Phalaenopsis Stem Propagation or Seed Pod

[Ki6bud]

Question: When replating the stem due to phenolic build up in the media, do you sterilize the stem with node in a 10% bleach solution prior to transferring it to a new flask or do you transfer it direct?

[dansyr]

For books, I'm not really sure, I put my protocols together from reading a mixture of online forums and academic journals. One "book" I found with some useful reference info was a Springer Protocols ebook edited by Thorpe & Young, Plant Embryo Culture. You might need a library to access it, I think it's behind a paywall.

For passing the nodes, don't resterilize as that will add further stress. Unless it is contaminated and you're trying to rescue it. Otherwise, just make a sterile vessel with fresh sterile media and plop it in (make sure to handle it only with sterilized forceps btw).

As to when, if the whole thing is dark and opaque, you definitely missed the window, but hopefully it's just slowed down some. As to how much gray you're OK with, I think it's a matter of how much time you have available and how much media you're willing to spend. Others may have a more definite answer. Flasking is my side hobby and I have quite a bit else going on, so it's more of when I get some spare time, I pass everything that I can. In general though, I aim for not letting it get to the point where it's black-dark around the node or the cut end of the stem. But again, the solidity of your media affects your rate of diffusion, so my specifics will very likely NOT be representative of your specifics. You'll get a feel for it with time.

[Ki6bud]

Thank you for your help. The media is still in the early stages of phenolic build up in the media so I have some time to "pass" the nodes. I would like to experiment on one of the four stems and try to create multiple shoots. I read that if you cut off the top 1/3 of the shoot with the stem intact, it produce more shoots. I also read to remove the shoot from the stem and "pass" it to new multiplication media. Any thoughts?

[dansyr]

Yep, have done both. At first, I usually passage flower stalk + new growth. Then as I start getting some nice undifferentiated growth at the base and some little leaves starting to pop up, I'll cut in roughly half, top and bottom, make sure you leave enough of the undifferentiated basal tissue on both sides. And put them both in contact with fresh P793 media and you'll usually get production of more PLBs. Which you can divide and subcultivate further, etc. I suggest you try wading through some papers on the topic (google scholar "phalaenopsis tissue culture" or "phalaenopsis micropropagation"), they'll offer a lot more of the specifics and details you'll be wanting to know, more than I can give you here.

[Ki6bud]

If the phalaenopsis stem in vitro exudes phenolics in the media, will the new leaf tissue on the bud (if used to produce PLB's) also exude phenolics?

[dansyr]

Originally Posted by Ki6bud

If the phalaenopsis stem in vitro exudes phenolics in the media, will the new leaf tissue on the bud (if used to produce PLB's) also exude phenolics?

You'll want someone with more experience than me here. My experience with thin sectioning leaves is minimal and marked with mediocre success. It's a combination of how appropriate your media is balanced to the leaf, how kindly you cut your section, etc. At least in my experience. More seasoned mass-multipliers may have better tips.

Since I'm usually aiming for smaller yields per orchid, I often just laterally section the base of the growing plantlet and place on fresh P793 agar. Usually get a couple new shoots off the remaining callous/meristem on the stem's side, and the callous/meristem base of the plantlet cut off usually pops out a couple other ones too. And you can repeat. Certainly not as crazy of a multiplication factor as doing the thin sections and fancy liquid setups, but it's worked for my purposes.

[Ki6bud]

My shoots are 21 days old and looks to be developing leaves. Question 1: At what size would you transfer them from the multiplication medium to the rooting medium? Question 2: When you transfer them to the rooting medium do you separate the shoot from the stem? Question 3: Can you separate the shoot from the stem and place it back in the multiplication medium?

[dansyr]

Expect the whole process to take ~1 year before you can have plantlets ready to be deflasked so don't be too eager to do stuff Plantlets are happier the less you mess with them, so err towards needing a reason to change before you do anything. Much like caring for adult orchids if you think about it...

Question 1: If they're happy, agar isn't dried out or phenolic, & plantlets can easily fit out of the neck of your jar, leave 'em be, especially if only 21 days. If you're looking to multiply in vitro, that has some timings you should look up/read about, otherwise, let em stay in P688 and see how many spontaneous shoots you get, and when it looks dry or the plant size is getting large, put them in new media. If you were to transfer immediately into P668, you wouldn't get roots and suddenly a ready-to-go plantlet. p793 is about nutritionally equivalent to P668, P793 just also has some hormones that encourage certain meristem activity. So it's not holding your plantlets back, certainly not after 21 days. Assuming the P793 is prepared correctly.

Question 2: I usually don't worry about removing the stem unless dividing as described earlier in this thread or the stem introduces problematic geometry.

Question 3: Kind of (not the stem, the stem is old, differentiated tissue, but if you leave callous or meristem on there, that can do stuff). Depends on your goals. See earlier posts in this thread talking about this, or let me know if they weren't specific enough and I can point you to some google scholar search terms, etc or else provide some clarification.

[Ki6bud]

I found a very interesting article on inhibiting the oxidation of phenolic compounds.

Inhibition of Phenylpropanoid Biosynthesis in Artemisia annua L.: A Novel Approach to Reduce Oxidative Browning in Plant Tissue Culture

[Ki6bud]

I have read many articles on the issue of phenolic oxidization and I have also contacted the supplier of my multiplication medium. The manufacturer stated that the synthetic cytokinin they use in their medium will combine with activated charcoal which will defeat the purpose the medium. I also read that the addition of ascorbic acid to the medium will reduce the phenolic oxidation significantly. I am waiting for the manufacturer to respond to my question if the ascorbic acid will interfere with their formula.