View Full Version : Going to buy the Tissue Culture kit
mrbungalow
11-07-2006, 03:51 PM
I have thought about it for a long time, but I am going to try the tissue culture kit available from Kitchenculture-Kit (http://www.kitchenculturekit.com)
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
JoeReal
11-07-2006, 04:31 PM
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
11-08-2006, 01:20 AM
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
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.
D'Andra
11-08-2006, 03:27 AM
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!)
D'Andra
11-08-2006, 03:38 AM
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!
JoeReal
11-08-2006, 09:59 AM
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
11-09-2006, 02:56 AM
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.
mrbungalow
11-09-2006, 06:24 AM
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. :2785:
Erlend
JoeReal
11-09-2006, 11:01 AM
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/techguide/bestinventions/inventions/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
11-09-2006, 01:17 PM
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.
mrbungalow
11-09-2006, 01:19 PM
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.
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! :0493:
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
Gabe15
11-09-2006, 01:28 PM
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.
JoeReal
11-09-2006, 02:19 PM
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.
Erlend,
Can you give us the URL ?
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.
PaulOdin
11-09-2006, 04:39 PM
http://aggie-horticulture.tamu.edu/tisscult/microprop/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
11-09-2006, 04:40 PM
Erlend,
Can you give us the URL ?
Sure thing, Wim; Eric the 6th grade Tissue-Culturer (http://www.kitchenculturekit.com/ericnews.htm)
:doggyandnaner:
JoeReal
11-09-2006, 05:12 PM
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:
http://www.bananas.org/showthread.php?t=55&highlight=micropropagation
JoeReal
11-09-2006, 05:14 PM
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.
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:
http://www.bananas.org/showthread.php?t=55&highlight=micropropagation
jeffreyp
11-10-2006, 04:18 PM
I have thought about it for a long time, but I am going to try the tissue culture kit available from Kitchenculture-Kit (http://www.kitchenculturekit.com)
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
Erlend,
I'd also suggest Carolina Biological...
https://www2.carolina.com/webapp/wcs/stores/servlet/ProductDisplay?jdeAddressId=&catalogId=10101&storeId=10151&productId=39591&langId=-1&parent_category_rn=&crumbs=n
jeffreyp
11-10-2006, 04:22 PM
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:
http://www.bananas.org/showthread.php?t=55&highlight=micropropagation
I remember Keith benson's posts and website on tc of banana plants back in 1999. But who really cares anyhow?
mrbungalow
02-22-2007, 04:48 PM
OK, it took some time, but now me & a friend are ready to TC. We didn't go for the TC-kit, but bought the stuff we needed from Phytotech-Lab. They shipped fast and cheap, and answered our questions the best they could. (Even though they probably got tired of hearing from 2 crazy Norwegians!) They have 2 different culture mediums specifically for bananas: Basal medium and Multiplication medium, the latter you have to add sugar. You also need agar, and they reccomended special lids for the containers, wich in our case are baby-food jars.
Apparently, it's possible to TC very young seedlings. I am still confused about wich part of the banana-plant you sterilize and use for your very first step-culture. I have read you can use almost any part of the plant with meristomatic cells, even the flower-stem(?) In that case you could theoretically TC grocery-store bananas if you take tissue from where they "hang together", but as I said, I am very confused. Some people talk about chopping the plant-material into a fine paste, and TC this. It seems some people also go inside the stem, just above the corm, and take tissue from here. If anyone can verify and/or explain further, I would be very grateful.
Erlend
chong
02-22-2007, 06:14 PM
I remember Keith benson's posts and website on tc of banana plants back in 1999. But who really cares anyhow?
For over 5 years now (I think), I've been engaged in email exchange with this forum started by Carol M. Stiff, PhD, who also owns "Kitchen Culture Kits" (www.kitchenculturekit.com ): hometissueculture@yahoogroups.com . This forum has wide following around the world, although most of them are here in the US and Canada. At the end of last year, she had a sale on her equipment and kits. I just don't know if the sale is still on.
Keith Benson is a consistent contributor in this forum and has discussed his home banana micropropagation techniques here. I thought I saved a copy of his article on banana TC culture on the web, but I couldn't find it. He made it look easy.
Most of the members of that forum will disagree with Joe's statement that "you don't need a sterile environment". I realize that he was refering to the culture of mushrooms. But in the case of bananas, which is akin to orchids, sterility is of outmost importance. Gabe has already decribed his experience on this.
As someone who has designed mechanical systems for clean rooms in the high-tech/bio-tech/pharmaceutical industries, I am very much impressed by the resourcefulness of many of the members in designing/making their own equipment, e.g., hoods/clean box, etc. Using anywhere from melamine laminate lined particle boards to rubbermaid plastic storage boxes for their hoods/clean box. Of course, they can't make your own HEPA filters, but there are other sources of HEPA filters than the high tech industry suppliers. Even home depot sells them. Then, if they have the correct size fan, depending on the level of cleanliness you want to achieve (the amount of cfm determines the Class), access gloves, and closures, they're all set.
