My newest tissue culture specimen and its grow log.
 

Hello everyone i just wanted to share my newest tissue culture specimen with you. I saw this flower and was instantly in love. I am also going to use this thread to document my tissue culturing procedure from start to finish so i felt that it belonged here if not could the moderator please move it to the appropriate forum? Thanks alot! :bowing
http://www.orchidboard.com/community...e_specimen.jpg

I asked if i could have a piece of the stem of this beautiful phalaenopsis and got given two branches :) a total of 4 nodes and 2 stem tips.
I forgot to take pictures while i was doing up these particular nodes but i will add them in when i do my next batch.

Okay back to the actual method. First i'm going to go into my sterilization technique because it is good to start sterilizing your explants before you make you medi as it takes 4 hours.

To sterilize node explants i make up a solution of:
100ml distilled water
.5ml Plant preservative mixture

This gives you a .5% solution of plant preservative mixture which can be used as a non-phytotoxic sterilization mixture for endogenous contamination. To use it simple place your explants in a small container and add just enough solution to cover the plants completely. Leave the explants to soak in this solution for four hours shaking vigorously from time to time to ensure complete exposure of the explants with plant preservative mixture solution.

While your waiting for your explants to sterilize it's a good idea to get started making your proliferation media so that by the time your done the explants will almost be done sterilizing.

My Phalaenopsis node proliferation media for this batch contained the following:
in 120ml of media i added,
1/3 Murashige and Skoog,
1.2 grams sugar,
20ml coconut water (this is the first time i have ever tried adding coconut water to my node culture and am not sure what the outcome will be but i read that it can decrease propagation times and mortality rates so i figured it worth investigating)
100ml water,
.2% plant preservative mixture,
.2mg BAP
.01 mg IBA
.2 grams gellan gum

All ingredients are mixed together in a mason jar and put in a pressure cooker at 15Psi for 35 minutes to melt the gellan gum and sterilize the media.
Once 35 minutes have elapsed the jars are allowed to cool until they have thickened and are cold enough so as not to hurt the explants.
At this point you just have to wait for the explants to finish sterilizing then you just take a sterile pair of tweezer's take the explants out of the plant perservative mixture solution without rinsing and quick place them horizontally in the jars with the nodes sticking out of the media. Seal the jars and place in a well lit room and wait for tiny plantlets to start forming. Once they do transfer them to hormone free media where the will grow to full size and develops roots so that you can plant them into compots in about 8 months.

I will have loads more picture coming soon and be updating this with more info as i go along. So if your interested be sure to check back because this thread is anything but finished
Best regards,
Kevin Disher

That is really different, nice of someone to give you the spikes...good luck...Jean

Thanks Jean :) i'm really excited it is just such a unique flower that i couldnt resist.

oh yea i forgot to mention the plant they gave me the spike from was really sicka nd had severe root rot to the point it was past saving by conventional means, and i am the guy that they have been giving there sick plants to anyways so really they were kind of just saving it and making sure it didnt die. It was incredibly kind of them though :) i couldnt be happier

That flower is beautiful! I'll definitely keep checking this thread to see your progress. Good luck!

one of the explants started to mold :/ i am re-sterilizing it though and hopefully it's ok