Flasked seeds and cells,- NOW WHAT?

[Royal]

Originally Posted by Connie Star

An inquiring mind is the best asset for a citizen scientist or anybody for that matter.

[Florida_guy_26]

Ok everyone, I have taken very small cell smaples from the newest growths on the dendrobium pierardii memoria katherine hatos and the Catt walkeriana 'estrela da colina' ( star of the hill ) using the newest tissues at the base f the breaking eye or newest lead in the rhizome. I have spread them along with a very small amount of seeds from den nobile wave king on 2 flask. I have notes but about the only thing I can do is thoroughly explain my process and results. So far, the flasks have maybe under 1- 1.5 grams medium /100- 120 ml of water and about .3 to .8 g of banana pulp freshly frozen and added. The gel was reconstituted using my microwave for roughly 3 minutes on high setting and then left to cool in the flasking case. I sprayed alcohol and disinfectant on everything including syringes and needles and even used hand sanitizer rubbed on the plant tissue to disinfect any outer contaminants. The syringe was sprayed again with alcohol and let to dry in the flask case while i worked on opening the jars and getting my tools ready to go. Cell samples of the plants were spread on a combination of motherflask medium and a small amount of W2 mixed together and the addition of banana pulp as this has all the nutrients for plant starts and should not require replating for around 6 weeks at least depending on growth. I will check both flasks periodically and check for cell replication/ growth and contamination every day. I will keep you all informed if anything happens at all. Thanks for following my experiment and lets all pray for sterile flasks and some great results.
-kevin-

[Florida_guy_26]

Ok - for anyone who was curious to see if the experiment worked or was successful, it was not. I spread the cells and things seem to be sterile in the jars- thank god, but there is absolutely no sign of cell growth. I am patiently waiting but so far no seeds are germinating and no cells are growing. I thought it was almost a given that meristematic cells from the base of the newest growths would start replication and proliferation, yet nothing has really worked so far. I will try cell samples from the leaf base (petiole) area and then the only other option for totipotent cells was root tips or apex of bulbs. I will keep the everything separated and labeled as I try other options of cell cloning, but this was just a follow up for anyone who was been following this thread. Thanks to anyone and everyone who has shown interest and I will post more results as they come. Thanks- kevin

[King_of_orchid_growing:)]

Sometimes the leaf tips of newly grown shoots have enough meristematic tissue to clone.

What kind of media are you using?

I find it unusual that you're not seeing even raised bumps on the surfaces of any of the tissue you've tried to clone.

How are you collecting the samples?

What's your method of disinfection?

Perhaps these are the things you might want to think about and readjust.

Are you growing them under lights?

[rogerman]

Maybe the time frame is wayyyyy too short...... Even for seeds here in Thailand to germinate in a professional lab it takes months.....Takes nearly a year from seed pod to getting them back from the lab to grow.

[Florida_guy_26]

Hey King of orchid growing- nice picture of the venus slipper. To answer your questions, 1 I am using a combination of motherflask medium and W2 from the western formulas (equal parts of each). The medium is mixed with about 3-5 grams of banana pulp freshly blended (with a little bit of the skin also) and then frozen. I only break up pieces of frozen pulp so i can mix them into the powder and water. I think 3-5 grams of pulp is ok to use and will not inhibit growth very much. I explained my explant samples in previous posts, but again I will explain it. First, I start by spraying everything with alcohol to disinfect the area of the plant i will take the tissue from, then I sterilize the needle and syringe. I take the newest growth or lead as the eye is literally very small ( catt walkeriana only had the start of 1 leaf coming up from the bract or sheath and the den pierardii katherine hatos was still just an eye that had swollen away from the base just a bit. I took meristematic tissue from the base of each new eye and the tissue was soft and very light. I pulled on the syringe until some liquid actually came away from the base and used a 25 gauge needle and 3ml syringe. I also got some very light tissue that came away with the needle so I immediately put everything in the flasking box. The filter was running for roughly 15-20 mins. and I used some more alcohol to disinfect the syringe so I could smear the cells onto the medium without contaminaton. I spread the cells with a small amount of sterile water and when I check the flasks, the water seems like it evaporated, but it left behind this light film on the medium. So far, there is an orangey- pink looking spot on the medium but nothing looks like contamination. I used hand sanitizer on the new growths but just enough to cover one side of the growth to sterilize it. I must have damaged the den pierardii because the mass that I extracted from did not continue to grow. So there you have my method of cell collection and application in each flask. Some of the tissue in my opinion should have grown but maybe it is too early to tell. The hand sanitizer seems to disinfect well as the cell samples do not appear to be contaminated so far. It also does not seem to hurt the plant that has tissue taken from it. I do think the samples I took should be viable and totipotent to the point of generating a new plant or at least new tissue. I did not take cubes of meristematic tissue as it disrupts the plant it is taken from and destroys one or more eyes as they break. I will try taking cells from leaf tips, leaf bases where they attach to the bulb or cane, and then apex bulb tissue and finally root tip tissue. I would much rather use root tip tissue than a whole eye or more. I will let everyone know if anything changes but so far, it has been about 2 weeks and still nothing. Anyway I will keep everyone posted if any other samples work. Thanks again guys and girls.

[King_of_orchid_growing:)]

1. I think you'd probably get better results from using a multiplication medium rather than mother flask medium or replating medium for cloning. Perhaps a combo of the mother flask medium and multiplication medium can work.

2. In my opinion, I think you'd get better results from taking an entire eye than just cells. You don't know you you're damaging the cells or not.

3. Root tips are fine, but according to what I've read, the best results come from those that are actively growing.

4. Like I said, meristematic tissue can sometimes be obtained from newly formed leaves on a new shoot.

5. If you're not getting the results you desire, it's best to play around with the concentration of the hormones you've got. You can always add more in the flask, but every time you do, you've got to make sure your work area is sterile.

[King_of_orchid_growing:)]

As for the seeds...

I don't recommend growing seeds in the same flask that you're attempting to clone stuff with (if that's what you're doing, if not, then ignore this).

It may also be that you're not giving them enough time to germinate. Like rogerman had mentioned, it can take up to a year.

[Florida_guy_26]

Right Philip- Like i said before, I do not have any type of replication medium, but I talk to kevin and helen western and have asked numerous questions about the medium. The only medium they seem to have for replication works best on catts and some of the catt hybrids and alliance. They do not have anything else specific to any species or species targeted replication medium other than some for catts and some for phal - meristem propagation that is for bloom stem cuttings. I will take samples from other cells and see if any grow, but I will also see if they have any kind of cell replication media unless you can refer a kind you use? Or if you know of any? I will keep trying and see. Thanks for the info.

[King_of_orchid_growing:)]

Have you checked here?

Plant Growth Research & Development | PhytoTechnology Laboratories

From what I've found, there are no specific multiplication media for each genera of orchid. There are just a few formulations designed for orchids that the industry feels the majority of the sales are in, namely Cattleyas and Phalaenopsis, perhaps a few others too, idk.

Generally speaking as long as you have multiplication medium of some kind, you're on the right track. From there you just play around with more agar or less agar, more x,y, or z hormone or less x,y, or z hormone.