarithmic...meh, semantics, i think we get what you mean!