TISSUE CULTURE and also called micropropagation

This is a my tutorial of tissue culture. Tissue culture is the technique to grow a new plant of little piece of the plant. It can be a very small piece plant or pollen. If you must grow it from a little piece of plant ,example 1mm *1mm then you have first grow callus and the callus is a multiple cell division and it´s looks like lump of green and pale yellow blob. Callus cells are those cells that cover a plant wound. Those cells are undifferentiated cells and you can grow a new plants of that. They called a explants. Firs you have grow callus and move to a different media and then you can grow multiple explants of it. That is cloning a plant.
If you have a piece of stem then you can stick straight on a media and then the stem will growth a multiple stems on it. That it called "shoot" you need in stem a meristems and those are things when the branch divided or growing a leafs and buds. Specifically, an active apical meristem lays down a growing root or shoot behind itself, pushing itself forward. Apical meristems are very small.  
In a last year i was interested of tissue culture and order my plant hormone and growth media from http://www.hometissueculture.org/ and those pages is good information and kits to order. It´s good start in there. Nowadays I order from ebay my plant hormones and growth media and it cheaper that way .But I haven´t yet test it. Is they a good for tissue culture? Let´s wait and see!  
Usually the growth media contain Gelling agent and I use pure agar for it. And It´s contain sugars, vitamins and plant hormones such like kinetin and Indole-3-butyric acid(IBA). There are a three basic hormone group , Those are Auxins, Cytokinins and Gibberellins.

Auxins produce roots basically, it´s good for a cuttings. Too much of this will kill your plants. Usually you use auxsin like this, indole-3-acetic acid (IAA)(can make DMT from this), indole-3-butyric acid(IBA), 1-naphthaleneacetic acid(NAA) And this is unusually used phenylacetic acid (PAA) you dont can´t find this because you can make meth from it. It´s banned in many country or you must have a company who use such chemicals. You need auxins for cell division. Auxins and cytokinins are present in equal levels you can make callus.

Cytokinins are the things you use cloning that promote cell division, roots and shoots. And it´s crucial componet for growing callus and you need it for everything. There are cytokinins like this, Kinetin, Zeatin and 6-benzylaminopurine (BAP). Zeatins are too much for my pocket, but they are powerful that normal cytokinins.  Stem cloning or plantlets growing ratio of cytokinins and auxins are the cytokinins are the leading hormone.

Gibberellins regulate growth and influence various developmental processes, including stem elongation, germination, dormancy, flowering, sex expression, enzyme induction, and leaf and fruit senescence. Gibberellins are good for germination and you can germinate seeds that is hard to germinate. percentage point is greater than typically. You can raise the seed germination from 20% to 80% , but you have to find good ratio of gibberellins PPM(parts-per-million) and water .  I use Ga3 for seed germination only and i don´t use in growth media. I Only diluted in water and soak seeds in there 10-30minute. And i have use IPA (Isopropyl alcohol) to dissolve Ga3. You need your IPA to be 70%-90% even pure to dilute Gibberellins, Then you can dissolve in water. I use usually 300-3000PPM ratio but it have to find good ratio for every type of plant. I think you have dissolve every plant hormone fist in 70% (or purer) IPA or another alcohol if is a powder and then you can mix with water.
There are a how many more hormones and growth regulators such i don´t need mention and i don´t use.

Now in the growth media . You need sugars in your media and you can use sucrose, dextrose, fructose and I think it can use glucose. Plants need Carbohydrates to grow . I use plain sugar (sucrose). Usually i use in a liter 20-60grams of sugar. I Used 20grams sugar in pereskiopsis and salvia.

Solid media are generally composed of inorganic salts plus a few organic nutrients, vitamins. I buy Murashige and Skoog basal medium with vitamins and there are the nutrients and vitamins what I need. There are several ready made-up compounds what you can buy. You can make your self if you don´t what to buy it. You can use magnesium pills and inositol pills(Vitamin B) and thiamine (Vitamin b1). You can start there but i think it´s better to put all nutrient in media. There are Macro-and Micronutrients, Macronutrients are C, H ,O ,P ,K ,N ,S ,Ca ,Mg( required content in the plant- 0.1%or%per dry weight). Micronutrients are Fe, Mn, Zn, Cu, B, CL, Mo (reguiment-ppm/dry weight) Some plants need Na, Se and Si.

Lets speak of gelling agents, there are agar agar that is a usual used stuff but there are others gelling agent that used. Agarose, carrageenan and gellan gum. Agar is a good stuff but you don´t want to use that what is in your local Chinese or Japanese shop or your markets because it´s not pure agar And you need pure. If is´n pure there are lot of things what you don´t want in the media. Something can fuck up your media. Too high Ph or low and Minerals what you don´t want. So turn your chemical supplier or order from internet. Ebay is good. But i have ripped off on ebay when i order pure 100% agar. I never got my product from there. I lost my money! So i think there are a few good online shops in net where you can order. Usually you use 6-9 grams of agar in the liter of media then you get it firm and gelly. When you use lower amount of agar then you can do liquid technique. I don´t know nothing about of it. I never have a acquainted at that.  But you need the media to be firm and jelly if you stick the piece of stem in it .  
Ph. must regulated and every plant have a own ph. where they like to live. So you must find it or try it , but usually its little bit of acid or neutral.  5-7 ph.

