Flasked seeds and cells,- NOW WHAT?

[Florida_guy_26]

Ok, So I just today flasked some dendrobium anosmum and den. udom sri seeds. I flasked Cattleya walkeriana var. tipo 'cambara cell samples I took from an older severed back bulb as well. The cells and seeds were placed on agar in my flasking case with the air purifier on so I hope everything was sterile.

I DO NOT have an autoclave/pressure cooker as I just moved and would not know how to use it anyway. For me, micorwaving is the easiest way to do the agar and just do my best to keep contaminants out.

I put everything in the case and sprayed it all with disinfectant including the needles and syringes with cells and the containers full of seeds. But my question now is, what do i do???

I flasked and now do I just cross my fingers and hope that everything is sterile and the seeds are still viable? I am hoping to see some growth soon, but I know it can take anywhere from 2 weeks to 6 weeks for the seeds to START germination and cells might be a bit less. I guess now I have to play the waiting game.

I have the jars inside the flasking case and will keep them there and just use flourescent lights for 8-10 hours a day until i see green in the jars- I hope the good green and not the nasty green. I am keeping my fingers crossed as this will be my last attempt at in-vitro orchid culture- if I do not have luck with the seeds germinating and cells growing, then i give the hell up.

[2ljd]

Originally Posted by Florida_guy_26

Ok, So I just today flasked some dendrobium anosmum and den. udom sri seeds. I flasked Cattleya walkeriana var. tipo 'cambara cell samples I took from an older severed back bulb as well. The cells and seeds were placed on agar in my flasking case with the air purifier on so I hope everything was sterile.

I DO NOT have an autoclave/pressure cooker as I just moved and would not know how to use it anyway. For me, micorwaving is the easiest way to do the agar and just do my best to keep contaminants out.

I put everything in the case and sprayed it all with disinfectant including the needles and syringes with cells and the containers full of seeds. But my question now is, what do i do???

I flasked and now do I just cross my fingers and hope that everything is sterile and the seeds are still viable? I am hoping to see some growth soon, but I know it can take anywhere from 2 weeks to 6 weeks for the seeds to START germination and cells might be a bit less. I guess now I have to play the waiting game.

I have the jars inside the flasking case and will keep them there and just use flourescent lights for 8-10 hours a day until i see green in the jars- I hope the good green and not the nasty green. I am keeping my fingers crossed as this will be my last attempt at in-vitro orchid culture- if I do not have luck with the seeds germinating and cells growing, then i give the hell up.

Don't give up just yet. You will have to play the waiting game at this point though. I've heard of some folks having success with the microwave to sterilize things, but I would really recommend getting at least a pressure cooker. It's not hard to use at all...believe me, that's what I use. I just flasked some Den. anosmum seeds about a month ago and they're already showing their first little leaves.

Quick question...what temperature are you keeping the flasks at?

[Florida_guy_26]

Well I only flasked the seeds and cells today- but I would guess around 80-85F day and 60-65F night. I think normal daytime and nightime temps for this time of year should work fine for them. What temps are you growing at day and night? I hope so far that I am doing things right but I am nervous.

[John D.]

I have had Dends germinate in a few weeks and had other variaties take months. I usually don't discard a flask for at least 9 months unless its contaminated.

[2ljd]

Originally Posted by Florida_guy_26

Well I only flasked the seeds and cells today- but I would guess around 80-85F day and 60-65F night. I think normal daytime and nightime temps for this time of year should work fine for them. What temps are you growing at day and night? I hope so far that I am doing things right but I am nervous.

I've found that the "sweet spot", temp wise, is mid 70's to 80F. I get the best growth in that range. Nights are just slightly lower.

[Royal]

Originally Posted by 2ljd

Don't give up just yet. You will have to play the waiting game at this point though. I've heard of some folks having success with the microwave to sterilize things, but I would really recommend getting at least a pressure cooker. It's not hard to use at all...believe me, that's what I use.

I totally agree here. You can buy one fairly cheap, find them at garage sales, or borrow them from a "mature" neighbor. If you are going through all the trouble and time of micropropagation, proper sterilization is minor and easy step in comparison.

My question is what media and protocol are you using for the cell culture? It sounds very interesting.

[Florida_guy_26]

My media is the western motherflask medium with about 2-5 grams of banana pulp added to each flask to ensure no browing out of protocroms and they also have a small hole in the top with band aids over them if they get to the point where they need air exchange. I wanted to make sure I do the least amount of work and have the plants grow as much as possible if they even germinate. I read that microwaving media for 8 mins is the equivalent of autoclaving for 15-20 mins but I just have not found a pressure cooker and there are no garage sales out west here. I will eventually get one, but for now the microwave seems to keep everything sterile enough. I use a plastic bin you can find at walmart with square holes cut in for my hands to go in, but i glued some cloth over each hole with a slit in the cloth wide enough to get my hands in. I spray everything with disinfecting spray and alcohol so bacteria and viruses and mold should all be killed. I use an air purifier- multitech s1000 with the added benefit of having not just a hepa filter but an air ionizer running on it as well. I think that is enough as there is enough air flow to push air out the holes in the flasking case when it is on low. I should have run it on high but I feel like it just does not filter the air as thoroughly that way. So now i play the waiting game- it has only been about 17 - 18 hours so far since I flasked yesterday. I hope this info helps all of you to understand and thank you all so much for reading and giving me your opinions and advice- as soon as i have the time and money i will buy a pressure cooker instead of microwaving the tools.

[Royal]

I'm not sure that western media will initiate growth from those cells. The seeds should do great but tissue culture requires plant hormones to induce callus formation, proliferation, and differentiation. Cell culture, while great for some plants, isn't a common method for cloning orchids. Most orchid cloning is done with meristem tissue placed on a special multiplication media.

I've had good success working in a homemade glovebox similar to what you described. I don't use an air filter at all, I just work very slowly and deliberately to avoid creating air currents. Seems to work well so far.

Good luck with the seeds and keep us posted.

[Florida_guy_26]

PS- I want everyone to know that i took the cells from the Cattleya Walkeriana var tipo 'Cambara' from a backbulb with a needle and syringe and then added some water and a bit of fertilizer to the syringe as well to keep the cells alive until i could spread them over the medium. I just hope that they grow and keep growing for a month or 2 before I have to replate them. I am praying things went well.

[Florida_guy_26]

I have needed to use an air filtration system inside the glove box because moving slowly and not creating air currents just HAS NOT worked for me. I tried in florida and the mold just was everywhere including the flasks, but so far I do not see any contamination in these jars. I read from kevin and helen western that even though the media usually do not initiate cell growth and callus formation, that it can be used with very average results and has been in the past. I do hope that the cells do at least start to grow and if I need to I can replate them to a different media in the future. I would imagine that people who use home made media still yield some decent results of cell growth and proliferation regardless of the hormone levels because you do not looks at it as a plant but rather a piece of a plat complete with its own cell initiatives and life cycle already programmed in. If I do not get any results from the cells, I will have to order the C3 medium which is designed specifically for catt multiplication and growth. 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 and if the results change, I will keep you informed.