PPM may be expensive, but if you consider that you can use ordinary materials with it, like distilled water from Walmart, compared to chlorinating/h. peroxide treatment, ozonating, UV irradiation (which if the water processors are supposed to do anyway) by yourself, it might be a trade-off. And PPM stays with the material longer than those other chemicals.
As a friend of mine would say, "My 2 cents."
D'Andra
02-23-2007, 08:29 PM
Hi Erlend,
Do you need to get both media or just one? Which did they suggest? What are you going to use as a hood? I didn't see any medium for bamboo on the website, but I haven't called them yet to ask.
I also am very close to doing some TC. I have a 2'x2'x4.5" 99.99% .3 filter and a motor from some old computer equipment (400-450cfm). I'm not sure what I'm going to use to build my flow hood yet. Somebody may suggest sizes of cabinet to build here (Chong?). I've got everything I need except the cabinet built, phyto-lids and some media. I only have around 200 small jars and 10 larger jars.
On the subject of where to get tissue. I've never heard of using bananas from the store but it really wouldn't cost much to try it. Have you ever read 'Plants From Test Tubes' by Lydiane Ryte? This and help from a friend who has done TC for years is what I'm using.
Good Luck!
Hi Chong,
Any help and guidance you can give me on materials and size to build would be greatly appreciated! If you can help me to get this set up right, it will just prove to the world you are the "King Of Clean!". Thanx!
mrbungalow
03-02-2007, 08:14 AM
OK, I tried my first tissue culture now. As explants I utilized the tissue located just above the corm, inside the stem. Common sense made me believe this is where meristomatic cells are located. Can anyone verify if this is right or wrong? Can you utilize other plant-parts in other techniques? Anyone? :2140:
Gabe15
03-02-2007, 09:29 AM
Erlend, yes that is where the meristem tissue is located. It is also located in whats called the infloresence-tip (where bracts are produced during flowering) and also for some varieties they have successfully used parts of the male flower.
mrbungalow
03-02-2007, 04:18 PM
Thankyou Gabe!
How far up in the pseudostem can you go? Just above the corm or even further up?
Erlend
bigdog
03-02-2007, 08:26 PM
Erlend, imagine if you were peeling away leaves and leaf bases from the pseudostem. Eventually, you would get down to a small nub in the center, just above the corm. That will be your apical meristem. On a more mature corm, you can use the lateral meristems that have formed, but haven't yet started to grow. This way, you don't sacrifice an entire plant. Good luck with it! I just had my first TC lab at school yesterday. African Violets and Crotons :( . Oh well! Bananas are next...
mrbungalow
03-03-2007, 03:16 AM
Thanks bigdog, I am starting to get it now. I believe I may have left a little too much leaf-bases on the ones I have in culture at the moment, but next time I will be certain about the explant!
Offcourse, I will let everyone know how easy or hard I found the whole process of TC.
Erlend
Mark Hall
03-03-2007, 02:04 PM
Erland do you know how long you will have to wait to find out if its started to grow is it days weeks or months ?
Are there any step by step (WITH PHOTOS ) on how to do it.
D'Andra
03-06-2007, 08:49 PM
Erlend, did you try making a paste out of any of the tissue? Which medium did you use? Keep us posted.
AnnaJW
03-06-2007, 09:12 PM
Erlend,
I am so interested in hearing about your progress on this!
Good luck, and I hope you have great success!!!
mrbungalow
03-07-2007, 01:30 AM
Mark: I found it very difficult to find beginners info on the net. There is Keith Bensons' page, but that one is down for the moment. I have been searching inibap.org for articles. None of wich have photos and all of wich use professor-language! I have heard 2 weeks is when you should start to see small shoots forming.
DAndra: No, did not make a paste. Just used tips of what I believe (hope) to be the atypical meristem. Used the banana basal medium from phytotechlab.com. They also have a multiplication-medium wich I will use for my first subculture. They said you don't need to add sugar to the basal medium, while the multiplication-medium needs sugar added. Offcourse, I also used PPM wich has a psycho price of diamonds.
AnnaJW: Thankyou! I will keep everyone posted on the progress. But I am afraid I will have to try a few more times. Right now we are "benchmarking", checking for things that have not gone optimally, and searching for "best practice"!
Erlend
RobG7aChattTN
03-16-2007, 10:15 AM
Look at www.une.edu.au/agronomy/AgSSrHortTCinfo.html. There is a lot of infor about creating your own medium and sterilization. I also remember reading that you can transfer sterilized plant material into a sterilized container of media on a cutting board over a pot of boiling water. The steam is supposed to kill any microbes in the air. Plant material is sterilized in a 10% bleach solution and the agar and container are sterilized in a pressure cooker.
RobG7aChattTN
03-16-2007, 10:21 AM
Sorry, it's changed to www.une.edu.au/agss/hor/horticultural-science.php and is the same article I was referring to.
RobG7aChattTN
03-16-2007, 10:24 AM
n/m
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