DIY Tissue Culture - The Planted Tank Forum
 
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post #1 of 7 (permalink) Old 08-23-2020, 05:48 PM Thread Starter
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DIY Tissue Culture

Does anyone make your own tissue cultures?

I don't try to sterilize the plants. That's a major PIA. I have bought some TC plants and moved them into bigger flasks to have more to grow out for when I set up my tank. I've also done it because some plants you can never find, and if I make my own, I always have it.

If you do... what media do you use?

I have used THIS for Monte Carlo but Pogostemon helferi and Anubias Pangolino didn't like it. P helferi did pretty good on THIS oddly.
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post #2 of 7 (permalink) Old 10-19-2020, 03:21 AM
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I've only done a few Eriocaulon spp., all from seed, but I have >20 years of tissue culture and transgenic plant experience.

If the P656 is working better for you than full-strength M5825, it's probably the salts. P656 is going to be something like half-strength MS salt base. MS salts was originally formulated for nicotiata (tobacco), and is hotter'n all get-out, just loads of nitrogen, with much of it coming from ammonium.

Been looking for madagascariensis seeds forever, I'd love to get it growing in tissue culture.
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post #3 of 7 (permalink) Old 10-20-2020, 12:28 AM Thread Starter
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I tissue culture orchids as a hobby and the P656 is an orchid media. P helferi liked it better. Monte Carlo liked the M5825 better, and I grew some anubius in a plain MS media once, but they are a pain for tissue culture. They grow roots and push themselves off the media.

I tried growing Amazon swords once, but they would only get about an inch tall before dying. I know the media wasn't exhausted because there was plenty and it happened in every culture I tried. Any idea on that?
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post #4 of 7 (permalink) Old 10-20-2020, 05:00 AM
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Never tried the genus; found this article:

https://link.springer.com/article/10...627-018-9938-6

Pretty generic:

Quote:
Surface-disinfected shoot-tips were cultured in submerged conditions in a solid–liquid bilayer medium, consisting of an upper, liquid layer (sterile distilled water) and a lower, solid layer Murashige and Skoog (MS) basal medium supplemented with 3.0% (w/v) sucrose, 0.8% (w/v) agar-agar, and plant growth regulators (PGRs) in different combinations and concentrations.
So, strong on the nutrients, but the basal medium has a pour of sterile water over it. I don't have access to the full article to see exactly how much water they mean.

The PGRs seem pretty routine:

Quote:
The combination of 2.5 mg L−1 6-benzylaminopurine and 1.0 mg L−1 α-naphthaleneacetic acid improved the multiplication rate to a maximum of 26.8 ± 0.51 shoots per explant after 60 d of culture. The number of multiplied shoots increased with each regeneration cycle, thus from only 26.8 ± 0.51 shoots per explant (first regeneration cycle), this number increased to 33.5 ± 0.58 (second regeneration cycle), and to 38.3 ± 0.62 for the third regeneration cycle with the same medium composition. The highest number of roots (8.3 ± 0.28) per shoot was induced in the presence of 1.0 mg L−1 indole-3-butyric acid, but further growth of these roots was stunted. The best rooting was achieved on PGR-free ½-strength MS medium, where 6.1 ± 0.21 roots per shoot were induced with 5.8 ± 0.35 cm length after 30 d of culture. The regenerated plantlets were successfully acclimatized to submerged underwater conditions, with 100% survival rate. The present protocol is suitable for the commercial propagation of Echinodorus ‘Indian Red’ for aquarium-industries.
If Amazon swords must be grown immersed, I suspect the lack of water over the agar is a contributing factor. I read a paper on growing madagascariensis, and the containers were also flooded.
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post #5 of 7 (permalink) Old 10-20-2020, 10:41 PM Thread Starter
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Interesting. With the ones I tried, I didn't use multiplication hormones. I was just trying to get them to live first. I'm not sure if I remember correctly, but I put in a single plant that grew a little, died, and then a mass of baby plants sprouted out everywhere. Being an aquatic plant, I guess it makes sense that an unusual (compared to most plants) amout of water might be needed.

The little bit I've tried has been interesting. Except for the Anubias, they all grew many times faster than orchids.
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post #6 of 7 (permalink) Old 10-21-2020, 04:59 AM
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Yes, everything will grow faster than orchids, especially aquatic weeds. Once coryanthes hit their log growth phase, they're pretty strong, and then larger Cyrtopodium punctatums- those can really grow fast. Still nothing in comparison to pretty much anything else you'd ever grow in vitro.


There is no reason that hormones would have kept your plants alive; they inspire certain types of growth- proliferation of shoots, production of roots, generation of undifferentiated callus material- but otherwise the axenic culture box is same as an aquarium or a greenhouse or a potted plant or whatever. In fact, if you take away the sugars (which we use to provide "liquid carbon" to the plants), you can go with heteroautotrophic culture (I think it is) by feeding them carbon dioxide, and the problems with sterility pretty much go away. Then it's just nutrients + water + hormones + CO2.


Water is undoubtedly needed as a "structural" support for just about anything that can't be grown emersed- Madagascar lace, for example, or perhaps vallisneria. The little eriocaulons did brilliantly on agar with no aquatic overlay. Just adorable little green urchins.


What happened to the "baby plants" you describe? I suspect because the species throws a lot of pups naturally, it tried to do so as a last-ditch effort to propagate before expiring.
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post #7 of 7 (permalink) Old 10-22-2020, 10:22 PM Thread Starter
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Quote:
Originally Posted by osp001 View Post
There is no reason that hormones would have kept your plants alive..
I didn't think they were needed to keep my plants alive. That is why I didn't use them. I used regular media with the intent of growing a healthy plant, and then I was going to experiment.



Quote:
Originally Posted by osp001 View Post
What happened to the "baby plants" you describe? I suspect because the species throws a lot of pups naturally, it tried to do so as a last-ditch effort to propagate before expiring.
The plant would grow up a little, then decline, then offshoots would grow, they would start to die off, and then more offshoots would grow. The process repeated until (I assume) nutrients were exhausted. Never did anything get more than around 2cm.
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