!st time Tissue Culturing with pictures

sddarkman619 · 08-11-2012, 11:09 PM

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.

Larry

sddarkman619 · 08-13-2012, 11:51 PM

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.

sddarkman619 · 08-14-2012, 04:38 PM

was curious if anyone could tell me if I should have my TCs under light or filtered sunlight?

sddarkman619 · 08-15-2012, 02:36 AM

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...

garymc · 08-24-2012, 03:14 PM

What's RO water?

sddarkman619 · 08-24-2012, 03:30 PM

reverse osmosis

trebor · 08-24-2012, 05:22 PM

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.....

sddarkman619 · 09-01-2012, 10:56 AM

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.

jmoore · 09-01-2012, 11:24 AM

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.

sddarkman619 · 09-01-2012, 12:06 PM

Quote:

Originally Posted by jmoore

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!

sunfish · 09-01-2012, 02:03 PM

So do the male flowers work ?

sddarkman619 · 09-01-2012, 03:10 PM

Quote:

Originally Posted by sunfish

So do the male flowers work ?

not sure yet Tony, it has a green bulb on it but no leaves yet.

sunfish · 09-01-2012, 03:58 PM

Quote:

Originally Posted by sddarkman619

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

sddarkman619 · 09-01-2012, 04:43 PM

Quote:

Originally Posted by sunfish

10-4 Got lots of male flowers if it works

any CG?

sunfish · 09-01-2012, 04:46 PM

Quote:

Originally Posted by sddarkman619

any CG?

Yes

jmoore · 09-02-2012, 02:45 AM

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

sddarkman619 · 09-06-2012, 07:32 PM

Quote:

Originally Posted by jmoore

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

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!!!

jmoore · 09-07-2012, 01:46 AM

No problem. Just the growing point

sddarkman619 · 09-07-2012, 02:39 AM

Quote:

Originally Posted by jmoore

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?

sunfish · 09-07-2012, 06:55 AM

Right

08-11-2012, 11:09 PM	#1 (permalink)
sddarkman619 Banana Man Find me on the map! Name: Larry Join Date: Jul 2012 Posts: 1,157 BananaBucks : 201,366 Feedback: 14 / 100% Said "Thanks" 340 Times Was Thanked 1,226 Times in 540 Posts Said "Welcome to Bananas" 1 Times	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. Larry __________________ Ca Licensed Nursery - Snail/Disease Free and Inspected: Bountiful Garden Nursery - A1469.001 Banana Plants for Sale https://www.bananatreesforsale.com Sign up to the Newsletter for Updates: Sign Up Here Last edited by sddarkman619 : 08-12-2012 at 12:37 AM.

08-13-2012, 11:51 PM	#2 (permalink)
sddarkman619 Banana Man Find me on the map! Name: Larry Join Date: Jul 2012 Posts: 1,157 BananaBucks : 201,366 Feedback: 14 / 100% Said "Thanks" 340 Times Was Thanked 1,226 Times in 540 Posts Said "Welcome to Bananas" 1 Times	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. __________________ Ca Licensed Nursery - Snail/Disease Free and Inspected: Bountiful Garden Nursery - A1469.001 Banana Plants for Sale https://www.bananatreesforsale.com Sign up to the Newsletter for Updates: Sign Up Here

08-14-2012, 04:38 PM	#3 (permalink)
sddarkman619 Banana Man Find me on the map! Name: Larry Join Date: Jul 2012 Posts: 1,157 BananaBucks : 201,366 Feedback: 14 / 100% Said "Thanks" 340 Times Was Thanked 1,226 Times in 540 Posts Said "Welcome to Bananas" 1 Times	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? __________________ Ca Licensed Nursery - Snail/Disease Free and Inspected: Bountiful Garden Nursery - A1469.001 Banana Plants for Sale https://www.bananatreesforsale.com Sign up to the Newsletter for Updates: Sign Up Here

08-15-2012, 02:36 AM	#4 (permalink)
sddarkman619 Banana Man Find me on the map! Name: Larry Join Date: Jul 2012 Posts: 1,157 BananaBucks : 201,366 Feedback: 14 / 100% Said "Thanks" 340 Times Was Thanked 1,226 Times in 540 Posts Said "Welcome to Bananas" 1 Times	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... __________________ Ca Licensed Nursery - Snail/Disease Free and Inspected: Bountiful Garden Nursery - A1469.001 Banana Plants for Sale https://www.bananatreesforsale.com Sign up to the Newsletter for Updates: Sign Up Here

08-24-2012, 03:14 PM	#5 (permalink)
garymc Location: Sikeston, Missouri Find me on the map! Zone: 7 Name: GaryMc Join Date: Apr 2011 Posts: 34 BananaBucks : 5,179 Feedback: 0 / 0% Said "Thanks" 4 Times Was Thanked 26 Times in 17 Posts Said "Welcome to Bananas" 1 Times	Re: !st time Tissue Culturing with pictures What's RO water?

