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Tissue Culturing & Other Propagation Techniques of Banana Plants This forum is for discussing propagation techniques of banana plants. Tissue culturing is the popular process of creating clones from a source plant. There are other techniques to propagate banana plants however, such as nicking corms or dividing corms. Learn more inside.


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Old 08-11-2012, 11:09 PM   #1 (permalink)
 
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Default 1st time Tissue Culturing with pictures

So I dug up a bunch of banana plants today, not sure what they were but I didn't mind chopping them up as this is my first try at TCing.

Cleaned off as much dirt, then put them in the sink to cut off a couple layers.

cut them down pretty good and tossed them in a jar of 10% bleach and RO water solution for 10 minutes to sterilize. Then put them in another bottle of water to rinse, then another bottle after that to rinse as well.

Moved said bottle of water into the Ghetto Gas Chamber for slicing and putting into medium.

Pulled them out of the bottle, placed them on a drying medium to absorb water, then sliced off some tissue to remove the poisoned sterile tissue with bleach.

Cut each tissue center with the meristem into quarters and placed into one batch of baby food jars sterilized at 15PSI for 1 hour in my pressure cooker.

Second batch went into a sleeve of petri dishes. I think I like doing this in petri dishes better than baby food jars. at least this part of it. The rooting part will be done in baby food jars.

The petri dishes were sealed shut with petri dish wrap so I can move them out of the hood to place into a sterile tub container.

Next is to wait a week or two to see what happens, unless somewhere my sterile procedure fails and I get contamination before they shoot.

BTW anyone else use the medium from phytotech? mine didn't fully solidify like agar does, is that supposed to do that? or is it supposed to solidify? If so something is wrong here.

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Old 08-13-2012, 11:51 PM   #2 (permalink)
 
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Default Re: !st time Tissue Culturing with pictures

WOW, it's only been 2 days and already what looks like new little shoots coming off the sides of my meristems. Some are also starting to turn a little green. This is crazy. Pictures in a day or two more after they get a little bigger.
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Old 08-14-2012, 04:38 PM   #3 (permalink)
 
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Default Re: !st time Tissue Culturing with pictures

was curious if anyone could tell me if I should have my TCs under light or filtered sunlight?
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Old 08-15-2012, 02:36 AM   #4 (permalink)
 
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Default Re: !st time Tissue Culturing with pictures

well, gotta redo my TCs. I forgot to sterilize my RO water and I have contams. However I do have some growth too. Will be doing it all over again tomorrow....errr later today...
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Old 08-24-2012, 03:14 PM   #5 (permalink)
 
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Default Re: !st time Tissue Culturing with pictures

What's RO water?
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Old 08-24-2012, 03:30 PM   #6 (permalink)
 
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Default Re: !st time Tissue Culturing with pictures

reverse osmosis
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Old 08-24-2012, 05:22 PM   #7 (permalink)
 
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Default Re: !st time Tissue Culturing with pictures

It aint easy being green !
But pretty soon youll be putting up all kinds of tissue cultures wondering how come every one else aint doing it.....
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Old 09-01-2012, 10:56 AM   #8 (permalink)
 
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Default Re: !st time Tissue Culturing with pictures

Well my third go at TC, and I have contamination in ALMOST all my jars, but a few are clean still. Did as most people say, soak in 10% bleach solution 10-15 minutes before placing in hood and rinsing with sterile water. Also not getting growth either. Using the banana tissue culturing medium from phytotech.
At least in my first attempt I was getting some growth, see here:
Some questions: Psuedostem, Meristem, where?

maybe someone could answer the question I posted in that post as well.

Here's the contamination I'm getting:


even though I 'm using a hood I use for mushroom cultivation, I'm still getting contams even though I am soaking the TC's and then slicing away the outer layers after rinsing in sterilized water.

I did however get some green going on this male flower :


this jar has no contams, but has some green going on.
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Old 09-01-2012, 11:24 AM   #9 (permalink)
 
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Default Re: !st time Tissue Culturing with pictures

Your previous linked post shows you are using the correct part of the plant.

As for the contamination. Could be anything.

Do you add a surfactant to your bleach solution, like tween 20? it reduces the surface tension and allows deeper penetration.
Do you rinse 3 times in sterilsed water?
I used a dip in 70% Iso Propyl alcohol before cutting the meristem in the hood.
Is the hood sterilised? Spraying with IPA before use is a good idea.
Do you sterilise your tools before and after each use? They should be run through a spirit lamp or dipped in 70 % IPA before and after each use.
Did you wash your hands and forearms?
Your arms should be bare so as not to introduce anything from your clothes.
You should disinfect your hands with an alcohol based hand cleaner or wear sterilegloves (whichever is the cheaper)
Do you add PPM to your medium? Plant preservation material is a broad spectrum microbicide and will help combat mild contamination. It should be added at 1 mg/l.
Do you use sterile paper towels. Cutting should be done on sterile paper towels.

