View Full Version : !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
http://www.bananas.org/gallery/watermark.php?file=50110&size=1 (http://www.bananas.org/gallery/showphoto.php?photo=50110)
http://www.bananas.org/gallery/watermark.php?file=50111&size=1 (http://www.bananas.org/gallery/showphoto.php?photo=50111)
http://www.bananas.org/gallery/watermark.php?file=50113&size=1 (http://www.bananas.org/gallery/showphoto.php?photo=50113)
http://www.bananas.org/gallery/watermark.php?file=50112&size=1 (http://www.bananas.org/gallery/showphoto.php?photo=50112)
http://www.bananas.org/gallery/watermark.php?file=50109&size=1 (http://www.bananas.org/gallery/showphoto.php?photo=50109)
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:
http://www.bananas.org/f260/some-questions-psuedostem-meristem-where-16429.html
maybe someone could answer the question I posted in that post as well.
Here's the contamination I'm getting:
http://www.bananas.org/gallery/watermark.php?file=50364&size=1 (http://www.bananas.org/gallery/showphoto.php?photo=50364&ppuser=13963)
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 :
http://www.bananas.org/gallery/watermark.php?file=50363&size=1 (http://www.bananas.org/gallery/showphoto.php?photo=50363&ppuser=13963)
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
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
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
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
10-4 Got lots of male flowers if it works
any CG?
sunfish
09-01-2012, 04:46 PM
any CG?
Yes
jmoore
09-02-2012, 02:45 AM
PPM: A Powerful Technology to Prevent and Eliminate Microbial Contamination in Plant Tissue Culture (http://www.kitchenculturekit.com/PPMwebsite/instructions.htm)
This supplier is British, should be a supplier in US
https://micro-science.co.uk/plant-tissue-culture/ppm.html
Polysorbate 20 - Wikipedia, the free encyclopedia (http://en.wikipedia.org/wiki/Polysorbate_20)
polysorbate 20 | eBay (http://www.ebay.co.uk/sch/i.html?_trksid=p3984.m570.l1313&_nkw=polysorbate+20&_sacat=0)
Have you read these posts?
http://www.bananas.org/f260/step-step-subculture-tissue-culture-bananas-9865.html
http://www.bananas.org/f260/step-step-subculture-tissue-culture-bananas-9866.html
http://www.bananas.org/f260/medium-ms-organic-substitute-medium-ms-9635.html
http://www.bananas.org/f260/tc-male-flower-bananas-11091.html
apologies if you have
sddarkman619
09-06-2012, 07:32 PM
PPM: A Powerful Technology to Prevent and Eliminate Microbial Contamination in Plant Tissue Culture (http://www.kitchenculturekit.com/PPMwebsite/instructions.htm)
This supplier is British, should be a supplier in US
https://micro-science.co.uk/plant-tissue-culture/ppm.html
Polysorbate 20 - Wikipedia, the free encyclopedia (http://en.wikipedia.org/wiki/Polysorbate_20)
polysorbate 20 | eBay (http://www.ebay.co.uk/sch/i.html?_trksid=p3984.m570.l1313&_nkw=polysorbate+20&_sacat=0)
Have you read these posts?
http://www.bananas.org/f260/step-step-subculture-tissue-culture-bananas-9865.html
http://www.bananas.org/f260/step-step-subculture-tissue-culture-bananas-9866.html
http://www.bananas.org/f260/medium-ms-organic-substitute-medium-ms-9635.html
http://www.bananas.org/f260/tc-male-flower-bananas-11091.html
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
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 :)
ImperialExotics
09-22-2012, 05:12 PM
I don't recall seeing this, so I'm going to ask.....
you DID sterilize your medium and containers in a pressure cooker or microwave right?
sddarkman619
09-22-2012, 05:50 PM
yes.
caliboy1994
10-02-2012, 06:05 PM
Saw your pictures in the gallery. Very nice! I think you should get your hands on Real Ice Cream and tissue culture it so we can all get one. :ha:
Abnshrek
10-02-2012, 08:00 PM
Saw your pictures in the gallery. Very nice! I think you should get your hands on Real Ice Cream and tissue culture it so we can all get one. :ha:
That's what I'm talking about.. :^)
sunfish
10-02-2012, 08:24 PM
:pics:
I haven't seen any TC's growing yet
trebor
10-02-2012, 08:54 PM
Saw your pictures in the gallery. Very nice! I think you should get your hands on Real Ice Cream and tissue culture it so we can all get one. :ha:
Caliboy That's a stellar idea! You aint been in collage long and your brain is swelling with good stuff! :) I bet selling them would be pretty darn easy.. I think I have one or two growing
one I got from (don't laugh) Wall Mart $ 7.50
one I got from Wellspring Gardens in Florida (gift from friend)
The Wall Mart one is doing great. the Wellsprings one is kinda wimpy
So back on topic .... Keep working on your TC project . Lots of peeps are watching and waiting to see some GREEN!
sddarkman619
10-02-2012, 09:51 PM
Been kinda busy lately and haven't really had the time to put together anything lately. But maybe I'll TC something this week. Depends on how tired I am from working at my friends nursery...
Bonita Creek Nursery...
Bonita Creek Nursery: Rare Fruit Trees in San Diego, Los Angeles and SoCal (http://www.bonitafruittrees.com)
vBulletin® v3.6.8, Copyright ©2000-2020, Jelsoft Enterprises Ltd.