mrbungalow

I have thought about it for a long time, but I am going to try the tissue culture kit available from Kitchenculture-Kit

Among other things, they say you can specify the mixture to be banana or other plants.

Can anyone verify that this is a good product? Also, I am not sure what part of the plant is the "meristem"? Procedures? Tricks? From what I understand, PPM is a necessity to avoid contamination of cultures.

Any general info or help would be greatly appreciated!

Erlend

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JoeReal

the meristem are equivalent to the "eyes" on potatoes, but instead on potatoes, these are located around the banana corms, you can easily spot them after you clean up the corm of roots, soil and dirt.

PPM, isn't that concentration abbrev. for parts per million? If you can reduce contamination to less than parts per million, then your kitchen hood would be sterile enough for tissue culture. But anyway, it is Plant Preservative Mixture biocide, just don't know what kind.

mrbungalow

I like the way you think Joe, that's what I thought too, "Parts per Million". But apparently it stands for "Plant preservative material". Some smart guy has invented it and now sells it at a blood-price. They say it's crucial for avoiding contamination.

Thanks for info on meristems.

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D'Andra

I'm going to do some more research on this. I just talked to a friend about PPM, and he said if you have a very sterile enviroment it is not usually needed. I'm not positive what he uses but it sounded to me like we have a couple of options. He gets antibiotics from a livestock feed store and makes a mix of something. I will find out in the next couple of days what exactly it is he uses.

I also read that you can use NaDCC. I'd imagine you can get this at a pool and spa store since it's used as a disinfectant for swimming pools. It may be sold as sodium dichloro-s-triazinetrione. I have no idea how to mix with your media though.

My friend has been doing MP of mushrooms for over 20 years and has almost no contamination ever. He doesn't use an expensive lab and other than a 40qt pressure cooker, I don't think he has too much invested in it. He mixes his own media. He said it's because when he started doing TC, he didn't know where to get pre-mixed media.

I'm going to be borrowing a book on the entire TC process, so I'll pass along any info I can.

We've got 3 - 50 gal aquariums, so I'm setting one up as my clean box.
A neighbor just gave me 50 baby food jars with metal lids. You can tilt or lean these jars before the medium gels, and after you put your tissue in it, store them with the medium level so your light source enters through the side of the jar. This will save you from buying photo lids for cheap or free jars.

I'll try to get back to you soon on the biocide/PPM/recipe.
Good Luck & keep me posted. (Pun intended!)

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D'.

D'Andra

Hey Erlend, JefferyP posted this link you may find useful.

www.quisqualis.com/tv03tc01p1.html

Be sure to click on "next page" tab.

Hope this helps!

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D'.

JoeReal

Based on my experience, propagating or culturing mushrooms, you don't need a sterile environment and don't even need to use tissue culture methods, it would be an overkill. For one, some edible mushrooms overpower other microbes easily, and next is that it is quite easy to harvest and select for the edible mushrooms visually.

Of course if you are doing it commercially, large scale when labor gets expensive, tissue culture would really improve quality and cut down on labor.

D'Andra

JoeReal is right about the mushrooms. I asked my friend why they do it, and it's to keep the plant true to type for commercial purposes. If you're selling under a brand name, your customers expect the same each time they purchase.

What I found out about the PPM is they put a small amount of sterile water/antibiotic/chlorine mix on top of the media when using new plants from other sources. The amounts I still haven't been able to find out, but it sounds like a very tiny bit.

I also found out that you use the apical meristem (undifferentiated tissue at the microscopic tip of a shoot/ root) because it is not yet joined to the donor plants vascular system. You are literally getting the cells before they can become contaminated by the donor plant. By doing this and proper sterilization of plant material to be used, there is not much need for PPM.

Now with that being said, I have not tried to do tc myself and there may be some reason why you would need a PPM.

When talking to my friend today, he told me to get the media, my jars, the donor plants and bring it over. He is not only going to show me how to do it, he said he'd do the first batch! I kinda want to wait until we get close to our first freeze before I pull any plants up though (mid 80's here today!). He also told me how to build a HEPA flow cabinet for about $150, and that you'll really appreciate not breathing all the bleach and alcohol that you get from a still air cabinet such as the aquarium I was going to use.

Let me know how it's going and I'll try to gain all the knowledge I can to pass along in this thread.

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D'.

mrbungalow

Thats excellent info D'andra and everyone else. I hope we can get a thorough description on TC of bananas from you in the future. I am also hoping for pictures.

I am going to use the winter to practice, so that I may have a load of plants this spring.

Interestingly, I found a publication on the internet from a 6th grader(!) who had a science-project about tissue culture of plants. He failed on all attempts, except the batch where he used PPM.
I am pretty sure I am no better than he is, and will probably use PPM.

