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Post by palotz on Aug 27, 2006 16:59:52 GMT -5
I was wondering if someone can help me out. I would like to try doing some Tissue Cultures and was wondering if the basic media on this website is where to start ? www.omnisterra.com/botany/cp/slides/tc/tc.htmHomemade media: 1/8 cup table sugar 1 cup water 1/2 cup stock solution (Miracid diluted 1/4 tsp in 1 Gallon water) 1/2 inositol tablet (125mg) 1/4 vitamin tablet with thiamin 2 Tablespoons agar flakes What do if anything do I need to add to this ? Do I simply follow the directions and add seeds to this media ? Did I read correctly ? Do you need to transfer to three different containers over time or does plant grow in this media and then potted once grown? Sorry for my lack of knowledge in TC, but it sounds very interesting so I wanted to try it. Cheers, Anthony
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Post by tom on Aug 27, 2006 17:22:27 GMT -5
you got the homemade recipe right (although I've never tried it), but you need some sterility informations (I think it is described latter on that page).
The recipe is what you mix and boil in a pan. Then you dispatch this in your jar (either Mason Type or baby food jar) and need to autoclave it (ie cook them in your pressure cooker, time will vary depending of the pressure (PSI) you can get into it).
Now that you have sterile media, you need to sterilize your seeds. The media is perfect for plants, but also for fungi, bacteria, yeasts... Everyone love it, but you want only your seeds/plants to enjoy it. So you need to give your seeds several baths of javel water solution, alcohol, peroxyde, etc (depeding of your protocol) and then rince them in 3x 5 minutes of sterile water baths. By this, you want to rince the sterilizants products of your material (to prevent it to kill your seeds/plants), but you dont want to reintroduce spores or others living things. Once the material is out of the sterilizing solution, you need to be into a very clean area, and work closely of a water or in live steam, for every next steps of the procedure, until you have introduced it (right after the 3 rinces) into jar and seal it tight. Everything used need to be sterile (tools are flamed, media and water have been pressure cooked, etc).
I'll try to find some more info on the procedure soon
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Post by palotz on Aug 27, 2006 19:37:01 GMT -5
Thanks Tom, I didn't think it was that simple. The most important part has to be the sterilizing. A couple more questions: How many baby jars do you think this will fill ? Also, I have a baby bottle sterilizer that goes in the microwave. It's a dome thingy that you add some water to the bottom. Could I use this to sterilize the jars with media ? store.babycenter.com/product/feeding/bottle_feeding/warmers_and_sterilizers/3261cheers, Anthony
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Post by tom on Aug 27, 2006 21:18:25 GMT -5
Well, it is not 'that' easy, but you have to try it to see it (and for fun!). Sterilizing seems to be one of the most difficult part, but it is not in most cases. Getting the plants multiplying at a sufficient rate to cover the 'running operation' cost (when you're comercial), and acclimatizing (which is a major problem with some species) are harder to overcome in specific case. Plus you have to find the 'right recipe' for a lot of things: the right javel concentration and time of sterilization, the right media (MS media isn't the only one), the right hormone balance and interactions, the 'touch' for manipulation without major contamination, several transfert to avoid phenolic compounds build up if there is any... and in the end, getting the plants doing what you want them to do (ie rooting, make a callus, growing, flowering, etc). The number of jar filled with media depend of how much you want to put it them 30 ml is a good average. Don't forget that if you use the original lid of baby food jar, that metal and microwave don't match at all... Look for the Kitchen Culture kit website, you'll find tons of informations there, and safety informations too. Microwave, pressure cooker and some hormones aren't inoffensive: some precautions must be taken to used them properly.
