So I'm going to try to do my first stem prop and was wondering how do you get more than one plant?

Will it do it all on its own or is there something special you need to do?


Thanks in advance!

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Judi

Judi it's all about the media you use. The growth regulators in the media induce how much proliferation does or does not happen. I use BAP and Coconut Water in mine. This is my protocol that usually induces a reasonable amount of proliferation without causing any mutation:

Base Ingredients for 1 Liter Final Medium:

20g/L P793 - PhytoTech Orchid Multiplication Medium without Charcoal and Agar
100 ml/L Coconut Water
10 mg/L BAP (10ml stock solution, see below)
4g/L A133 - Agargellan
890ml Distilled Water

Directions:

Add the ingredients to the distilled water and bring to a rolling boil to make sure the Agargellan has dissolved fully. You can then pour the liquid medium into stem prop tubes or other autoclaveable containers. Then autoclave as you normally would any other flasking medium to sterlize them.

Notes:

This brings the BAP level up to 12mg/L, P793 just does not have enough on it's own. You can play around with the amount of BAP. 10mg/L is generally the amount most people use. I've had no problems pushing it up to 12mg/L and I'm usually getting more plants per node. You can also use O139 if you want more control over the BAP. But you may have to add some NA back in.

No matter what basal medium you use, you will have to play with the amount. The recommended amount of 25g/L of the P793 is too hot for me. So I tend to drop down to 20g/L.

I have had very good luck working using Agargellan from Phytotech as my gelling agent. I like it more than the other gelling agents I've tried.

I use a large volume of Coconut water in my medium. The amount of Distilled water above is adjusted for the amount of other ingredients to get you very close to 1000ml of final medium. If you change the amount of BAP and/or Coconut, be sure to adjust the amount of water you add to bring the final volume up to 1000ml.


Directions for making BAP Stock Solution (1mg/ml)

100mg BAP (Benzylaminopurine)
1ml 30% KoH (Potassium Hydroxide)
99ml Distilled Water

BAP will not dissolve in water, so KoH is usually used as the solvent. Add the 100mg of BAP to 1ml of KoH. Carefully stir the mixture until the BAP has fully dissolved. The slowly add the 99ml of distilled water.

At this point I usually place 10ml of the stock solution into small vials and freeze any of them that I am not going to immediately use. Then when you need to make a batch of stem prop medium, all you need to do is thaw one vial of the stock BAP solution.

Thank you so much Rob.

Right now I just want to experiment to see how successful I can be stem propping, proliferation would be the next step. Have to get stem propping down first. Lol

I have O753 from Aaron Hicks which says it contains 2.0mg/L BA. According to your formula this is not enough. Using the O753 do you feel it would be benefical to use say 20ml/L Coconut water (1/5 amount) to be equivalant to the BA? Again not looking for proliferation, just want to make one stem until I get it down, if ever.

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Judi

Hey,

I do it a little different. Busy here, so I haven't had a chance to reply. This one is likely to be brief as well.

I use the O753. It comes from Phytotech. I am guessing Aaron just resells it. I mix it the same way as Rob does his with 900ml of distilled water and 100ml of coconut water, that I also get from Phytotech.

I also add additional BAP, but not as much as Rob. I add 3 more mg for a total of 5mg. I don't bother with the NaOH and the BAP powder. Phytotech sells it already mixed up in a 1mg/ml solution. It is a little more expensive, but probably evens out once you have bought the BAP powder, NaOH, and paid their hazard shipping fee for the NaOH.

I'm not sure how stable BAP is, but I always try to use fresh media. That said, I have gotten proliferation on old media, but I never use media more than a couple months old.

The 5mg of BAP has always worked well for me. I don't always get multiples, but usually I do. Sometimes it is 2 extra. Sometimes it is 40 extra. Trust me, 40 extra is not really a bonus. As far as the difference between Rob using 12 and me using 5... everyone often does this stuff a little different and you might find that 7 or 8 or 2 works better for you.

I am not sure it is mentioned anywhere, but be aware that the BAP inhibits root formation of your new stems. You might get 10 stems, but none will have roots. You have to prepare media without the BAP, chop up your mass of plants into 10 separate plants, and move them to the final media so that they will grow roots. This media can be your basic media such as P668 or P656. Herein lies an advantage somewhat of not using the BAP. You might only get 1-2 plants, but they start forming roots quickly, the plants grow faster, and you often have ready to pot out plants well in advance of stems initiated with the hormones.

There is a hormone that does not inhibit root formation. Phytotech was promoting it a few years back, but we never knew how much to use, and I never tried it.

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Carpe Diem

Thanks for the additional info Ben.

Since you use the O753 have you ever tried it just as it is without any additional BAP or coconut water? If so, what were the results.

Aaron says it can be used just as it is and get stems. After reading both your and Rob responses I'm beginning to wonder.

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Judi

I have never used it without the extra BAP or coconut water. I was taught about the modifications (by Rob and a couple others) early on. I'm sure it will. Give it a try and if you are not getting the results you wanted, you can always change.

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Carpe Diem

Without the modifications you will likely only get one plant per node. It will vary a lot with different phals. Some just tend to produce more plantlets than other do.

Relative to root formation, if I get a lot of plantlets off a node, I tend to take them out of the stemprop media fairly early, cut them up under sterile conditions and then place them out in a replate flask with my normal replate media.

Am I interpeting this right? You can use P668 or 656 for stems as well, but it would only yield 1-2 plants?
So you would only be using the BAP & coconut water to get multiples. Correct?

Reason I ask is I have P668 that I just made up for flasking and if that will suffice for just one plant/stem I think it would be easier for me than trying to deal with multiples , having to cut up the mass and replate into P668 anyways. For my first try I'm thinking the least amount of steps the better.

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Judi

P668 does not have any cytokinin in it to induce the stem to produce a plantlet. You still may get a single plantlet. But some stems may just sit there and do nothing.

Thanks Rob just wanted to be sure.

So save the P668 for flasking and go with the 0753 for stems.

I appreciate the help and all the info both of you have provided.

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Judi

While waiting for the lids to the baby food jars to come the few stems I had turned yellow and started drying up so I only have a stem from a 3N plant.

Are 3Ns' any more difficult to stem prop than 2N or 4N?

Wondering if I should even bother now.

Thoughts anyone?

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Judi

I always stem novelties, so I think 3ns are wonderful to stem because they usually have nice big fat spikes that are easier to work with. They are no different IMO as far as ability to make a stem prop.

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Carpe Diem

That's good to hear. cause if they weren't I'd be SOOL!

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Judi