Maybe crazy tissue culture experiment with Nepenthes seeds

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So I read a lot of research papers on TC because I was wanting to try it. I don't have a pressure cooker that is large enough OR a microwave OR an autoclave. Clearly I'd spend a lot of time dating the pressure cooker or I wouldn't be able to do TC. Or so I thought.

Then a large number of seeds I'd ordered arrived....

I came across some papers describing chemical sterilization of media in tissue culture using bleach as a part of efforts to enable low cost tissue culture that could be used in more rural communities, without laminar flow hood, for cultivation and propagation of potato, sugarcane, etc. There are several papers describing various agents tested for sterilizing different explants. The results with bleach caught my eye, mostly because I have bleach and the results seemed to be better than a lot of other alternatives they tried. Not listing any single procedure mostly because what I ended up doing didn't *exactly* match any of the research papers.

I actually have very accurate weighing scales and such, but given that I was following methods to make TC accessible in rudimentary situations, I figured I'd also attempt doing things by volume while I was at it.

The process I followed:

Media: 1/2 strength M&S salts + 4 spoons of sugar + 8g of agar + 200ml of coconut water + water to make 1 liter approx (one thing led to another and I think it was closer to 1.25 liters.

Boil while stirring on medium to low flame in an open pot. Turn off when boiled for a minute or two. Solution is slightly brown (probably impurities from sugar and agar)

Prepare TC containers: I used disposable transparent plastic containers with lids. Immersed in approx 10% solution by volume of bleach. Take out just before using, do not rinse, drain on clean surface.

Just before using = enough time to drain slightly, but not dry.

Back to media:Let cool. When cool enough to comfortably touch outside of the steel utensil, (50-60 degrees C I imagine) add one spoon of bleach. Solution turns clear-white. All that amber-brown tinge from the sugar (presumably) is gone.

Dispense media into TC containers - TC containers are not fully dry. Allow to set. Doesn't take too long, start working with seeds.

Used variation with psyllium husk to set the media in addition to agar. Worked well, but the media set really fast and I suspect is too thick.

Sterilizing seeds: My method was quick and dirty - used a plastic tea strainer and a small bowl filled with bleach solution. 5-10% or so. Plastic strainer should sit comfortably on the rim of the bowl, so that seeds in it are immersed in the bleach.

Place strainer in bowl, add seeds. Dip a couple of times (think tea bag), let sit - maybe for a minute or so. Less for seeds that were really delicate. I eyeballed it to something like "do they seem well soaked with the bleach solution? Ok good, take out". Rinsed with solution of hydrogen peroxide while in strainer - just held the strainer over the sink and ran the solution from a bottle with a nozzle over the seeds - about 10% solution of 6% hydrogen peroxide.

Use tweezers to grab "tails" of seeds and place onto solid media. I placed them well initially, all nice and separate. Later I was too exhausted and I had too many seeds and there were clumps and what not - I will probably regret these if they are the only ones to end up contaminated). Place lid (also not dry) and close firmly. Noting here that the containers seem fairly air tight.

The containers are kept in a relatively bright part of my normal bedroom.

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3 days in, so far, so good. But I suspect contamination and stuff usually starts later.

Sterile technique issues:

Space was never really fully sterile. Worked on kitchen counter wiped clean with bleach solution. Heated medium on cooking gas in open pot right next to dinner (but it was cold and not actively boiling and sending vapour into the air).

Didn't sterilize tweezers or anything other than occasional dip in bleach while retrieving seeds. That said, I didn't get them exceptionally dirty either, like touching them with my fingers or putting on dirty surface, etc.

May have wiped tweezers on shorts to remove media stuck to it. Same old shorts I wiped hands wet with bleach solution on. This was toward the end - we'll see if that created problems, I've noted the containers that came after it.

This probably is a HUGE test of how well chemical sterilization works.

TC technique issues:

Used coconut water, since one of the papers said an advantage of chemical sterilization was that you could use things that could lose nutrients with autoclaving - no idea of contents and will probably vary from coconut to coconut

Have weighing scales, but used domestic measures like spoonful of sugar, etc. This is reliable for reproducing in my home, but may create problems to publish technique. Your spoons may vary, for example. (But if it works, I can probably measure out from my spoons and give rough weights)

Fan switched off, working surface wiped with bleach and no discernable "flow" of air in room, but no laminar flow hood or other protection from contaminants either. Containers were damp with bleach solution, but left open while the agar set and were covered only after seeds were put into them.

I suspect the pH of the media is going to drop once the bleach wears out. I tried to factor this in by keeping the media pH a bit on the high side 6.5-ish, but I'll test the pH of the first jar to be found with contaminants.

Results so far:

The agar seems to be a lot more solid than I anticipated. Must use less next time.

