Sterilizing plants - Tissue Culture - The Planted Tank Forum
 10Likes
  • 4 Post By Kehy
  • 2 Post By Seattle_Aquarist
  • 2 Post By Malakian
  • 6 Post By Darkblade48
  • 4 Post By cbachmann
  • 2 Post By adkaquascaping
 
LinkBack Thread Tools Display Modes
post #1 of 12 (permalink) Old 05-09-2017, 02:36 PM Thread Starter
Planted Tank Obsessed
 
Malakian's Avatar
 
PTrader: (0/0%)
Join Date: Aug 2014
Location: Norway
Posts: 422
Sterilizing plants - Tissue Culture

I'm planning to try and propagate some plants via tissue culture aka In Vitro. I got all the stuff and read up on the subject, but I need some help with sterilizing the plant tissue.

All guides I can find is for terrestrial plants, and usually suggest a 20% (made from 6% household bleach) bleach solution with the addition of some dish soap, 10-15 min soak. This seems like a pretty strong solution to me, won't most aquarium plants be killed by such a wash? I only need a part of the plant to survive, either it be leaves, stem, rhizome etc. Would think leaves are the first to go when sterilizing though. Would something like 10% for 30 min be easier for the plant to handle?

Any suggestions would be appreciated.

Edit: I have potassium permanganate, glutaraldehyde, hydrogen peroxide, bleach and alcohol sterilizing agents at hand if anything else would be a better alternative.


To view links or images in signatures your post count must be 10 or greater. You currently have 0 posts.



To view links or images in signatures your post count must be 10 or greater. You currently have 0 posts.
Malakian is offline  
Sponsored Links
Advertisement
 
post #2 of 12 (permalink) Old 05-09-2017, 02:53 PM
Wannabe Guru
 
Kehy's Avatar
 
PTrader: (14/100%)
Join Date: Feb 2012
Location: Sumner, WA
Posts: 1,495
This might help? It has sterilizing instructions

http://www.notulaebotanicae.ro/index...File/7520/7019
Kehy is offline  
post #3 of 12 (permalink) Old 05-09-2017, 03:18 PM
Planted Tank Guru
 
Seattle_Aquarist's Avatar
 
PTrader: (64/100%)
Join Date: Jun 2008
Location: Seattle, WA
Posts: 6,279
Hi Malakian,

The AGA (Aquatic Gardener's Association) had a great speaker on the subject of tissue culture (Dr. Mike Kane) at their 2010 convention. You can purchase the DVD on their website.
Malakian and Malakian like this.

Roy_________
To view links or images in signatures your post count must be 10 or greater. You currently have 0 posts.

To view links or images in signatures your post count must be 10 or greater. You currently have 0 posts.

To view links or images in signatures your post count must be 10 or greater. You currently have 0 posts.

75 Gallon, 2X55W AH Supply CF 8800K, 1XFluval F&P 2.0; 45 Gallon Tall, 96Watt AH Supply CF 6700K; 30 Gallon Long; Fluval F&P 2.0; 20 Gallon, 1X26W AH Supply LED; all with CO2 and (Calcined) Montmorillonite Clay
Seattle_Aquarist is online now  
 
post #4 of 12 (permalink) Old 05-09-2017, 03:23 PM Thread Starter
Planted Tank Obsessed
 
Malakian's Avatar
 
PTrader: (0/0%)
Join Date: Aug 2014
Location: Norway
Posts: 422
Quote:
Originally Posted by Kehy View Post
This might help? It has sterilizing instructions

http://www.notulaebotanicae.ro/index...File/7520/7019
Good find!


To view links or images in signatures your post count must be 10 or greater. You currently have 0 posts.



To view links or images in signatures your post count must be 10 or greater. You currently have 0 posts.
Malakian is offline  
post #5 of 12 (permalink) Old 05-09-2017, 04:26 PM
Algae Grower
 
PTrader: (1/100%)
Join Date: Jan 2015
Location: Cleveland, OH
Posts: 110
Don't we usually sterilize plants from the store at 1/20 bleach solution or 5% ?? 20% is significantly higher.
SeaCur is offline  
post #6 of 12 (permalink) Old 05-09-2017, 04:39 PM Thread Starter
Planted Tank Obsessed
 
Malakian's Avatar
 
PTrader: (0/0%)
Join Date: Aug 2014
Location: Norway
Posts: 422
Quote:
Originally Posted by SeaCur View Post
Don't we usually sterilize plants from the store at 1/20 bleach solution or 5% ?? 20% is significantly higher.
That's what I thought, why I asked. But the paper on the Anubias uses 10% with a bit of soap for 15min. I'm gonna try that on some S.repens and AR mini nodule stems. We'll, they first use Mercuric chloride as the first step, before a 15 min 10% bleach wash, followed by a 5 min 5% bleach wash. I don't have the Mercuric chloride so I'm gonna skip that, and I don't really see much benefit in doing a additional 5 min wash in weaker solution so skipping that too. Hopefully the one wash is enough.