Erlend

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JoeReal

PPM sells for $1 per ml. There should be a lot of better alternatives. Tissue Culture has been very successful long before PPM is. For one thing, there is a new invention that generates dissolved ozone in water and anything you rinse with the ozonized water practically become sterile on their surface contact. That machine is featured in one of the best inventions of the year, and its primary usage is for cleaning store-bought fruits and vegetables of pesticide, pests, and microbial contamination. It is called the Lotus Sanitizing System, but I think it can be used to soak your meristems or source material for initial decontamination:
http://www.time.com/time/2006/techgu...ns/meals4.html

The antibiotics that the makers of PPM are using, and if one is not careful, it could encourage the development of even stronger microbial contaminants. So limit its use or alternate with other methods of sterilization or use other antibiotic types. I am just worried that the use of such things will bring anew the viscious cycle of developing new antibiotics. The use of alcohol sprays, pressure cooker for sterilization are old reliable standard ways of sterilization without encouraging the microbial antibiotic's resistances. A nice enclosed hood with adjustable positive or negative pressure is a very good workbench for a lot of tissue culture. They are easy to build if these are pricey.

PPM appears to be a very expensive TC shortcut but in my opinion, it will not be doing us any favor in the long run. Practicing to be clean at each step, thinking of ways of minimizing contamination and accuracy in the measurements are some of the very good habits I have observed from a lot of my friends involved in TC. It also says that you will have to try various concentrations of PPM for each type of plant cultivar that you are trying to propagate, and that would be a lot of trials and expense.

Gabe15

I have done embryo rescue germination with bananas before, which is basiclaly TC but instead of meristem you use embryos. Contamination was my biggest problem and immediatly kills the cultures. Everything needs to be sterile, it is very important because the medium that is used will grow mold overnight if not sterile.


D'Andra,
Its really not a matter of wheather your meristem is sterile, its everything else you use, your tools, your medium, your growing environment. During my trials last year I used a UV lamp and anhydrous ethanol on everything except the embryos and even then, mold took over everytime. I am doing a similar project this year and have already purchased over 100ml of PPM, it seems like it will really help.

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Growing bananas in Colorado, Hawaii and Washington since 2004.

mrbungalow

I have worked in a modern bio-technology lab before, and remember using some of the lab-equipment for my private plant-hobby. (Don't tell anyone!)
Anyway, I used the autoclave to sterilize soil before I used it for planting palm-seeds etc. I still got mold on some of the boxes, even though I dipped seeds in bleach-solution first! I think it's probably impossible to get everything sterile, unless you have a perfectly sterile environment. Modern sterile-benches have a flow-hood that heats and cools air coming out. I can guarantee you, that my kitchen is far from sterile, in spite of all that good scandinavian cooking!

A smart professor once said "Never underestimate the power of microbes".

This sixth-grade students' experiment I mentioned earlier was interesting. Such a dramatic difference with PPM. And kids have a way of putting things simple.

But offcourse, I understand what you are saying. The TC-industry has obviously done well before the PPM-era, and there probably are alternatives.
And I am not thrilled about using antibiotics needlessly.

Erlend

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Gabe15

I dont think PPM is an antibiotic per-se, they really dont tell you what it is because they want to keep it secret, they call it a biocide. Its not limited to bacteria either, it also kills mold. Also looking at definitions of antibiotic, many of them state that an anbiotic is created by microoganisms, I doubt PPM is, but really it comes down to you dont know what it is at all.

I should also say that with my trials last year, there was absolutly zero bacterial infection, but that did not stop mold spores from getting in.

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Growing bananas in Colorado, Hawaii and Washington since 2004.

JoeReal

I come across some references that PPM contains antibiotics as a major ingredient but as to what type, we can only speculate.

A positive pressure hood is quite easy to construct. And if I were to design this now, I would need a hepa filter whose intake air are first bubbled through UV bombarded water (can buy this equipment now), then ozonized water (can also buy this now). The air being sterilized and humidified keeps pushing out through the leaks and you can work easily in a reasonably sterile environment (will have to sterilize the whole enclosed area once in a while with strong biocides), as there will be no backdrafts or accidental intake of air through other means. Without positive pressure with a sterilized air, there will always be contamination. The hood is very important if I were to do TC in the future, that will be my major workbench.

wim

Erlend,

Can you give us the URL ?

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With kind regards
Wim

PaulOdin

http://aggie-horticulture.tamu.edu/t...microprop.html

Maybe you've all seen this page, since it is accessible through Agri-Starts, but it is a list of links for tissue culturing. The kitchen kit is on it, and so is a how-to slide show for tc, and other links.

Paul

mrbungalow

Sure thing, Wim; Eric the 6th grade Tissue-Culturer

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JoeReal

I was the very first person in this forum to post about tissue culture or micropropagation of bananas. The same kits were also sold at that time. Check this out as proof. First posted July 2005, and no one else before that:

Micropropagation of bananas

JoeReal

Only one of the old links remain active. The one that has the link to the kit is now long gone. That was July 2005.

jeffreyp

Erlend,

I'd also suggest Carolina Biological...

https://www2.carolina.com/webapp/wcs...y_rn=&crumbs=n

jeffreyp

I remember Keith benson's posts and website on tc of banana plants back in 1999. But who really cares anyhow?