[Malakian]

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.

 

[Kehy]

This might help? It has sterilizing instructions

http://www.notulaebotanicae.ro/index.php/nbha/article/viewFile/7520/7019

 

[Seattle_Aquarist]

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]

This might help? It has sterilizing instructions

http://www.notulaebotanicae.ro/index.php/nbha/article/viewFile/7520/7019

Good find!

 

[SeaCur]

Don't we usually sterilize plants from the store at 1/20 bleach solution or 5% ?? 20% is significantly higher.

 

[Malakian]

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

 

[Malakian]

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

 

[Darkblade48]

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!

 

[adkaquascaping]

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?

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[Subconfish]

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

 

[cbachmann]

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?

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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!

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[adkaquascaping]

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!

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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.

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

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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!

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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.

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