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How to improve resiliency of in-vitro plants?

_Maq_

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In-vitro plants are tiny and fragile and often succumb to parasites soon after planting. It's almost impossible not to damage them while handling. Can we use any commercially available systemic fungicides (and bactericides, possibly) to increase their resistance?
 

Wookii

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Must admit I've never encountered fungal or bacterial issues with in-vitro plants. I have found they take a lot better in a mature substrate though, and they are particularly more at risk in fresh aquasoil and will frequently melt due to exposure to excessive ammonia levels, which is why I always advocate at least a weeks dark start with water changes to flush out excess ammonia before planting them.
 

_Maq_

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With all respect, I think that this widespread obsession with ammonia is grossly exaggerated. Ammonia is sometimes dangerous to fish and shrimps, that's true. The rest is unsubstantiated.
 

Wookii

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With all respect, I think that this widespread obsession with ammonia is grossly exaggerated. Ammonia is sometimes dangerous to fish and shrimps, that's true. The rest is unsubstantiated.

There’s no obsession, it’s a commonly experienced issue with new tanks and in vitro plants.

Perhaps another experiment for your list, tank A with fresh Amazonia and immediately planted invitro plants, and tank B with the same soil, but matured using your normal methods and then planted.
 

Nick potts

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As above, never experienced any bacterial or fungal issues with in-vitro plants. Melting I have, whether that was from the new aquasoil i don't know.
 

_Maq_

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As above, never experienced any bacterial or fungal issues with in-vitro plants. Melting I have, whether that was from the new aquasoil i don't know.
And what do you think is the cause of melting? :rolleyes:
 

KirstyF

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With all respect, I think that this widespread obsession with ammonia is grossly exaggerated. Ammonia is sometimes dangerous to fish and shrimps, that's true. The rest is unsubstantiated.

Also a little curious about this @_Maq_ . It would seem that plant melt in new tanks using fresh aquasoil is experienced frequently enough by hobbyists (including experienced ones) to suggest some correlation. If you do not believe that this is as a result of ammonia leaching, what other cause would you apply.
I know that you like to mature your tanks somewhat before planting so would you put the issue down to microbial activity (or lack of it) rather than ammonia.
If so, would you therefore expect to find the same level of melt in a new tank with inert substrate (no excessive ammonia) as you would in a new tank with substrate that leaches?
 

ElleDee

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I'm also not sure what problems you are describing, @_Maq_. I have pretty extensive tissue culture experience outside of the hobby, and in vitro plants just are fragile by their nature. They are totally babied in an unnatural environment and haven't developed a lot of the protective morphological characters that a conventionally grown plant would have. They may also not be growing normally - tissue culture routinely uses plant growth regulators (plant hormones or their synthetic counterparts) in the gel media and the plants can do some weird stuff.

Bacterial and fungal contamination is a death sentence in vitro, and mold can overtake tissue culture plants in a dry start, but I think you are talking about issues in a submerged tank? I haven't really experienced any issues that I would contribute to plant pathogens, fungal or bacterial, and I attribute most issues with melting to the trials and tribulations of converting from and emersed to submerged state. The plant has to grow new, better adapted plant organs, while at the same time their unadapted plant organs are not functioning as well as intended. Obviously the species we're talking about are evolved to be able to make that transition, but it must still be resource intensive and tissue culture plants don't tend to have a lot of extra gas in the tank.

Neither of us inject CO2, so that's going to always put us at a disadvantage as well, but honestly I have had pretty good success with TC plants in the hobby, though I think with some stem plants it would have been faster if I had just gotten emersed stems - the just start out sooo tiny and take forever to get up to size. TC crypts have been fantastic a fantastic value in my experience, with the exception of one cup of C. parva that came with really disordered growth and hyperhydrosity and unsurprisingly I was unable to get the plants to recover.

I also would be very concerned about using fungicides in any system where there is going to be livestock. Most of those are really nasty substances and very toxic to aquatic life specifically.
 
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Perhaps another experiment for your list, tank A with fresh Amazonia and immediately planted invitro plants, and tank B with the same soil, but matured using your normal methods and then planted.
Correlation is not causation. For example, one of the possible causation might be bacterial bloom due to highly available nitrogen source and lot's of organic waste emited from the soil. Plants can easily deal with 100+ ppm of ammonium, but not deionized NH3, by the way, similarly to fish/shrimps. So it all depends on PH mainly.
I personally found that cycling without plants and without soil is way easier than other options. The reason is simple: I can do cycling at relatively high PH/KH and still be more or less safe when adding soil (thus lowering PH significantly) and plants after cycling.
 