Also when it comes to tissue culture, it has to be 100% sterile. Just killing algae and pests isn't enough, might explain the higher concentrations. If I manage to succeed I want to keep a small "library" of plants. It will be interesting to see how plants like Subwassertang, moss and HC will fair against the sterilization. If I fail at the sterilization stage, I can resort to buying an already in vitro plant, as that will already be sterile and culture from that.

I'll try and document everything and put up a guide/how to for others if it works out.

Plants are in the jars. MS Media with 3mg/L BAP and 3% sucrose.

Next couple of day will be exciting to see if it's a sterile culture or not. Had 3 jars, so tried 3 plants. AR mini, H.Siamensis 53B and S.Repens. Nothing to exciting to look at, but here are some images.

S.Repens

H.Siamensis 53B

AR Mini


To view links or images in signatures your post count must be 10 or greater. You currently have 0 posts.



To view links or images in signatures your post count must be 10 or greater. You currently have 0 posts.

Last edited by Darkblade48; 05-31-2018 at 08:37 PM. Reason: Please use the edit function for back to back posts to keep threads cleaner
Malakian is offline  
post #7 of 12 (permalink) Old 05-31-2017, 10:59 AM Thread Starter
Planted Tank Obsessed
 
Malakian's Avatar
 
PTrader: (0/0%)
Join Date: Aug 2014
Location: Norway
Posts: 422
Update: 2 out of 3 jars got contaminated and did not show any signs of new growth. The AR "mini" has no contamination as of this far, and is actually growing. Only 3 pairs of new leaves, but I see a TON of shoots coming in. Exciting

To add to the contamination part, I used submersed plants and from what I have gathered with In-vitro and aquarium plants is that it's almost impossible to get submersed plant parts properly sterilized. So I'm quite amazed that even just 1 jar didn't get contaminated, beginners luck I guess :P
Dman911 and Dman911 like this.


To view links or images in signatures your post count must be 10 or greater. You currently have 0 posts.



To view links or images in signatures your post count must be 10 or greater. You currently have 0 posts.
Malakian is offline  
post #8 of 12 (permalink) Old 06-01-2017, 03:45 AM
Moderator
 
Darkblade48's Avatar
 
PTrader: (3/100%)
Join Date: Jan 2008
Location: Toronto/Singapore
Posts: 11,441
Quote:
Originally Posted by Malakian View Post
We'll, they first use Mercuric chloride as the first step, before a 15 min 10% bleach wash, followed by a 5 min 5% bleach wash. I don't have the Mercuric chloride so I'm gonna skip that, and I don't really see much benefit in doing a additional 5 min wash in weaker solution so skipping that too.
Definitely a good idea in the end! Mercuric chloride is a pain to work with, and you'd have to dispose of it as hazardous waste.

In general, it is best to avoid mercury compounds if at all possible!

Anthony


To view links or images in signatures your post count must be 10 or greater. You currently have 0 posts.
and
To view links or images in signatures your post count must be 10 or greater. You currently have 0 posts.

Eheim Pimp #362 - Eheim 2213 x2, Eheim 2028, Eheim 2217, Eheim surface skimmer and Eheim autofeeder.
Victor Pimp #33 - HPT272-125-350-4M
Darkblade48 is offline  
post #9 of 12 (permalink) Old 05-15-2018, 03:17 AM
Algae Grower
 
PTrader: (0/0%)
Join Date: Apr 2018
Location: Upstate NY
Posts: 87
How'd this work out for you in the end?


I'm actually standing in the lab I've almost finished building for this exact purpose. I invested half my 401k in myself. It's what I went to college for and I hate my job. So, time to invest in me and not the markets. Properly stocked, quite well equipped. About $1100 in plants coming in mail in next 48h. Everything from DHG and HC Cuba to Anubias stardust and A. Snowwhite and Iguazu 2009. My MS salts and PGRs are on the way and should be here by this weekend along with another 400 tissue culture vessels and lids.




Did you ever have any luck with your project or did you abandon it or just kinda burn out with it?

Sent from my SM-N950U using Tapatalk
adkaquascaping is offline  
post #10 of 12 (permalink) Old 05-19-2018, 02:21 PM
Algae Grower
 
Subconfish's Avatar
 
PTrader: (0/0%)
Join Date: May 2018
Posts: 50
Seems like he was afraid to kill his plants. I've used a 15% bleach solution dipped for 3 minutes fine. Then prime for 5 and redipped for 1 min then culture. Some plants are more sensitive than others
Subconfish is offline  
post #11 of 12 (permalink) Old 05-19-2018, 05:03 PM
Planted Tank Obsessed
 
cbachmann's Avatar
 
PTrader: (6/100%)
Join Date: Aug 2013
Location: New York
Posts: 436
Quote:
Originally Posted by adkaquascaping View Post
How'd this work out for you in the end?


I'm actually standing in the lab I've almost finished building for this exact purpose. I invested half my 401k in myself. It's what I went to college for and I hate my job. So, time to invest in me and not the markets. Properly stocked, quite well equipped. About $1100 in plants coming in mail in next 48h. Everything from DHG and HC Cuba to Anubias stardust and A. Snowwhite and Iguazu 2009. My MS salts and PGRs are on the way and should be here by this weekend along with another 400 tissue culture vessels and lids.




