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By jkochuni
Posts:  625
Joined:  Sun Mar 22, 2009 8:19 pm
#13430
Below are photos of my progressing TC experiments...

Sawtooth

Image

Dingley Giant

Image

Pink Venus- Callus is forming rapidly, will probably develop plantlets soon

Image

Fused Tooth

Image

Yellow- You can see the callus starting to develop under the medium

Image

Jaws- Might have a callus starting to develop

Image


I also have stable stalks in TC for the following (no signs yet of calluses)

-Crossed Tooth
-Fuzzy Tooth
By kevinqwe
Posts:  1840
Joined:  Tue Mar 31, 2009 3:04 am
#13432
looks good. hope to see more development on the last pic.
By jkochuni
Posts:  625
Joined:  Sun Mar 22, 2009 8:19 pm
#13444
Matt- Write up your entire process for culturing stalks and PM it to me. I'll check over it to see if we have any differences in our process.
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By Matt
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Posts:  22523
Joined:  Mon Apr 21, 2008 11:28 pm
#13448
jkochuni wrote:Matt- Write up your entire process for culturing stalks and PM it to me. I'll check over it to see if we have any differences in our process.
Sounds good Jason. I'll do that soon. Yes, I use 1ml of BAP in most of my cultures, but not all. Are you using BAP?
By jkochuni
Posts:  625
Joined:  Sun Mar 22, 2009 8:19 pm
#13450
All the ones pictured above, except the Jaws culture, used no BAP. My new cultures (Fuzzy tooth and Cross Teeth) are split between medium containing BAP and medium containing no BAP. It will be interesting to see which develops calluses sooner.
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By Steve_D
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Joined:  Tue Nov 18, 2008 5:06 pm
#13472
jkochuni wrote:Matt- Write up your entire process for culturing stalks and PM it to me. I'll check over it to see if we have any differences in our process.

Please post the results (the differences) so that all of us can learn, OK? :D Although I don't currently do tissue culture, I find the subject very interesting. :)
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By Matt
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Posts:  22523
Joined:  Mon Apr 21, 2008 11:28 pm
#13475
Steve_D wrote:Please post the results (the differences) so that all of us can learn, OK? :D Although I don't currently do tissue culture, I find the subject very interesting. :)
You bet Steve. Here's my procedure:
1 liter of media is made with
- 1/2 strength MS with full strength vitamins
- 1 ml PPM
- 30 g table sugar
- 1 ml BAP
- pH 5.6

Sterilization:
1) Wash with antibacterial soap (similar to Dial Complete) that has 0.6% triclosan.
2) Rinse under running water for quite a while (usually around 30 minutes)
3) 10 to 20 second dip in 90% alcohol.
4) 5 to 8 minutes in H2O2
5) 8 to 10 minutes in 10% to 20% bleach (I vary it and lately I've been doing mostly 20%)
6) Two 3 minute rinses in sterile water

Jason's:
Media is essentially identical to mine.
-30 seconds in 70% alcohol
-5 minutes in H202- 3%
-6 minutes in Bleach- 10%
-5 minute rinses in 3 separate sterile water containers

Basically I think I'm over sterilizing a bit. Though the stalks don't die on me, they just don't form callus. I'm planning on cutting my sterilization time down to match Jason's. In fact, I used to do shorter soaks in the H2O2 and bleach, but I still didn't have any flower stalks strike other than up near the pedicel.
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By Steve_D
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Joined:  Tue Nov 18, 2008 5:06 pm
#13494
Thanks for posting the differences in sterilization protocol. When you rinse in sterile water, do you rinse only one bit of cut tissue at a time, and do you throw away the sterile water after rinsing something in it? Does one need to make quite a lot of sterile water? Should the flowerstalk or other tissue be left to soak in the sterile water, or should it be agitated? :)
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By Matt
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Posts:  22523
Joined:  Mon Apr 21, 2008 11:28 pm
#13496
Steve_D wrote:When you rinse in sterile water, do you rinse only one bit of cut tissue at a time, and do you throw away the sterile water after rinsing something in it? Does one need to make quite a lot of sterile water?
I usually do pieces of tissue in "batches" and I'll rinse an entire batch in sterile water, but I won't reuse the same sterile water for another batch. It's one and done with the sterile water. And yes, one does need to make a fair amount of sterile water.
Steve_D wrote:Should the flowerstalk or other tissue be left to soak in the sterile water, or should it be agitated? :)
I usually agitate it the same way I do when it's in the H2O2 and bleach.

Good questions!
By jkochuni
Posts:  625
Joined:  Sun Mar 22, 2009 8:19 pm
#13498
Steve_D wrote:Please post the results (the differences) so that all of us can learn, OK? :D Although I don't currently do tissue culture, I find the subject very interesting. :)
I definitely will. In fact I just pressured cooked all my media this evening to culture all the stalks you sent. I did half in plain medium and half using BAP. I should have enough cultures going now to give us a definitive answer in 5/6 weeks.

On a side note- A bit of good news...I have been able to trade for a Bohemian Garnet flower stalk, and I also have been able to acquire a flower stalk from a Dracula VFT. They both should be here next week, and I really, really hope I can get these going.
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By Matt
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Joined:  Mon Apr 21, 2008 11:28 pm
#13500
jkochuni wrote:On a side note- A bit of good news...I have been able to trade for a Bohemian Garnet flower stalk, and I also have been able to acquire a flower stalk from a Dracula VFT. They both should be here next week, and I really, really hope I can get these going.
I hope so too! Good luck with both of them, especially the Dracula!
By jkochuni
Posts:  625
Joined:  Sun Mar 22, 2009 8:19 pm
#13501
I'll keep my fingers crossed. Hopefully our order comes in soon, so I can get my trade sent off.
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