_Maq_

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Recently, I've got a cup of in-vitro Rotala indica (aka Ammannia Bonsai). They looked like healthy. I've divided that batch between one of my aquaria and a paludarium. In both of them, the Rotalas started to melt, and from them the melting spread further, to unrelated plants which were healthy until then. In the paludarium, I could have drawn maps as the melting spread inch by inch from those damned Rotalas to my Crypts, Lagenandras, Rotala macrandra, Eriocaulons, Ludwigia glandulosa, Lindernia etc. Under water, the boundaries were not that clear, but the situation was quite the same.
I don't see a reason why submerged plants could not suffer from fungal and bacterial attacks just like terrestrial ones. Do you really think there are no fungi and bacteria in our tanks?
Also, there's no connection with ammonium/ammonia, as all farmers (incl. submerged rice) worldwide know. They don't fear to fertilize with manure, urea and ammonia, but no matter what nitrogen fertilizer they use, they still spend enormous sums for fungicides.
In-vitro plants are created in laminar boxes and sealed carefully - why? Not to let in any ammonia? Don't you know that ammonium is often part of the nutrition formulae in agar cultures?
 

seedoubleyou

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@_Maq_ so is it plausible that these in vitro plants being so delicate succumb to the waterborne parasites and bacteria as they’ve had zero prior exposure?
If so, why is this not being experienced so much once a dark start method is being done?
Could this be because the dark start entails a 100% waterchange that would remove a lot of the bacteria and parasite? Would most of it if been dealt with by the filter in that week or two prior to planting?

It’s great that you’ve pointed out that the ammonia is not the cause, so what exactly would be the cause and what could mitigate this?

Cheers,
Chris.
 

_Maq_

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I think we can never get rid of potentially harmful microbes. I'm not a pro in microbiology but I've been trying to learn, reading books and scientific papers. Common sense tells me following:
(1) Healthy and robust plants routinely eliminate fungal and bacterial attempts.
(2) In-vitro plants are tiny and it's almost impossible not to damage them while planting. So their protective tissues are damaged. Generally, only fungi can penetrate healthy tissues. So, freshly re-planted in-vitro plants are exposed both to fungi and bacteria.
(3) In-vitro plants are kept in sterile conditions. Maybe I'm wrong, but I believe their immunity reactions are retarded.
(4) Everything that promotes large fungal / bacterial populations increases the risks. All these 'Japanese' fertile soils be damned. They contain a lot of degradable organic matter, and thus support proliferation of bacteria and fungi. A secondary effect - increased oxygen demand - further weakens plants' resistance to pests.
(5) Whatever is the source of stress, dealing with it requires energy. Energy means respiration. Respiration means oxygen consumption. Lack of oxygen means death.

Originally, I believed that @Simon Cole could possibly suggest some fungicide which could be applicable, at least in newly established tanks without any fauna.
 

ElleDee

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Recently, I've got a cup of in-vitro Rotala indica (aka Ammannia Bonsai). They looked like healthy. I've divided that batch between one of my aquaria and a paludarium. In both of them, the Rotalas started to melt, and from them the melting spread further, to unrelated plants which were healthy until then. In the paludarium, I could have drawn maps as the melting spread inch by inch from those damned Rotalas to my Crypts, Lagenandras, Rotala macrandra, Eriocaulons, Ludwigia glandulosa, Lindernia etc. Under water, the boundaries were not that clear, but the situation was quite the same.
I don't see a reason why submerged plants could not suffer from fungal and bacterial attacks just like terrestrial ones. Do you really think there are no fungi and bacteria in our tanks?
It's not that there aren't any bacteria or fungi, it's that that very few bacteria or fungi are pathogenic, and even when they are they have specific infection courts on a limited range of species, often just one. I don't know what the cause of your melt is, and it sounds like it was a real crappy experience, but to have a pathogen rampage through multiple plant families in both emersed and submerged environments... well, I think if that was possible you'd see it affecting the major growers it'd be a big problem for the hobby. There are pathogens with wide host ranges, and they are devastating.
 

erwin123

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In-vitro plants are tiny and fragile and often succumb to parasites soon after planting. It's almost impossible not to damage them while handling. Can we use any commercially available systemic fungicides (and bactericides, possibly) to increase their resistance?
i have never had in-vitro plants "succumb to parasites soon after planting". .. maybe you can show some photos of what you think are in vitro plants succumbing to parasites and the experts here can give their opinions whether your observation is correct?
 