Next thing is very important. You must to do everything in a sterile place and sterile equipments. You must be sure of chemicals at they are sterile.  You have clean every surface whit bleach or alcohol 70%( ethanol or isopropanol) Or purer. Bleach usually comes 5% and i think is a good for cleaning surfaces. You need milder bleach if you using to sterilizing plants. Below 1 % is good to clean plants and you have stirred it to 5-20 min. Then you rinse it in distilled water and then you put the piece of plant in the 70% alcohol in a few minutes. Then rinse three times of distilled water. You have to figure out how long you can disinfect the plant your are using .Trial and error. Some plants are durable and some plant don´t handle too much bleach or alcohol. Try to get the piece of a plant from cleanest place and newest growth. Try to keep the plant in a one peace when the cleansing project happened and then you can cut it for suitable pieces.

Materials what you need in this project is easy to obtain and does not cost too much. Things you need :
Scalpel, long Tweezers, Baby food jars(or tissue culture jars), Plastic caps( transparent), plates, Pressure cooker(or autoclaves), Aquarium (terrarium or transparent plastic box), Micropore tape (or breathing tape), Glove box (or laminar flow hood), Breathe masks( dust mask), Plastic bottles (glass bottles), Measuring cups (measuring glass), Syringes 1ml,2ml,10ml,50ml, Ph-meter (Ph-paper), Fluorescent lamp(natural light), Kettle, Automatic timer(optional), camping burner (alcohol lamp), Heat mat(optional), Thermometer,

Chemicals you need:
Bleach 5%, 70-99% Alcohol (Ethanol,IPA), Hydrochloric acid (Muriatic acid), potassium hydroxide (KOH)(Sodium hydroxide NaOh), 100%Agar, Sugar (sucrose, dextrose, fructose & glucose), Plant hormones(Kinetin,BAP,NAA,IBA),  Murashige and Skoog basal medium with vitamins (Or another medium), distilled water (sterilized, PPM(optional) Muriatic acid you can change acetic acid.  

Now is the basics have learned . Next we do the media example for pereskiopsis spatula. Work with salvia too. I don´t know is it a best formula but i have a succeed. First you take liter distilled water and stick the ½ Murashige and Skoog basal medium with vitamins. This is the place to put the agar and the hormones in the kettle. But I put the agar only and it is a 6grams per liter. Next you check the ph. You can use ph-paper or a ph-meter. I modify ph whit the potassium hydroxide (KOH) or Hydrochloric acid (Muriatic acid). Acetic acid is good for this and safer. NaOh (lye) is good as well. Be careful whit them. You can burn your skin or turn in to a soap . Yo gonna fuck up your sight seen if you spill the stuff in your eyes. Ok. Right now I put the ph. on 5.6.
Then put the stove on and let water to gentle boil. I usually let the mixture boil 5-10min low temperature.  Next step is a divide the mixture in a ten 100ml portions , this is not must. You can do all in a one portion but I want to different concentration of plant hormone. So I take 100ml in flask and the take a syringe and suck the plant hormone what i want. Then push the piston down and I shake the mixture and divide in a three babyfood jars, I use 50ml syringe in this, it´s help a lot. Approximately 33ml per jar and put the plastic cap on and when you have done all of this, you put all jars in the pressure cooker for a 15minute. Sterilize all the media. Let it chill of before you put anything in the jar. I do a usually ten different concentration of mixture of plant hormones. I have seen that is a good thing to do and you see what compound is a best. I have seen the best is the where are kinetin and 6-benzylaminopurine (BAP) equal amount 5ml per liter . A few drops of NAA and IBA in 100ml. So it´s a 0,5milliliters (Kin,BAP) per 100ml. When jars are cooled of you moved in a clean sterilized place. Somebody uses laminar flow hood or cabinet. I use a self made glove box .I fist made for a magic mushrooms. I have daydream about it. Hmm” Own flow hood” Ok.( It´s good for mushroom inoculation). I put the jars in the sterilized glove box. I use to sterilize 70% IPA or stronger . Soak the box with alcohol and let the alcohol float in the bottom, then move the jars in the box fast and clean. This time I have been a cut the stem of pereskiopsis and put it in the 0,5%-1% bleach solution, few drops of dish soap and stirred for ten minutes , then I have rinse with distilled water and then soak in the 70% IPA and stir it a few minutes, Rinse three times with distilled, sterilized water. You don’t want to contamined your product with bacteria or anything shit. You can expect some mould and bacterial growth in a few jars. Don’t give up if you fuck up your first trial. Learn where your contaminations arrived. Try to void that in a next time.  There are stuff called PPM (Preservative for Plant Tissue Culture) and you can use it for a disinfect your plant and put it in a media to avoid contaminations. It´s very priced stuff and one liter can cost like 1500$. 10ml solution is about 10$. You can preservative a plant piece in 50% PPM a long time. If you are in a trip and you find a plant what you want tissue culture, Take a piece of plant and put it a PPM. This way you can save good plant tissue for a longer time.    
Now you can cut the plant in the right sized pieces with the sterilized surgical blade and tweezers. Every time you cut with surgical blade different plant or touch anything you have to sterilize it in a burning flame to red hot. Same with the tweezers!!! You must remember it always!!!  I think the right size is a approximately over one centimeter. But longer piece can product more stems. Then you put plant pieces in baby food jars and put the plastic cap on and moved in to aquarium or transparent tank. I have a 40 liter aquarium. If you have a place where sun can shine you don’t need a lamp. I use a 45cm 25w fluorescent lamp (natural white light) and little pit of natural sunlight can shine in my growing chamber. I have a automatic timer to switch the lamp on and off. I have it on a twelve hour period. Ten in a morning to ten in the evening it´s on. I have a heat mat but I don’t need it right now because is weather is so hot right now but in a winter time I gonna need it. The lamp gives heat also. I try to keep the temperature in 25 degree Celsius. I don´t know is this best degree. Works for me!

