Adventures in Flasking
 

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.
Attachment 148750Attachment 148751Attachment 148752Attachment 148754Attachment 148755Attachment 148756Attachment 148757Attachment 148758Attachment 148759Attachment 148760

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.

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! :dance:

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.
Attachment 148799Attachment 148800Attachment 148801Attachment 148802

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
Attachment 159712

Cap parent
Attachment 159713


Cap at 6 months
Attachment 159714

Excess water poured off
Attachment 159715Attachment 159716

Attachment 159717

Possible germination after 15 days
Attachment 159718

Homemade laminar flow station
Attachment 159719

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.

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

David

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.

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

Germination all around was pretty poor, probably around 1-5% but there are several plates with lots coming along. The medium with the higher pH with coconut water had the lowest rate of germination so far. Couple pics of progress.

Attachment 159947Attachment 159948

Hey, small success at the beginning is still success! I'm sure you'll narrow down the variables and have better germination the next time you try.

David

Thanks, David! I recently read that seed germination for phals and cattleya are greater on medium with a lower pH, ~5.2, though it is a very old paper and details weren't very clear. I've seen good success for both those on pH of 5.7 so will likely shoot for around 5.4-5.5 on my next batch. Temps could also be a factor as it's a bit cool in my space with a high of only 73-74F. At the end of the day, even with such a low germ rate, there are still hundreds of seeds developing so still a success for sure.

It's fascinating how a change in pH can have such a drastic effect on seed germination. I'm going to eventually get into flasking, but I want to get settled for a bit longer just keeping the plants thriving before I take those next steps :) ah, the inevitable path! In the meantime, I will simply appreciate your work!

Received a Sophronitis brevipedunculata capsule from a fellow member; Thank you! The capsule busted open in transport so the seeds were sterilized in 10% bleach for 25 min, transferred to a sterile coffee filter and rinsed multiple times with sterile water. The paper was transferred to 10ml of sterile water and plated up on 4 petris of P723 pH 5.5. I also plated remnant unsterilized seed on an additional plate to test the efficacy of the PPM biocide used in the medium as well as some of the phalaenopsis seed that has been in solution in the fridge since 7/29 to check viability.

Couple plates of the phalaenopsis at day 36.

Attachment 159996Attachment 159997

Eagerly waiting...

Me too! I may have oversterilized the seed which could be detrimental or beneficial in that it could have fried them or on the other hand increased germination probability by removing the outer seed layer. Moving forward I’m going to use a 5% Clorox solution for a much shorter time. But hey, this is a learning process!

Starting to see some chlorophyl development in the sophronitis plates but it’s been slow going. I just got a new inkbird and heating element so will maintain a temp of 76F in the tent with the hopes that germination will pick up. The phal seed that was stored in sterile water in the fridge for 30 days and then plated has germinated quite well. I’ll plate up the rest at 6 months to check viability. Here’s one of those plates:

Attachment 160332

Phal plbs slowly moving along. I’ll probably replate them in the next month or so. Unfortunately, I only see 2 germinations on the soph plates.

Attachment 160887Attachment 160885Attachment 160886

Nice. I hope more of the Sophronitis seeds germinate!
The Encyclia capsules I planned to send to you both aborted - not quite sure why - but I'll continue pollinating and sent you seeds as I can.

David

That would be nice but it’s been a sec so not sure any more are gonna pop. No sweat on the encyclia! I crossed these back on Oct. 2 so should be ready in the spring.
Attachment 160891Attachment 160892

Attachment 160893

Nice cross, both parents are beautiful!

David

Phal protocorns still plugging. Starting to see some leaf development. Will likely replate next month.
Attachment 161133

Just plated up some Orange Cattleya, Orange Guarianthe aurantiaca…capsules courtesy of Roberta; Thank you!!! They were still a bit green and the seed mass was still pretty wet but we shall see how they do!
Attachment 161134

Well if they don't make it, there will be more in a year or so I'm sure... these seem to get knocked up pretty regularly.

:dance:

I sowed these plates a lot thicker…3 plates per capsule with each plate getting 5ml of seed solution. They are covered! The large waxy-looking chunks are masses of seeds…shake as I might, they just didn’t want to break up. I might scoop them out at some point but really didn’t want to fiddle with it at the time. The seeds look nice and plump through the loop so hopefully they have some good viability.

Attachment 161148

The Orange Cattleya, Orange Guarianthe aurantiaca are looking great! Seeds are swelling and chlorophyll development is beginning.

Additionally, just crossed Fdk. Frank Smith with Ctsm. Dapper Dots.
Attachment 161329Attachment 161330
Attachment 161331

Just plated up some Inobulbum munificum (thanks R!). A spike with 3 capsules had been knocked off of the plant by a cat and were, I believe, only 5 months old (please correct me if I’m wrong, Roberta). Per Roberta’s suggestion, I stuck the spike in water and let it go for another month. The capsules were starting to crenate so I figured I better get them going. One was compromised with rot and another ended up getting completely soaked with bleach water inside when sanitizing due to an unseen hole, so only used 1 capsule. The seeds looked pretty dry upon opening but looked awesome under the loop with nice fat creamy yellow embryos. Made 6 plates sowed crazy densely.

Attachment 161367Attachment 161368Attachment 161369

Glad that the plant gave an "insurance policy" with the 3 capsules! You're right on their age. It looks really promising!

No doubt! I probably could have salvaged the other 2 but this was a ton of seed. Also, it would appear that the orange catts are getting ready to explode!

Good call on the Inobulbon. Once they start growing, not small plants. You're going to have fun finding room for all those babies!

Hopefully will carry through to deflasking and will send out to all members who want them!

this is really neat. I'm so glad you're documenting your flasking adventures!

David

Orange catts moving along. Interestingly, some of the seeds in clumps, especially on this plate, are developing faster than the individual seeds on the medium.
Attachment 161459Attachment 161460

These Inobulbum are gonna be a lawn.
Attachment 161461

First true root poppin.
Attachment 161462

Wow, spectacular! I'd say that the Inobulbon plantlets are going to need replating SOON! And probably more than once before they're big enough to live in the outside world. I think that the one capsule had enough seed...:biggrin:

Congratulations on several successes!

David

ship a flask over this way?!

:waving

i know, i know, it ain’t worth it :evil:

but keep up the great stuff!

You never know! :evil:

First replates of the 6 month old phals onto maturation medium with banana.
Attachment 161599Attachment 161600Attachment 161601

Scooped some of the orange Catt seed clumps, suspended in sterile water to separate and put them to replate medium as well.
Attachment 161602Attachment 161603Attachment 161604

Attachment 161954Attachment 161955Attachment 161956

Attachment 161957
Attachment 161958

Definitely a lawn or two, or maybe enough for a golf course??