Simon Cole

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This is one of those conversations that I have been hoping for for some time. Very interesting. I sometimes ponder how lucky I was to find the UKAPS community :thumbup:. To me, the financial burden of failed in-vitro plants is a major concern and a real set-back to the hobby. It is a discussion well worth having, so thank you @_Maq_ for starting this thread.
tissue culture routinely uses plant growth regulators (plant hormones or their synthetic counterparts) in the gel media and the plants can do some weird stuff.
I have had some <weird experiences> with the synthetic phytohormone triacontanol. Would I use it to achieve well-branched in-vitro Bucephalandra - hell yes, especially if I was working with mother plants. There are quite a few others that I have used over the years that are more natural, and I would presume that agar jelly is packed with them given its constituent ingredients. You may have used liquid seaweed for the same purpose.
...a cup of in-vitro Rotala indica ...the Rotalas started to melt, and from them the melting spread further, to unrelated plants which were healthy until then.
I have <always felt> that the plant hormone abscisic acid (ABA) plays a role in these instances. Whether it is applied as a plant growth regulator during in-vitro cultivation, is actually a rather interesting question to ask. In theory, if it was applied to in-vitro cultures, then in my eyes that could enable producers to artificially shut the stomata of these plants. It seems highly counterproductive, because how would they achieve air exchange in plant tissue. But it is still possible that unscrupulous producers could be doing this to avoid the in-vitro plants from wilting when they reach the point of distribution and sale. I still think it is far more plausible that in-vitro plants generate this hormone naturally, and release it through cut tissue into the aquarium water, and that this is a major cause of melt spreading into nearby established plants, and I am fairly convinced that these types of Rotala are more susceptible to abscisic acid (ABA) based on our <shared experiences> growing them. But this possibility is not off the table. Hopefully none of the producers check this forum, or they might just double their sales after reading this.
...in vitro plants being so delicate succumb ...Could this be because the dark start entails a 100% waterchange that would remove a lot of the bacteria and parasite?
I cannot find the thread, but a member suggested recently that water conditioners have a significant impact on microbial survival and growth. I don't really have an opinion on this. I would imagine that a good microbial balance favours in-vitro plants establishing.
In-vitro plants... retarded
I beg you for forgiveness for my limited English. Please, have mercy with me, I don't want to be rude.
I am honestly never the kind of person that tries to trip people up on their terminology. Sorry to everyone I have <done this to in the past>. My psychologist thinks that I am on the autistic spectrum :crazy:. But since you write such beautiful English, and because I love reading everything you write, please allow me to imply the term "suppressed". I am retarded, but my plants are just suppressed :D
I believed that @Simon Cole could possibly suggest some fungicide which could be applicable
@seedoubleyou : @JoshP12 is one of those people with whom I just seem to have the most interesting and enjoyable conversations. We looked at the <31 most common biological control agents (BCAs)> that are used in terrestrial and hydroponic plant cultivation. BCAs can replace fungicides. I have used Trichoderma harzianum T-22 in terrestrial cultivation before, and I do keep a bit lying around in my fridge. In my opinion, they are all very cheap and exceptionally valuable in protecting in-vitro plants from microbial disease. T22 is one of the best, but it may well be genetically engineered, so bear that in mind if you don't like that kind of thing, or if you are worried about <people in white biohazard suits turning up on your lawn with placards spray-painting biohazard symbols on your front door>.
Other options might include chemical treatments: I have used potassium sorbate in the past to stop mildew, and feel that it is sometimes rather useful <during a dry start>, and quite cheap. Unlike the more harmful fungicides, potassium sorbate is naturally occurring in plant tissue and fairly harmless. On that note, I expect that poor supplier biocide/fungicide choice could be a factor attributed towards the melting of nearby established plants. I don't think this is at all likely, but it is remotely possible, because producers ask us to wash it off first, and there could be an unknown variable interacting with established plant health.
to have a pathogen rampage through multiple plant families in both emersed and submerged environments... well, I think if that was possible you'd see it affecting the major growers it'd be a big problem for the hobby
I think that the industry is probably starting to struggle with <nematode plant pathogens>. I am inclined to view other microbial plant pathogens as both highly opportunistic and rather common, but I do come down on the side of the argument that views most associated melting as a water-column plant hormone impact. My view is that once the plants starve from reduced air exchange, and their tissues start to die, then at this point their <immunity> begins to suffer as a consequence. This is most possible if there is a pH drop in plant tissue, or if fewer biochemical or biological defences are left intact and functional to ward off opportunistic pathogens. I get a bit freaked out thinking about things like infected gangrene in humans, but it seems similar. Opportunistic and common plant pathogens rarely result in disease unless there is dead or dying plant tissue available for them to exploit. Like you, I do not think they are particularly contagious otherwise.
 