I put them in the containers on 25th. Today is 28th. So far no sign of contamination or anything growing - seed or fungus. I suspect the fun will start once the effect of the bleach wears off - either with fungus or my blood pressure in excitement.

I am not actually expecting a lot of success here, but hoping for enough observations to tweak this technique to arrive at something that works "well enough" - or give in and buy a pressure cooker (unlikely) or return to my sphagnum moss (which I've sowed seeds into anyway).

I'm almost wary of calling this technique TC given the relatively careless way I worked compared with the descriptions I read.

Learnings:

1 liter is a hell of a lot of media requiring a hell of a lot of containers requiring a hell of a lot of space. Luckily I had a hell of a lot of seeds, so ok, but as an experimental process, I'd probably have been better off with a much smaller quantity. Contamination in 4 cultures is just as learning an experience as contamination in 25 cultures :(

And if there is no contamination, I have no idea how I'll grow that many seeds further. But I suppose I will have plenty of material for the next learning curve.

This probably worked even to this small extent because I was putting seeds into TC. I will have to be more precise with quantities of bleach if I am working with growing tissue - like if this thing works (I'm defining "works" as seeing germination before fungus in even one container) and if I end up having to take seedlings out of one container and into fresh media. Excess bleach will fry them, I suspect. Being conservative with bleach will automatically require at least more effort to improve (maybe I should say begin) sterile technique.

I suspect, if I don't actually kill these, for transplanting them to new media, I'll skip the bleach in the medium or keep it really low and only sterilize containers with it and pressure cook the media. I may also make some kind of an enclosed space (was thinking of sticking an air purifier into a window cut out into a surface sterilizable box/compartment)

Regardless, however this pans out, I'll update here, just so anyone else thinking of these crazy shortcuts knows what happened.

Please don't consider this to be a protocol anyone should follow, unless you're fine killing all your seeds as well or unless I report wild success and revolutionize TC :p
 
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There are also some containers with drosera seeds. Mostly because I had too many containers with media, drosera seeds to spare as well, and... well, as a sort of reality check, given I have a better idea of how fast those germinate and grow, in case the neps go too long without any growth - fungus or germination.

I didn't actually sterilize the drosera seeds at all, so they are more fungus candidates than germination, but I suspect all the chlorine could take care of any minor contamination on the seeds.

I am also realizing that I may have to figure out a way to get a sterile working space sooner than expected - if any of the containers show contamination, I'd like to at least try to save the non-contaminated seeds - by taking out the contamination or replating those seeds - which will be trickier than this, I suspect.
 
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The seeds right now. Some already look swollen. Not sure if they appear swollen because of some effect of the gel or whether they are supposed to swell this early. No fungus so far. There are specks and stuff that can be seen, but they are not fungus, I promise. The psyllium husk had some... stuff and I'm a bit worried about germinating psyllium now :p And there are some air bubbles in the medium too. But so far, nothing looks "off".

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Some of the seeds look a bit faded though. I think the bleach may have "got" them? Was not able to capture them on camera properly. Some of those too appear swollen, but the color is quite faded apart from slight darkness in center of seed.
 
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Interesting write up. I don't have too much experience with tc but from what I can see your methods were sound. All the seed you have seemed to have large, viable embryos so I imagine that you'll have some germination before too long. Keep us updated, it'll be very exciting to see if you do have success with this.
 
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One week. So far, so good. No contamination or germination in any of the 24 containers.

Getting slightly worried even by the lack of contamination. If I have nuked the contaminants so thoroughly with relatively primitive methods, WHAT HAVE I DONE TO THE SEEDS? I'm worried that I may have fried them. At this point, almost hoping for contamination to reassure me that it is possible for something to grow in that media I made. But I will hang on and hope for germination instead. Even one damn seed will be good.

But I guess one week is too early. The fastest nepenthes germination I've seen to date was 13 days for the Nepenthes Echinostoma (this isn't saying a lot, since I've never had any nepenthes germination other than those few Nepenthes mirabilis var echinostoma and another few of Nepenthes Rafflesiana yet) and that was not in vitro.

Next time I'm going to do tomato in one container just to reassure me.
 
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So far, so good with the Tissue Culture containers. No contamination, no germination either. It is not yet two weeks, so not too alarmed.

OTOH, the same seeds sown on sphagnum moss had quite a bit of fungal contamination. Removed obviously contaminated seeds, sprayed H2O2, keeping an eye. May use some fungicide if more contamination appears. (Edit: The fungus is growing on the seeds, the sphagnum seems fine)

This is strange, because I rarely get fungus on my seeds, and was fully expecting my rudimentary TC to get contaminated. The opposite has happened!!!

