Stem Propagating Phal In class

[Htsorchid]

I hope that the pictures come through. In my Plant Propagation class we finally arrived at tissue culture. In the lab we were told to get a cuttings from the schools greenhouse. I have always want to learn more about growing orchids and had once planned on trying to germinate orchids from seed. I decided I would cut off the stem of a Phalaenopsis in the Greenhouse and give it a go. The rest of my classmates were using cutting from Wandering Jew and Bridal Veil and I thought them way to easy to propagate.

Most of the Orchids in our schools greenhouse don't have names and are NOIDs and when they have plant sales on Fridays they really don't care much about the species name. Just that they're pretty and that they're tempting to buy.

We used four different medias, one is MS Media and one is plain Agar Base media and then I think one has fertilizer as well. The last one as you probably can tell, just has activated charcoal in it. As for the others I can't remember which has MS media and which is just plain Agar. So I will have to look at my class notes to tell you which is growing the best and which media had the greatest success.

The the other picture of the mason jars are my own experiment of stem propagating. I just got a Keiki that I bought and it had part of the stem with it. SO I thought why not just Stem Propagate it! It look like the stem was still alive and so I hope that it will work. I will post more pictures as things progress.

And of Course since this is my first attempt...Wish me Luck!

[Zoi2]

What a cool experiment....GOOD LUCK!
Keep those pictures coming, can't wait to see your success.
Joann

[Ben Belton]

Good luck! How did you attempt to sterilize your stems?

[Blueszz]

Oh I wish I could do this at home
Good luck and show us pictures when you have something to update!

Nicole

[stefpix]

Can you describe the process of tissue culture?
do you get one plant per piece?
how does a plant grow from a piece of stem?

[Htsorchid]

To start off! Happy Thanksgiving!

I hope that I can explain this well and if not i might just share the lab note or the instructions to repeat the experiment.

The Media I found out are as follows. White Lid-MS media, Blue lid-MS Media with BA(Benzyladenine;Cytokinin), Red lid-MS Media with 2, 4-D(herbicide, Auxin), Black lid-MS media with activated Charcoal.

The Lab Procedure is as follows:
1-Collect 5 nodal sections froma plant of your choosing and store in RO water.
2-Soak in 70% Ethanol for one minute
3-Soak in Bleach(concentration is dependent-However my teacher suggested 1.5% Bleach)and then add .1% Tween 20. Leave and shake in solution for 10 minutes
4-Move to flow hood and rinse sections three times in sterile distilled water
5-Store in sterile distilled water
6-Place forceps and scapel in Bead sterilizer for 3 seconds(its like 250 degrees)
7-Remove and place across petri dish to cool
8-When cool enough that you can touch tool on back of gloved hand for 3 seconds, proceed
9-Place one section on sterile petri dish with the forceps
10-cut back the burned(white) end of the stem
11-Place the sample in the middle of a test tube of media #1 with the section about 1/2 way into the media(I just push it in tell its node touch the media)
12-Repeat steps 6-11 with medias 2-4
13-warp with parafilm, and label with initials
14-place all four test tubes in a rack in the growth chamber
15-Record any contamination and plant observations each week for three weeks

Well that's our lab and I have checked on them three days later and no contamination. Fingers Crossed, I did notice that some other peoples test tubes with wandering Jew and Bridal veil had that brown out stuff in the agar by the stem in the tube. I think they will die, but mine have no browning or discoloring of Agar.

The Test sterilized the Test tubes for us in the Autoclave and had them at an angle so when the agar set it would not pool water in the bottom. Pooled water can Kill or damage the stem that you cut.

When it comes to phalaenopsis all you need is a Old flower spike or Inflorescence with nodes on it. You have to check to see that they are still alive and that the meristem inside is alive. The best way to tell is if the cover is still alive and if so the meristem is alive. As well for every cutting or stem you cut that has a node on it can be turned into a new plant.

I hope this gets it all and if not, I too am learning how to do this and I hope the sterilization process works.

[Ben Belton]

When I do stems, they seem to contaminate within the first week or at about 3 weeks. I don't know why, but that seems to be the pattern.

I soak mine in 15% Clorox solution. Your teacher said 1.5%. That's interesting. I think Clorox is actually something like 3% sodium hypochlorite, so maybe that is what your teacher was referring to.

For anyone interested in doing this, if you don't have Tween 20, you can use regular liquid dish soap.

Good luck with your stems! Let us know how it turns out!

[John D.]

Regular Clorox is 6% sodium hypochlorite so 1.5% is one part Clorox added to 3 parts water, or a 25% Clorox solution. That's on the high end of concentration commonly used for disinfection of stem props. Ben's 15% is one part bleach added to six parts water, works well with less chance of damaging the tissue. 10% (one part bleach + nine parts water) is often reccommended for seed disinfection.

[stefpix]

thanks!
And what is the result?

Like keikis growing from the stem nodes?
1 plant per node?
stefano

[Ben Belton]

It depends. Sometimes you can get many plants per node. I can take some pics when I get home if anyone is interested.