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_Maq_

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@Simon Cole, if I understand correctly your response, you seem not to be much concerned with parasites, but rather with the way in-vitro plants are treated by their suppliers. Fytohormones which are beneficial as long as the plantlets remain sealed but effectively harmful after opening. Right?

This is a field virtually unknown to me. I've never realized that fytohormones can be harmful under some circumstances.

My aim is practical. I'd like to find some kind of bath to wash in-vitro plants in before planting them into the tank. Or some similar treatment. And I've used the adjective 'systemic' since I hope the plants would 'suck' it into their tissues and gain lasting protection.

Many fungal parasites are species selective, of course, but what about those which regularly attack seedlings of virtually any family (Pythium, Phytophthora, Thanatephorus, Botryotinia, Fusarium, Aphanomyces)? My experience with terrestrial plants led me to suspicion that these opportunistic parasites may be active in underwater conditions as well. Especially when soil substrates are used, and in-vitro plants - resembling true seedlings - are concerned. Is my assumption wrong? Then, why? I'd like to know.

I have potassium sorbate. Should I use it as a bath, or apply directly into the tank, perhaps? What duration and concentration would you suggest?

Or whatsoever. The topic seems to be broader than I assumed. In any case, the simple fact is that we often lose in-vitro plants. What can we do to avoid it? Please, pardon my down-to-earth approach. I like to learn new things but I'm a layman in biochemistry.
 

ElleDee

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@Simon Cole Your ABA idea is interesting, but don't think that it would actually confer an advantage. Humidity and wilting isn't usually an issue in TC since the cups are like little terrariums. They can dry out eventually, but it takes a while.

Now, about growth regulators affecting the tank generally... Hmmm. I think if you washed off the media you wouldn't be adding much to the tank, not enough to really affect anything, but different species react differently to exogenous hormone application. So I'm'm not really sure. I think there's a Seachem product (can't recall which atm) that includes a suite of PGRs and I have no idea what they are trying to achieve with that.
 

Simon Cole

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@_Maq_
Pythium, Phytophthora, and Aphanomyces - oomycetes (water moulds) - they are more closely related to algae, many cause root damage.​
Thanatephorus - true fungi, they can damage all parts of plants, usually those in contact with soil.​
Botryotinia, Fusarium - true fungi, associated with damaging leaves and green stems.​

As you mention, many adapt to reproduce under water (hydrophytes), damp conditions (hygrophytes), and many are fully aquatic. For instance, most Botryotinia probably prefer to sporulate in the air, whereas we know that most Oomycetes prefer water.

Baths can sterilise in-vitro plants, but rarely will you find a chemical fungicide that will persist once they have been planted or submerged. There have been a few persistent and systemic chemical fungicides developed over the years, but they tend to have associated toxicology. If you did start using synthetic chemical like etridiazole or tetrazole, then we might not be able to help you in terms of how safe this is for your aquarium. Certainly these chemicals are effective against oomycetes and may not harm your plants at the right concentrations, but it is hard to know how persistent they will be, or whether they will harm your microbial balance and aquarium animals. A wide variety of non-persistent chemicals are actually quite suitable for sterilising baths, but they probably do not achieve future protection. You can certainly try sprays like dilute potassium sorbate if you are doing a dry start, and want some level of interim protection against mildew and fungi that attack leaves and stems while they are exposed to the air. But when you submerge them into your tank this will be washed away. I would not suggest dosing your tank with potassium sorbate - I have to confess to @seedoubleyou that this could probably harm other microbes, like your aerobic filter bacteria (at certain concentrations), so really this is only suitable for dry starts and emergent growing.