But then, I haven't grown a lot of nepenthes and I hear nepenthes seeds get fungus easily, so... I suppose it shows how TC can prevent contamination or something. Or it shows how ignorant TCers can sterilize everything - fungus AND seeds? ???
 
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I also planted most of the leftover seeds into (well rinsed) cocopeat yesterday to avoid storing them for too long and experiment with yet another substrate and have very very few seeds of one or two species left. Am wondering if I should try taking off the seed coat and putting some each on sphagnum, cocopeat and TC. Working week ahead is too hectic. Maybe next weekend.
 
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It's been my experience that fungus will only grow on non-viable seeds. I've never seen it spread from those seeds either. It's normal to happen to a few after a week or two, but hopefully that won't happen to too many of them.
 
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It's been my experience that fungus will only grow on non-viable seeds. I've never seen it spread from those seeds either. It's normal to happen to a few after a week or two, but hopefully that won't happen to too many of them.

Oh! ok. This is very reassuring. Thank you! You are right in the sense it doesn't appear to be infecting all seeds - there are completely untouched seeds right next to ones fully covered - and it has appeared after a week has passed. So it is ok to do nothing? Or still a good idea to at least keep an eye and remove any infected seeds?
 
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Oh! ok. This is very reassuring. Thank you! You are right in the sense it doesn't appear to be infecting all seeds - there are completely untouched seeds right next to ones fully covered - and it has appeared after a week has passed. So it is ok to do nothing? Or still a good idea to at least keep an eye and remove any infected seeds?

Yeah I usually don't bother doing anything when that happens, because it doesn't seem to matter.
 
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It's been my experience that fungus will only grow on non-viable seeds. I've never seen it spread from those seeds either. It's normal to happen to a few after a week or two, but hopefully that won't happen to too many of them.

I have seen viable and non-viable seed succumb to fungal infestation over the years; and it is reckless to risk the remaining culture. Subculture any flask that shows contamination. You'll be sorry otherwise . . .
 
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I have seen viable and non-viable seed succumb to fungal infestation over the years; and it is reckless to risk the remaining culture. Subculture any flask that shows contamination. You'll be sorry otherwise . . .

Pretty sure Nimbulan was referring to seeds planted in vivo, not in vitro.
 
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I have seen viable and non-viable seed succumb to fungal infestation over the years; and it is reckless to risk the remaining culture. Subculture any flask that shows contamination. You'll be sorry otherwise . . .

BigBella, these are seeds sowed on the same day on sphagnum. The in vitro ones are now almost two weeks in the container and there is neither fungus nor germination.

I mentioned it here, because the seeds would be a sort of "control" I suppose, for germination, but instead of the contamination I was expecting in vitro, I have seeds on sphagnum getting contaminated (never happened before this), while my clumsy tissue culture attempt seems to be fine so far - all 24 containers!

Edit: Since you have done extensive TC, do you know if I can consider my cultures to be reasonably sterile and safe from contamination if nothing has shown up in 13 days? Also, should I worry about germination also resulting in contamination from inside the seed? Does that sort of a thing happen? (I am asking in the sense of whether I should be ready to sub culture at a moment's notice even now or I can breathe relatively easy...)
 
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Edit: Since you have done extensive TC, do you know if I can consider my cultures to be reasonably sterile and safe from contamination if nothing has shown up in 13 days? Also, should I worry about germination also resulting in contamination from inside the seed? Does that sort of a thing happen? (I am asking in the sense of whether I should be ready to sub culture at a moment's notice even now or I can breathe relatively easy...)

You can be reasonably certain that the cultures are "clean" after almost two weeks. Contamination is usually obvious well before then.

If the seed was intact, there should be no issue; though, you should have some fresh media in hand, in case you need to subculture any of the plants. Contamination, if any, can rapidly spread.

Be patient as well. The fastest germination that I had ever experienced was just short of two weeks; and the longest, several months . . .
 
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I have germination in the drosera seeds planted on sphagnum. Nothing in the TC. Neither contamination nor germination. No germination of nepenthes in sphagnum either.

Anyone know what kind of a difference in germination time is normal between regular media and TC?
 
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I have germination in the drosera seeds planted on sphagnum. Nothing in the TC. Neither contamination nor germination. No germination of nepenthes in sphagnum either.

Anyone know what kind of a difference in germination time is normal between regular media and TC?

In my experience, the in vitro seeds were the first to germinate, more than 75% of the time -- by an average of twelve days. Nepenthes seed generally have a short shelf-life, especially lowland species; but it also takes quite a bit longer than most Drosera to germinate . . .
 
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4 seeds of nepenthes maxima "giant wavy" germinating on sphagnum. Nothing in TC yet. No fungus, no germination. But some seeds seem.... swollen.

Several other nepenthes seeds are also starting to germinate on sphagnum - very early hints of green.

Nothing in TC :suspect:
 
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