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Post by sdeering on Aug 27, 2006 21:41:06 GMT -5
Go to the new and used store and find a pressure cooker for sterilizing, you can find one for 5 to 10 dollars you will know the media is sterile, if the mike doesn’t do it its lost time and media. 15 pounds for 15 min. after it is up to pressure is my rule. I sterilize all my seed and tissue with 10% household bleach 90% water 1 drop soap for 10 min + or-. Remember once things are sterile you have to keep them sterile while working with them. I call them weeds (mold,fungus,bacteria) are everywhere, hands, air, breath, even inside some plant tissue. I get my media from www.phytotechlab.com in the US. Shipping is more than the media. The media is cheep like 1 to 3 dollars per liter. By all means if you have the stuff try it. It may work but the real stuff may work 10 times better and faster. I use 10 to 20 mills/ small baby jar. Watch out for mites getting in the jars= contamination, they drag in spores. TC is quite labor intensive. I find if I take shortcuts I get contamination. It is very frustrating after 2 months of looking after stuff to have to through it out. It is almost impossible to keep everything 100% sterile, so plan on throwing some out. There is lots of info on the net now on the whole procedure if you look for it. Good luck, it is fun when it works. I have found I have way better luck germinating seed in TC than normal processes. Stephen
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Post by palotz on Aug 28, 2006 8:07:50 GMT -5
Thanks for all your help guys any other input would only help. I didn't mean the process was simple. I was refering to the media, I didnt think a simple recipe like the one above could work. I know the process could be frustrating and keep up quit a bit of time thats why I still haven't tried it. It would just be a small operation to satifiy my curiosity. cheers, Anthony
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Post by tom on Aug 28, 2006 8:36:26 GMT -5
Ah, i got it wrong . As sdeering told you, commercial media should work best, as they made their proof and are more accurate. The sources of N, P, and K in them has been chosen especially for their usefulness in vitro (and in a few special media, such as Vacin & Went, they are speciafically chosen to have a low salt contents, etc), and the fertilizer you'll mix in your homemade mix will not necessarily have the same compounds as a source of those macronutrients. Give it a try anyway, but don't be discouraged if you fail the few first times, it is normal (even if you have commercial media ). Keep us posted on your progress! For procedure, you can also take a look here: www.world-of-carnivores.com/tissue_culture.htmlI haven't tried his protocols but by what i know, most seems reliable.
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Post by sdeering on Aug 28, 2006 20:56:14 GMT -5
If you look up www.phytotechlab.com they list all of the ingredients for all their media. There is very small amounts of alot of stuff. If you have the home made ingrediants you should give it a try anyway. You nver know it may work. Stephen
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Post by palotz on Aug 28, 2006 22:00:25 GMT -5
If you look up www.phytotechlab.com they list all of the ingredients for all their media. There is very small amounts of alot of stuff. If you have the home made ingrediants you should give it a try anyway. You nver know it may work. Stephen I would get some but I'm not sure which one to get ?? Which one do you use ? cheers, Anthony
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Post by tom on Aug 28, 2006 22:16:30 GMT -5
MS media (Murashige & Skoog) is the most widely used media in plants tissue culture, it is reliable, good for a wide variety of species and is cheap. A good start for most CP is 1/2 of the dose, but some plants like it even lower (1/4 to 1/5).
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Post by palotz on Aug 28, 2006 22:33:19 GMT -5
MS media (Murashige & Skoog) is the most widely used media in plants tissue culture, it is reliable, good for a wide variety of species and is cheap. A good start for most CP is 1/2 of the dose, but some plants like it even lower (1/4 to 1/5). There are many kind's of MS media when I go to the website. Which one would it be ? Also, does it already contain gelling agent ? (agar or whatever) cheers, Anthony
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Post by tom on Aug 29, 2006 20:32:28 GMT -5
I would go for the basal salt MS (containing both macro and micro nutrient) "M524 MURASHIGE & SKOOG BASAL SALT MIXTURE CONTAINS THE MACRO- AND MICRONUTRIENTS AS DESCRIBED BY MURASHIGE & SKOOG (1962). PLANT TISSUE CULTURE TESTED"
By its description, neither sugar or gelling agent are present.
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Post by palotz on Aug 29, 2006 21:11:24 GMT -5
Thanks Tom, Just as I thought. I have been looking for agar flakes but I'm having a hard time finding some locally. The media is cheap but shipping is very expensive. I will try the media out when I can convince myself the shipping is worth it. cheers, Anthony
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Post by tom on Aug 29, 2006 22:20:54 GMT -5
I'm sure this seller also sell gelling agent: either agar or Phytagel. If not, or if you prefer finding it locally, check for the health/organic foods stores. Agar is used as a gelatin (Knox) replacement in food and work well in TC too
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Post by lloyd on Oct 10, 2006 10:56:50 GMT -5
I'm getting ready to get into TC. So far I've been checking out the websites mentioned here and the recent article in the CPN on TC of VFT flower stalks. The CPN article suggests these "specialty" items: Basal MS salts, Casein, Inositol, NAA, 2iP, and agar. I have a few questions.
Phytotech sells 1 L of Basal MS salts M524-1 litre. This is the smallest amount. Is it a reasonable amount to buy?
Are the casein, inositol and agar available from health, drug or grocery stores just as good as the Phytotech stuff?
Do I need more or different hormones than NAA and 2iP?
Does anybody use the biocide PPM and if so has it proven useful?
Has anybody bought from Phytotech? It seems they ship UPS which adds outrageous handling charges to get across the border. If anybody has bought from them what were the S&H charges in total?
Thanks.
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