Your best method for protecting in-vitro plants is going to be to inoculate them with a broad-spectrum formulation of Biological control agents (BCAs) before planting. The fastest and most effective way to do this is to shake your BCA powder into a suspension, and then spray it over the plants at least 72 hours before planting. If your in-vitro plants are coming in tubs as opposed to packets, then you can leave them to inoculate for a lot longer, and in some circumstances you might be able to simply sprinkle your dry BCA powder over the top of them and wait (days or even weeks). The longer the better, because you get higher rates of inoculation. However, remember that many BCAs are susceptible to chemical fungicides/biocides, and this is significant if the producer has treated them with these beforehand. There are thousands of BCAs to choose from, and those in the Trichoderma genus tend to consistently get the best results, and can offer the best all-round protection against all of the plant pathogens that you mentioned. It does get very species/variety-specific, both in terms of which pathogen is at play, and which variety of BCA defeats it. Greenhouse producers will often find that they need to try numerous strains before they find the right one to counter a resistant disease outbreak. For that reason, many commercial BCAs are mixtures of different strains and species.

There are a few BCAs that have been developed specifically for hydroponics that may be of interest. There is a lot of anecdotal support that hydroponic applications are effective, but I have seen very little research if any that has demonstrated that this is the case: One researcher feels that a <lower water pH is beneficial>, but I would take that opinion with a big pinch of salt until it has been corroborated, because it seems like a post-hoc ergo propter-hoc assumption. Other research is really still in it's infancy, so it's up to you. Potentially you could add BCAs to your water column, or mix them in with the aquarium soil, but sadly I have no idea whether this might work. The best "terrestrial" results usually come from spraying, irrigation, or mixing BCAs into the soil (usually in that order of precedence). Nobody seems to have developed any BCA products suitable for treating submerged aquarium plants. Even if they did, there isn't enough research to substantiate it would work, and it would be like trying to hit the bullseye on a dart board blind-folded. You best bet for now is to simply find a way of inoculating them before you plant them, perhaps in emergent growing tubs or with a dry start.

It would be reassuring if in-vitro aquarium plant producers considered inoculating their cultures with BCAs beforehand, and let us know what they included.

Other results I have seen indicate that some sort of UV light is effective, but this has personal health risks; and to be honest, you could just add a chemical fungicide to your water column and ride it out, aiming to establish your microbial balance and biological filtration at a later date, while the plants adapt to the stresses of growing under water. A holding tank would be rather useful for this purpose.

The point about ABA as a phytohormone is that it could potentially be added as a preservative. This would keep the in-vitro plant in a dormant state and avoid wilting until opened, but conversely could cause existing plants to shut their stomata and suffer stress if it is released locally into the water column in high concentrations, because in-vitro plants could be positively laced with ABA for all we know. This does sound like a crazy conspiracy, so I apologise, because it is exactly that. It comes from the theory that ABA can lead to odd events in aquariums after plants are cut and this hormone is released from existing natural plant tissue. On a related note, auxin seems to do <odd things> to aquarium plants. The truth is that nobody can really prove that phytohormones (PGRs) are a big issue in aquariums, but to me they seem to be the missing link to resolving a whole host of problems. Organic molecules are like that... always a bit side-lined ...but to some of us very significant. I try not to mention them too much because it gets a massive debate going about allelopathy, bioremediation, and algal blooms; and hopefully the theories will become more commonplace if they appear in future aquarium plant books.

@ElleDee - PGRs are quite easy to add to hydroponics at known concentrations. I guess that it would be easy to add them to aquarium water. To me, Purigen seems to be the antithesis: I would imagine it works by removing them from the water, which I have always felt is why people feel it works well against algal blooms. But I do think that some of the synthetic PGRs have huge potential for delivering very interesting results in terms of plant growth, and possibly no impact at all upon algal growth.
 
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Andy Pierce

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Lately I've been solving my in vitro plant challenges by purchasing potted plants instead. My main objection to the in vitro plants is morphological "wrongness" - no strongly determined "roots go down and leaves go up" - and fragility upon planting. I also have vague worries about genetic non-diversity of clonally propagated plants.
 

Tim Harrison

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In vitro can be a bit hit and miss. My experience is ammonia burn from fresh AS is definitely a thing. For instance, I”be had Rotala stems rot below and just above substrate level. Rest of the plant pretty heathy. Can be a similar story with HC and MC.

With regards actual culture harbouring pathogenic microorganisms I very much doubt it. Plants in contaminated pots would decay pretty quickly. Bad batches would be caught before leaving the lab. But it’s not to say they could be more vulnerable to infection once planted.

Ammonia burn or some other stress factor might render them more susceptible to infection. Which once it gets ahold could plausibly spread through what might appear to be healthy plants.

But never really had a problem growing them to maturity in old AS. Nor dry start.
 
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