Micropropagation of Orchid, my final year project - Orchid Board

Bell_bella · #1 10-02-2008, 02:44 PM

well, I need some advice. URGENT. I juz got myself a big pod of vanda dearei last two days. I'm goin to culture it tommorow in my lab. Unfortunately it had matured yesterday. so what should I do for the treatment stage? I mean the sterilization stage. It a bit cracked. I'm worried for contamination. anyone pls?

Swamper · #2 10-02-2008, 02:58 PM

I see you are looking all over for help and I wish I could help from experience. Since I dont knwo first hand , here is a link that may be able to help you. Good luck and keep asking.
Ruben In Orchids Flasking Service you can also email them with Q's, they are very nice and helpfull
Good Luck

Bell_bella · #3 10-02-2008, 03:14 PM

thank u .. its just I don't expect my vanda dearei pod is splitting dat soon..
my seniors ask me not to propagate/culture them if the pod has matured(burst) because it fungus contamination might occuc. but mine is only a bit cracked. so I don't know whether I should just cancel my plan tommorow or not. the pod is quite big and i feel a lost if not trying to culture it. but as the same time, I dun want to waste the media I made earlier coz I have other pods waiting.

Royal · #4 10-02-2008, 03:20 PM

Quote:

Originally Posted by Bell_bella

thank u .. its just I don't expect my vanda dearei pod is splitting dat soon..
my seniors ask me not to propagate/culture them if the pod has matured(burst) because it fungus contamination might occuc. but mine is only a bit cracked. so I don't know whether I should just cancel my plan tommorow or not. the pod is quite big and i feel a lost if not trying to culture it. but as the same time, I dun want to waste the media I made earlier coz I have other pods waiting.

If your senoirs don't want you to sow any seed from a mature capsule (matured and split), then it's too late. A crack, even a minor one, is enough for contamination to occur. But you must have some sanitation equipment and chemicals, right? Seed is easily disinfected. For me, I find it harder to disinfect green capsules. Ask them for permission to use free, mature seed for a "trial" and see if they'll let you. Worth a try.

Swamper · #5 10-02-2008, 03:28 PM

Thanks Royal

Bell_bella · #6 10-02-2008, 03:34 PM

yeah. thats what I heard and been told. "A crack, even a minor one, is enough for contamination to occur". but last early august, I did try to propagate matured seeds of Dendrobium parthenium. It even burst open (matured) I took them and put in an envelope. I culture them :
1. directly put it (the seeds only not the pod) inside chlorox (30% v/v) + tween 20 (2 drops) --in a beaker/blue cap bottle and wait for 20 minutes.

2. Later wash 3-4 times with sterile distilled water. of coz i did this under Laminar Flow Cabinet in school Tissue Culture Lab. I know the chances of survival is very low and contaminatiom is very high. But I did it for trial.

3. Result. after a month, 3 petri dish (plate) out of 6 plates were free from contamination. (other turn green but contaminated)

4. but then at the end, I only have one now that had survived. others severely contaminated. As for the only plate, soon the protocorm will be formed. I'l post the image of it if I can. but I still have problem with attaching image in this forum. I mean it required URL.. kinda difficult.

John D. · #7 10-02-2008, 03:48 PM

The clorox treatment should sanitize the seeds. It is stronger and longer that I use. The problem could be the culture containers. Petri dishes do not seal well, if they are not being grown in sterile area they may contaminate from the slight air exchange. Usually contamination from the seed will show up quickly.

Bell_bella · #8 10-02-2008, 03:58 PM

yeah..I agrree. I use parafilm to seal them..not so promising right. But then, it also the remaining water that cause the cross-contamination easily. I mean, when I poured the seeds on the media, it was still with water. (eventho it is sterile distilled water)

i'm afraid too strong chlorox [ ] might damage the fragile/vulnerable orchids seeds.

Bell_bella · #9 10-02-2008, 04:09 PM

Here it is:

So, shoud I continue with my plan?

Royal · #10 10-02-2008, 04:13 PM

If the water is sterile, that's not the problem. Use the normal strength 6% bleach to make a mixture of at a ratio of 1:10 (10% by volume). That should disinfect most seed types. If they float, they haven't been fully wetted, so they are more likely to be contaminated. Just use some of your Tween.

If you find this isn't adequate, increase contact time - not concentration. This ensures minimal damage to seed. The beauty of collecting dry seed is that if you fail, you always can have more in the refrigerator.