Stigma Turning Brown?

[Becca]

Well I will just leave it as is, but I will take a picture if I get a chance this evening.

[Royal]

Oh yeah, you know I'm all about the trades! (plants, flasks, seed, whatever!)

Usually, if everything is sterilized properly (including the seed) there shouldn't be any contamination issues. Mold doesn't just appear from nowhere given enough time. It must be introduced. Your problems probably had more to do with technique or an inadequate seal. Just something to keep in mind for next time. Good Luck!

[vmax3000]

My first time through was with a pod off of a phal that had been "donated", as many of my NOIDs are, by a friend who knew I raised orchids and was afraid she would kill it. I got them off to a great start in petri dishes wrapped in parafilm. I transferred them to "flasks" (Pace salsa jars with a hole drilled in the lid that was covered by a stick on filter). All was well and then it came time to transfer them into a new "flask" and I got lazy/ distracted/ busy and waited too long and the mold that is inevitably around took hold and I just watched. Mental note to self - don't wait when flasking is at hand!! It was really fascinating to get as far in the process as I got and rather successfully, too!!

I am taking the pod in today to see about scheduling some time for one of the professor's autoclaves and laminar flow hoods! I'll keep you apprised of my progress, if you are interested!

[Royal]

Originally Posted by vmax3000

All was well and then it came time to transfer them into a new "flask" and I got lazy/ distracted/ busy and waited too long and the mold that is inevitably around took hold and I just watched. Mental note to self - don't wait when flasking is at hand!! It was really fascinating to get as far in the process as I got and rather successfully, too!!

That's kind of what I'm talking about. Mold doesn't just appear over time. A sealed, sterile flask should remain sterile - forever. The plants will die eventually from not being re-plated, but time is only an issue for the plants, not sterility. If you got mold, it either got in after the fact, or was introduced during flasking.

Try sowing multiple mother flasks. You may even want to do two seed disinfection runs, with different exposure times. If you are working in a LF hood and using an autoclave, contaminated seed might be the culprit. If they float, disinfect/agitate more rigorously. If they sink, standard disinfection times will probably be fine.

Let me know when you are ready for more seed!

[vmax3000]

Originally Posted by RoyalOrchids

mother flasks

What's that? Seriously, I followed recommendations from another orchidist who had successfully plated. His instructions were, loosely, as follows: Sow the sterile seed petri dishes until the first hint of "green", then, transfer them to a "flask" (pace picante jar), then, when they had gotten a plant-like structure, transfer them to their final "flask"....I had gotten to the plant-like structure phase with the phals. The angrecoid seed molded in the petri dish. These were both green pod, so this time will be different in that the pod has begun to open. So, I will be sterilizing seed this time. Should the 5% bleach soln be rinsed off the seed? Should the seed be sowed wet? You guys have me thinking......stand back!!

[Royal]

Originally Posted by vmax3000

What's that? Seriously, I followed recommendations from another orchidist who had successfully plated. His instructions were, loosely, as follows: Sow the sterile seed petri dishes until the first hint of "green", then, transfer them to a "flask" (pace picante jar), then, when they had gotten a plant-like structure, transfer them to their final "flask"....I had gotten to the plant-like structure phase with the phals. The angrecoid seed molded in the petri dish. These were both green pod, so this time will be different in that the pod has begun to open. So, I will be sterilizing seed this time. Should the 5% bleach soln be rinsed off the seed? Should the seed be sowed wet? You guys have me thinking......stand back!!

Your petri dishes were essentially mother flasks. I'm sure they work great, but I just use baby food jars. They seal well, cheap or free, and are made to take autoclaving. They are also tiny, so you can cram a bunch into a small pressure cooker.

The process you describe sounds about right. Just remember that there are very few absolutes. Some won't require replating twice. Some might need it more to grow out longer. Some people sow seed on a multi use media and don't replate at all. Really, the reason we replate is because germination requires a different media composition than is optimal for growth of roots and leaves. They'll just turn green on a lean media, but replate them on a richer substrate and they go nuts.

A glove box is key to maintain sterility. You can just cut holes in a big clear rubbermaid storage bin and attach gloves to the inside. In a clean room with still air, you may not even need the gloves. In a dusty drafty room, you may want to tape the cracks shut. Only open flasks inside a disinfected glove box. Disinfect it with a strong bleach solution or 70% alcohol in a spray bottle. I've used both with good results. I recommend thermal disinfection (autoclave or p-cooker) for your tools and glassware.

I try to rinse the seed after sterilizing it. I think I really just dilute it a little by adding water. I try to decant as much water as I can, then dump it in or use a dropper.

[gixrj18]

I've found that patience is the best way to fight contamination. When you are soaking (tools, seeds, flasking chamber, etc.) in the bleach water, just leave them in there for an extra ten minutes. Also, for your tools, chamber, outside of the seed pod, and anything going into the chamber, you should use 25% bleach water....only use the 5% on the seeds.

[Becca]

Well I am sad to say that the other parent with the second pod and no problems with the stigma has started to turn yellow at the base of the pod...the stem is still green and the stigma is still green.....but I know what this means...just barely over a month and it has aborted. Now I will be keeping my fingers crossed that the other pod with the stigma that turned brown will not abort.

[vmax3000]

Originally Posted by Becca

Well I am sad to say that the other parent with the second pod and no problems with the stigma has started to turn yellow at the base of the pod...the stem is still green and the stigma is still green.....but I know what this means...just barely over a month and it has aborted. Now I will be keeping my fingers crossed that the other pod with the stigma that turned brown will not abort.

Becca,
I am so sorry. I, too, have experienced the premature death of a pod. Life does take care of itself as it sees fit. I am doing the same for your other pod! It's a fascinating process and the whole thing, even the failed plating and flasking attempts I had caused me to step back and recognize another absolutely stunning aspect of my chids' lives. Hang in there!

[gixrj18]

Vanessa,
When you transfer your germinated protocorms to the jar, are you making the exchange in a sterile box, or laminar flow cabinet? If not...this is your problem, most likely.