Flasked seeds and cells,- NOW WHAT?

[Royal]

Originally Posted by Florida_guy_26

I do not have any catt bloom stems so the only thin i can do is take the back bulb, chop it into cubes and try cloning that way if it will work as I just do not have anything else to use. Please let me know what you think...

A Cattleya inflorescence does not have dormant meristems like a Phal stem. Neither does a back bulb. You really want a new growth while it's still just a swelling eye. There is a meristem in there which does have undifferentiated tissue. A backbulb, leaf, root, flower, etc. are already differentiated and rarely (if ever) used to culture new orchid plants.

"Cell culture" is a different concept from "meristem culture." I wouldn't be surprised to see some growth planting a meristem on W2.5 but I'd be shocked to see any growth of differentiated cells on any media, including C3.

I don't mean to be contrary, just trying to help. I know we learn from experimentation. It sounds like you are persistent and that's a good start. Keep at it and I'm sure you'll succeed.

[Florida_guy_26]

WOW that just bums me the frig out... So I guess there is no use for cells regardless of where they come from as they are differentiated and probably wont grow no matter what they are on. I just dont have the time and effort and plant tissue to cut the sh*t out of a 500-1000$ orchid either, nor would I even if I did as It just would not yield the results Id like. It sounds like other than chopping up buds the only other way to get enough plants would be to try self pollinating it if it ever blooms.

[Florida_guy_26]

Well I have to say that the flask with the cells from the back bulb is green- like it might be utilizing light for photosynthesis. I do not know for sure if it is growing, but I do see green in the flask where I spread the cells on the medium and the cells have formed a raised patch. I hope the seeds start to grow but so far they look like a bunch of dust still. When do they start to green and form the green ball of cells?

[2ljd]

Originally Posted by Florida_guy_26

Well I have to say that the flask with the cells from the back bulb is green- like it might be utilizing light for photosynthesis. I do not know for sure if it is growing, but I do see green in the flask where I spread the cells on the medium and the cells have formed a raised patch. I hope the seeds start to grow but so far they look like a bunch of dust still. When do they start to green and form the green ball of cells?

Well, to give you some idea...the current batch of Den. anosmum flasks I have going, started to green up after about 1-2 weeks. For this batch, I timed it just right and caught the pods just before they split. Other times it's taken longer as the seeds weren't as mature or they had been stored for a while prior to being flasked.

[Florida_guy_26]

i stored some seeds in peroxide and hope i didnt kill them by doing that. I had them in it for months so i think i killed them. Anyway, i learn as i grow and i have done stupid things- I just hope i dont do anything so stupid like that in the future.

[2ljd]

Originally Posted by Florida_guy_26

i stored some seeds in peroxide and hoep i didnt kill them by doing that. I had them in it for months so i think i killed them. anyway i learn as i grow and i have done stupid things.

Sorry to say, if the seeds were soaking in peroxide for anything longer than 10 minutes, they're probably dead. Even when you do soak them, it should be only about a 10% solution. Oh well, try finding more seed and try again.

[Florida_guy_26]

well do you mean a solution of 3% peroxide mixed at the rate of 1 cup peroxide to 10 cups of water?

cuz my peroxide is 3% no dilution but its as weak as it gets- plus after being opened, it weakens another 1/3 of that strength. as far as i read.

[2ljd]

Originally Posted by Florida_guy_26

well do you mean a solution of 3% peroxide mixed at the rate of 1 cup peroxide to 10 cups of water?

cuz my peroxide is 3% no dilution but its as weak as it gets- plus after being opened, it weakens another 1/3 of that strength. as far as i read.

Sorry, my mistake. You did say peroxide, but I was thinking bleach. Either way, I haven't left the seeds soaking in any kind of solution for longer than 10 minutes to sterilize them. I know some folks soak the seeds overnight in a sugar solution to help bloom any fungal spores before sterlizing them, but not longer than that. It's always a good idea to store the seeds in a dry, cool environment...i.e., the fridge.

[Florida_guy_26]

from now on, ill store them in a ziploc bag and just use some kind of spray sanitizer to clean them. Then ill wash them in water and transfer in a pipette. I just was not clear on how you rinse anything off the seeds when in the flasking box as it is very hard to do without actually being able to see crystal clear which I cant in my box. I will just rinse the best i can. I am tired of all these stupid things I need to do to have a sterile environment. I still had 33% contamination rate as 1 of 3 jars had contaminants, but it must have been from the peroxide solution i poured out with the seeds onto the agar. God this all sucks.

[Royal]

Store seed dry, moisture is the enemy as it encourages microbial activity. Plastic baggies are not a good idea as they hold the moisture in. I just harvest the split pod, snip the tips off, and stick it in a coffee filter. The coffee filter stays on a shelf at room temp until I get around to sowing it. If it's a really big pod, I also pry apart the sections of the capsule to aid in drying. No need to spray or clean the pod, just dry it quickly.

I know there is a lot of info to sort through when researching all this. Some is just technique or preference (like bleach vs. peroxide). The basic principles are most important. It sounds like you understand the process, but are just getting caught up with all the details. There is more than one way to skin a cat. You just have to find what works for you.

A pre-soak in plain distilled water for 12-24 hours may help "soften up" the seeds a bit, which will help make them sink and and make good contact with the sterilant (bleach or H2O2). If the seeds sink, you can decant the sterilant by carefully pouring it out. Open another tube of sterile distilled water and pour some in with the seed to rinse. Let them sink again and decant out most of the water. You are left with seed that has been disinfected and rinsed, just dump it in the flask. If there are lots of seed stuck in the tube, add a bit more sterile water, swirl, and dump in another flask.

My technique is similar to this. I prefer a 10% bleach solution (1 part houshold bleach to 9 parts water). I also use eye droppers to transfer liquids instead of pouring. I'm no pro, but I do have success. I get contamination too. 33% really isn't that bad for a makeshift setup like yours or mine. I know it's tough, but don't get frustrated. Just make sure you are using sound, proven techniques, then adjust from there. There are many opinions out there, and lots of dogma, don't put your full trust in any of it - not even mine. Do your own research then try it for yourself. You'll be teaching others here in no time.