Flasked seeds and cells,- NOW WHAT?

[Florida_guy_26]

I wanted to post this so you guys could see why I took pseudobulb cells and tried to clone them. I came across this article on cloning and it is pertaining to orchid culture.

"When cloning plants, one takes advantage of the fact that all plant cells are totipotent cells, meaning that the cells are able to differentiate to non-specific cells and then divide further as such totipotent cells. The cells form a callus. Hormones are used to start and keep up these processes in vitro."

I also got the very small bag of BAP on friday from canada so I hope to start taking cell samples to clone some orchids again soon. I was wondering if anyone here can help me with 1 or 2 things tho- what sized needle is best for taking cells from developing buds/eyes? I am using 25 gauge needle and 3ml syringe but wonder if diabetic type syringes and needles ( 1cc syringe and 28 gauge needle would work as well. I do not know if 28 gauge needles will still extract cells liquid from new buds but I know the needle is less invasive to a new bud/eye. Will taking cells from a new bud or eye kill that lead? I have damaged some buds in the past and they never grew right or even stopped all together. Just wondering if using a needle to extract tissue would do the same thing as cutting up beds or shoots. I mean I know that with meristems, you cut up one bud or shoot and get many with it, but would taking cells with a needle stop the bud from growing?

[Royal]

Originally Posted by Florida_guy_26

I also got the very small bag of BAP on friday from canada so I hope to start taking cell samples to clone some orchids again soon. I was wondering if anyone here can help me with 1 or 2 things tho- what sized needle is best for taking cells from developing buds/eyes? I am using 25 gauge needle and 3ml syringe but wonder if diabetic type syringes and needles ( 1cc syringe and 28 gauge needle would work as well. I do not know if 28 gauge needles will still extract cells liquid from new buds but I know the needle is less invasive to a new bud/eye. Will taking cells from a new bud or eye kill that lead? I have damaged some buds in the past and they never grew right or even stopped all together. Just wondering if using a needle to extract tissue would do the same thing as cutting up beds or shoots. I mean I know that with meristems, you cut up one bud or shoot and get many with it, but would taking cells with a needle stop the bud from growing?

I'm still wondering where you got the whole syringe idea. The meristem in a developing growth is enveloped in layers. Picture the cross -section of an onion. To obtain that center piece, you must peel the layers back. That one little piece is so small in relation to the whole that the chances of you hitting it with the tip of a needle are small. Plus, from all that I have read, the entire meristem is taken, not just meristem cells.

I would suggest starting with established standard protocol, then adjust from there. Then maybe you will have some measure of success along the way instead of being met only by frustration.

If you where able to extract the growing tip out of a developing growth with a syringe, the growth would be damaged and would not form a pseudobulb.

[Florida_guy_26]

I read from numerous articles that any tissue from a developing bud/eye can be used, even the simple epithelial cells that cover or sheath the growth. I do not know if that is right, but I guess that is part of trial and error with these little b*tches, so I will continue to try simple cell samples and see if any of them work. I did read that even the epithelial cells that make up the sheath around the meristematic tissue itself are totipotent and would yield a good, stable, regulated growth with the right amount of cytokinins and auxins for proper root and stem growth as all the tissue has the same hormones circulating through all of it. I can try using the sheath tissue and see what that yields and if it does not work, then I can take cells from dormant buds or whatever. I do not want to risk royally wrecking a plant just to make a few new little ones. I will try using the syringe and needle to extract the cells that are totipotent and see what that gets me. I did also read that cells near the apex of a stem and the leaf petiole and even root tips have totipotent cells that can produce new plants. Let me know what you have read or heard also. it does help.

[Connie Star]

Hi Florida guy,
I've been following this with interest- I have a master's in Botany from U of N. Carolina and I worked with tissue cultures for my thesis research.
Totipotency was demonstrated with carrot cells originally. While it theoretically applies to all cells of all plants, that doesn't mean it's easy. The process that has been developed for cloning orchids was arrived at with a little theory and a lot of trial and error, mostly error.
The cell cultures I worked with were "tumorized" and relatively very easy to grow but exacting requirements still had to be met.
Keep us posted on how it's going. I would suggest keeping careful research style notes.

[Florida_guy_26]

I already am- I am just doing much trial and error and I am happy to see that you are as interested in this as I am. I did read all the same things about most cells of a plant having totipotency and the ability to grow from even leaf vein cells. I did read that the apex of orchid stems, canes, or pseudobulbs do contain cells that will generate new plantlets as the cells of roots and even leaf sheaths will as well. As far as I have read from most postings on the web and even in some books, there is the possibility that many types of cells in orchids will generate callus or calli, but the only sure parts are around the leaf base, root tips, apex of bulbs, and lastly around the new buds or eyes or shoots or whatever people want to call them. I will try extracting cells from the new buds as those cells have the right mix of cytokinins and auxins but the basic theory or proven flasks have all used simple epithelial cells which is what most plant cells technically are. You just have to find a spot where the cells can be extracted and will be totipotent. I will try smaples from different parts of the plants- i just do not want to damage any plant beyond repair. I will keep records, but so far, just normal cells from the pseudobulb of the catt walker did not grow but were contaminated by the fluid used to spread them. Hope that helps guys.

[Connie Star]

Hello again florida- I don't know why but I get the impression you are a medical doctor? I am too. Or, are you a research scientist? This stuff still fascinates me after all these years. I want to get into hybridizing and flasking when I retire. I've just gotten my feet wet with a flask of "baby orchids" that I bought in Costa Rica. So far, so good but much can go wrong.

[Connie Star]

By the way, I've only been growing orchids for a year- up to 50+ you get hooked fast. I'm a senior member just because I post a lot, not that I am an expert so take what I say with a grain (65mg) of salt.

[Florida_guy_26]

Sorry to disappoint you Connie, but I do not even have a normal 4 yr degree in plant biology or botany and I hope that does not change your opinion of me. I do however have years of growing experience with not just orchids, but also ficus and other members of the Morus genus like mulberry trees, some other exotics and cactus. I have learned much just by trial and error but my mother was very smart and showed me grafting techniques for pairing branches of oranges trees onto magnolia trees. I learned alot just from what she taught me about plants but also n my own as I have had orchids since I was 15 off and on. Anyway, I did not have luck with cells from the catt walker pseudobulb so I will try the leaf base and see if any cells around there can generate new plants. If not I will try a root tip and see and if not, then I will try the bud/eye tissue as one breaks. but I will try 2 methods- taking cells just with a syringe to see if single cells can become protocorms or if the whole bud is required. I will post notes periodically on here about it. and maybe collaborate with anyone else who would like the info.

[Connie Star]

Not disappointed at all! An inquiring mind is the best asset for a citizen scientist or anybody for that matter. I'm very curious to learn how this all works out.

[bodaciousbonsai]

floridaguy. just because you do not have a 8 year degree in bontany does not mean you havent done enough research to be a well known name in the plant world. I myself am in the works of growing orchid seeds through mycology a.k.a. mushroom fungus. Mycology is the World Wide Web in nature.( Paul Stamet. I am currently growing spores from a moonlight mushroom & saprophytic mushrooms. I believe orchids are very in tune through symbiosis through out the entire growth cycle of the orchid. All I have is a G.E.D ( good enough diploma) and a 2 year degree from B.O.C.E.S. in conversation. I have been growing orchids for about 15 years now and am just starting to get to the good stuff. I love brain food. lol, You do not need a doctrine in bontany to go advanced. I also owned a coral reef tank for 20 years. Everything in a coral reef is very complex and this has alot to do with symbiosis.