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08-24-2012, 03:30 PM	#6 (permalink)
sddarkman619 Banana Man Find me on the map! Name: Larry Join Date: Jul 2012 Posts: 1,157 BananaBucks : 201,366 Feedback: 14 / 100% Said "Thanks" 340 Times Was Thanked 1,226 Times in 540 Posts Said "Welcome to Bananas" 1 Times	Re: !st time Tissue Culturing with pictures reverse osmosis __________________ Ca Licensed Nursery - Snail/Disease Free and Inspected: Bountiful Garden Nursery - A1469.001 Banana Plants for Sale https://www.bananatreesforsale.com Sign up to the Newsletter for Updates: Sign Up Here

08-24-2012, 05:22 PM	#7 (permalink)
trebor trebor's Photo Gallery Location: Florida Find me on the map! Zone: POMPANO BEACH, FL Zone 10 Join Date: Jul 2010 Posts: 759 BananaBucks : 110,899 Feedback: 0 / 0% Said "Thanks" 845 Times Was Thanked 1,329 Times in 483 Posts Said "Welcome to Bananas" 256 Times	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..... __________________ If ya can't eat it, it ain't growing in my back yard !

09-01-2012, 10:56 AM	#8 (permalink)
sddarkman619 Banana Man Find me on the map! Name: Larry Join Date: Jul 2012 Posts: 1,157 BananaBucks : 201,366 Feedback: 14 / 100% Said "Thanks" 340 Times Was Thanked 1,226 Times in 540 Posts Said "Welcome to Bananas" 1 Times	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. __________________ Ca Licensed Nursery - Snail/Disease Free and Inspected: Bountiful Garden Nursery - A1469.001 Banana Plants for Sale https://www.bananatreesforsale.com Sign up to the Newsletter for Updates: Sign Up Here

09-01-2012, 11:24 AM	#9 (permalink)
jmoore jmoore 's Photo Gallery Location: Lowestoft, UK Zone: 8a Name: James Join Date: Aug 2008 Posts: 766 BananaBucks : 121,765 Feedback: 4 / 100% Said "Thanks" 192 Times Was Thanked 799 Times in 373 Posts Said "Welcome to Bananas" 71 Times	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.

09-01-2012, 02:03 PM	#11 (permalink)
sunfish Banned sunfish's Photo Gallery Location: San Diego Find me on the map! Zone: 9-11 Name: Tony Join Date: Apr 2008 Posts: 18,429 BananaBucks : 1,012,274 Feedback: 8 / 100% Said "Thanks" 3,210 Times Was Thanked 20,592 Times in 7,760 Posts Said "Welcome to Bananas" 2,716 Times	Re: !st time Tissue Culturing with pictures So do the male flowers work ?

09-02-2012, 02:45 AM	#16 (permalink)
jmoore jmoore 's Photo Gallery Location: Lowestoft, UK Zone: 8a Name: James Join Date: Aug 2008 Posts: 766 BananaBucks : 121,765 Feedback: 4 / 100% Said "Thanks" 192 Times Was Thanked 799 Times in 373 Posts Said "Welcome to Bananas" 71 Times	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 Last edited by jmoore : 09-02-2012 at 02:48 AM.

09-07-2012, 01:46 AM	#18 (permalink)
jmoore jmoore 's Photo Gallery Location: Lowestoft, UK Zone: 8a Name: James Join Date: Aug 2008 Posts: 766 BananaBucks : 121,765 Feedback: 4 / 100% Said "Thanks" 192 Times Was Thanked 799 Times in 373 Posts Said "Welcome to Bananas" 71 Times	Re: !st time Tissue Culturing with pictures No problem. Just the growing point

09-07-2012, 06:55 AM	#20 (permalink)
sunfish Banned sunfish's Photo Gallery Location: San Diego Find me on the map! Zone: 9-11 Name: Tony Join Date: Apr 2008 Posts: 18,429 BananaBucks : 1,012,274 Feedback: 8 / 100% Said "Thanks" 3,210 Times Was Thanked 20,592 Times in 7,760 Posts Said "Welcome to Bananas" 2,716 Times	Re: !st time Tissue Culturing with pictures Right

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