Did you ever have any luck with your project or did you abandon it or just kinda burn out with it?

Sent from my SM-N950U using Tapatalk
I'm sure you're busy as hell, but is there any way you could do a journal for this? I'd love to.follow it!

Sent from my SM-G928V using Tapatalk

My tanks:

To view links or images in signatures your post count must be 10 or greater. You currently have 0 posts.


To view links or images in signatures your post count must be 10 or greater. You currently have 0 posts.
cbachmann is offline  
post #12 of 12 (permalink) Old 05-20-2018, 09:39 PM
Algae Grower
 
PTrader: (0/0%)
Join Date: Apr 2018
Location: Upstate NY
Posts: 87
Quote:
Originally Posted by cbachmann View Post
I'm sure you're busy as hell, but is there any way you could do a journal for this? I'd love to.follow it!

Sent from my SM-G928V using Tapatalk
Only journals I'm really keeping are my log books. The one for media batches (exact tracking of everything used, let's me see if there is issues or success with variations where those things arose from), and one for my protocols. A lot of the plants I have are not currently in TC at any sort of volume, and steralizing explants from submerged or underground can be a nightmare. So tracking each variation of the steralizing protocols I use is again essential. I'll also hopefully before long be testing effects if varying concentrations of each of my plant growth regulators on (hopefully) at each genus. Then I can optimize media recipes for each plant species and each phase (establishment and/or callus induction/multiplication/rooting). And then again also tracking how successful my attempts at using callus in a liquid culture is vs normal multiplication methods. It sounds like a lot, but in all honesty is not that much. At least to me, since part of why I loved my biotech program so much (shout out SUNY ESF!) Is I very much enjoy data analysis, so the precision and tracking and trends and such.... They just clicked with me as second nature.


Once I'm in a real space...and not my spare bedroom/office in my apartment I'm going to actually keep my Facebook page updated (see: Adirondack Aquascaping, I think counting my wife and me, you'd be #4 to like it haha). It's empty except for a logo I made last night after taking my narcolepsy meds hahaha. And I plan to do YouTube stuff. Make a channel that shows a little about a TC lab and hopefully genetic work eventually, little bit about plants and care and soil and co2 and all that goes with it, some scaping guides, and hopefully even a little 'you can try it at home!' series. Showing hobbyists how they can do propagation of plants (be it aquarium, garden, house, w/e) in TC at home without the thousands of dollars needed to set up even a modest lab space. How to use Walmart sugar and bottled distilled water to propagate a cool rare begonia or Cryptocoryne and such. Spread scientific literacy and show people it's not scary or hard or intimidating, help foster interest and excitement about it. Maybe inspire some people to go to school for biotech like I did.


Oh, and if I get large enough to be traveling the world to collect new plants.... You better believe that'll be on there too.



For now tho... It's just my lab notebooks and working to establish and start multiplying 90 species, while trying to hunt down about another dozen that I really want, while working a full time job, 12.5h shifts 3 to 4 days a week in a semiconductor facility. No time to blog it and such. My wife and my cooking blog has even been shelved for now. This ... This is my free time. I'm taking a break from mythic raiding in wow. This is now a second full time job. So while there's no journal or log or blog ATM ... There WILL be a YouTube channel in the future, should this be successful. But if updates and knowing when those things are coming and happening, and when I start selling in a few weeks. ... Check the fb page and watch on there. It'll be the first place I update along with my as-yet-not-made website, domain name is parked just nothing in it yet haha.

Sent from my SM-N950U using Tapatalk

Update, there is a thread on Barr report and on APC forums. Identical initially, comments more updated on APC

Sent from my SM-N950U using Tapatalk

Quote:
Originally Posted by cbachmann View Post
I'm sure you're busy as hell, but is there any way you could do a journal for this? I'd love to.follow it!

Sent from my SM-G928V using Tapatalk
Couldn't tell you what section it's in, but I did start that journal page. Check profile and threads I started, you'll find it now.

Sent from my SM-N950U using Tapatalk
swarley and swarley like this.

Last edited by Darkblade48; 05-31-2018 at 08:37 PM. Reason: Please use the edit function for back to back posts to keep threads cleaner
adkaquascaping is offline  
Reply

Tags
None

Quick Reply
Message:
Options

Register Now



In order to be able to post messages on the The Planted Tank Forum forums, you must first register.
Please enter your desired user name, your email address and other required details in the form below.

User Name:
Password
Please enter a password for your user account. Note that passwords are case-sensitive.

Password:


Confirm Password:
Email Address
Please enter a valid email address for yourself.

Email Address:
OR

Log-in










Thread Tools
Show Printable Version Show Printable Version
Email this Page Email this Page
Display Modes
Linear Mode Linear Mode



Posting Rules  
You may post new threads
You may post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

 
For the best viewing experience please update your browser to Google Chrome