I said it could be anything.
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Old 09-01-2012, 12:06 PM   #10 (permalink)
 
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Default Re: !st time Tissue Culturing with pictures

Quote:
Originally Posted by jmoore View Post
Your previous linked post shows you are using the correct part of the plant.

As for the contamination. Could be anything.

Do you add a surfactant to your bleach solution, like tween 20? it reduces the surface tension and allows deeper penetration.
Do you rinse 3 times in sterilsed water?
I used a dip in 70% Iso Propyl alcohol before cutting the meristem in the hood.
Is the hood sterilised? Spraying with IPA before use is a good idea.
Do you sterilise your tools before and after each use? They should be run through a spirit lamp or dipped in 70 % IPA before and after each use.
Did you wash your hands and forearms?
Your arms should be bare so as not to introduce anything from your clothes.
You should disinfect your hands with an alcohol based hand cleaner or wear sterilegloves (whichever is the cheaper)
Do you add PPM to your medium? Plant preservation material is a broad spectrum microbicide and will help combat mild contamination. It should be added at 1 mg/l.
Do you use sterile paper towels. Cutting should be done on sterile paper towels.

I said it could be anything.
I do all of those as I do mushroom culturing for gourmet and edible mushrooms EXCEPT:

"Do you add PPM to your medium? Plant preservation material is a broad spectrum microbicide and will help combat mild contamination. It should be added at 1 mg/l."

and
"Do you add a surfactant to your bleach solution, like tween 20? it reduces the surface tension and allows deeper penetration."
and
"Do you rinse 3 times in sterilsed water?"

I rinsed once, I don't know what a PPM is, can you send a link?
and the tweeter heard of it.

as for the other post with the pictures. can any part that has merristem be used or does it have to be the very middle where the "growing point" is? or is the entire merristem considered the growing point?

Thanks Jmoore!
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Old 09-01-2012, 02:03 PM   #11 (permalink)
 
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Default Re: !st time Tissue Culturing with pictures

So do the male flowers work ?
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Old 09-01-2012, 03:10 PM   #12 (permalink)
 
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Default Re: !st time Tissue Culturing with pictures

Quote:
Originally Posted by sunfish View Post
So do the male flowers work ?
not sure yet Tony, it has a green bulb on it but no leaves yet.
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Old 09-01-2012, 03:58 PM   #13 (permalink)
 
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Default Re: !st time Tissue Culturing with pictures

Quote:
Originally Posted by sddarkman619 View Post
not sure yet Tony, it has a green bulb on it but no leaves yet.
10-4 Got lots of male flowers if it works
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Old 09-01-2012, 04:43 PM   #14 (permalink)
 
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Default Re: !st time Tissue Culturing with pictures

Quote:
Originally Posted by sunfish View Post
10-4 Got lots of male flowers if it works
any CG?
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Old 09-01-2012, 04:46 PM   #15 (permalink)
 
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Quote:
Originally Posted by sddarkman619 View Post
any CG?
Yes
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Old 09-02-2012, 02:45 AM   #16 (permalink)
 
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Default Re: !st time Tissue Culturing with pictures

PPM: A Powerful Technology to Prevent and Eliminate Microbial Contamination in Plant Tissue Culture
This supplier is British, should be a supplier in US
https://micro-science.co.uk/plant-ti...lture/ppm.html

Polysorbate 20 - Wikipedia, the free encyclopedia
polysorbate 20 | eBay

Have you read these posts?
Step by Step Subculture In Tissue CUlture Bananas (part 1)
Step by Step Subculture In Tissue Culture Bananas (part 2)
Medium MS Organic as Substitute Medium MS
TC Male Flower Bananas
apologies if you have

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Old 09-06-2012, 07:32 PM   #17 (permalink)
 
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Default Re: !st time Tissue Culturing with pictures


JMoore, I just want to say thanks for your help and direction. I received my PPM and polysorbate 20 today, I'm washing my jars, and I am waiting for my phyto caps for the baby food jars.

I really appreciate it!! You'll be the first to get some TCs from me if they work.

One more question in reference to my other post with pictures. So we were looking at the side view of the corm and you can see the Growing tip, now if I look down at the corm, a top view, let's say the corm is 6" in diameter. Can the whole top of the corm's merristem be used for TCing or only the very center part where there is a growing point?

Thanks!!!
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Old 09-07-2012, 01:46 AM   #18 (permalink)
 
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Default Re: !st time Tissue Culturing with pictures

No problem. Just the growing point
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Old 09-07-2012, 02:39 AM   #19 (permalink)
 
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Default Re: !st time Tissue Culturing with pictures

Quote:
Originally Posted by jmoore View Post
No problem. Just the growing point
well then that explains why I got no shoots this time or the remaining leave bits didn't grow this time.

But get one good growing point and that can turn into a bunch...right?
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Old 09-07-2012, 06:55 AM   #20 (permalink)
 
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Default Re: !st time Tissue Culturing with pictures

Right
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