If you have contamination in a jar, don’t throw it away. You can try to save that but don’t put it in the same place of others. Quarantine . Then you can drop a few drops of 5% hydrogen peroxide and 70% IPA. Let it work a few minutes .Then pour access liquid of. I can save a few plants with this method. If you see a little bacterial growth it´s time to try save it. That don´t work if there are too much of bacteria- , mould growth. If you have PPM solution you can try it. I never try. But next time I try if a have a contamination. Somebody uses a antimicrobials such like antibiotic penicillin and other stuff. I think if you are doin this for a business then you should buy it.  
Let the jars stand in the bottom of aquarium and put the lid on and tape the corners shut whit the micropore tape and the dust mask . Now you can look what happened in there and do some notes how things goes on when time goes past .

Now the shoots have emerge and it´s growing almost over the top. Next get your tweezers and junk the plant out off the jar and put it in a sterilized plate. Now you can cut the plantlets(stem) off. Then you moved those in the new jar where are a new media and hormones. Now hormones have to be majority auxin and gibberellins to grow roots. Or you can put them in the rockwool or water to grow the roots. When all have done and plant have a roots it´s time to move in the soil. You have sterile your soil before you put the plant on it. It may have a shock for a bacteria of soil if not sterilized. Put the soil in the microwave oven and heat it hot(few minutes) and took it out and stir it and put it a gain and do it for a several time and check temperature and try to keep it over 70 decrees of Celsius for a 20-30 min or put in the pressure cooker (autoclave).
Next let the soil cooled off and then you can plant it. Now you have keep the plant in the humid place or it will shock and die. This time you can use aquarium for humid tent. Put the jar of water in there and keep it moist. You can use a clear transparent plastic jug 0,5liter and sprayed with the water everyday for a few weeks. I think is a good keep the humidity over a week and then little by little introduction on a natural air.      
Next time Im gonna document the whole thing. Take a pictures and video and sent it for you to learn it.
Here is a Pages you want to visit.

http://www.hometissueculture.org/    (you can order products in here)
http://www.phytotechlab.com/        (you can order products in here)

INFo
http://www.kitchenculturekit.com/tcinfo.htm#Training  
http://www.planttissueculturemedia.com/
http://www.omnisterra.com/botany/cp/slides/tc/tc.htm
http://www.quisqualis.com/tv03tc01p1.html

Nice YouTube videos:
http://www.youtube.com/watch?v=KYvJByYrSPg
I have learn a lot of this guys videos. So look and learn!!!
http://www.youtube.com/user/fbt2007


I HOPE THIS HELPS SOMEBODY AND BECOME INTERESTED TISSUE CULTURE.

And my english not good so i think there will be a typos but i hope that doesen´t disturb enybody.

Very nice write up! Looks very helpful, I'll be sure to use the resources you've posted and thank you for all the tips and tricks.
I am interested too see any pictures of the pereskopsis you've started, as well as any other plants you've done via micro-propagation.

You say it is possible you can grow it a plant from pollen? If that is true, that is amazing.

In one jar reads Lophophora williamsii. No it not a peyote. It´s pereskiopsis spatula

And my english not good so i think there will be a typos but i hope that doesen´t disturb enybody.

Absolutely not I could tell you where not native speaking english while reading but it did not detract from the quality of the post one bit.

Very nice write up can't wait for more. I was going to attempt something simular with sassafrass rootbark but it got put on the back burner sadly.

I know many casual readers will turn there heads away at some of the names of chemicals used thinking they can not aquire them but indeed almost everything here is easy to get at your local gardening store or wal-mart for that matter. The agar in some cases can be substituted with "magic crystals" which is a gelling agent used for plants to make a clear soil. This should be ground down before adding water.

Quote

And my english not good so i think there will be a typos but i hope that doesen´t disturb enybody.

Absolutely not I could tell you where not native speaking english while reading but it did not detract from the quality of the post one bit.

Very nice write up can't wait for more.

It´s nice to hear that i can write like that. I self have allways a doubt my english and it´s took so long and it´s time-consuming. Im glad that my looked movies and played games, they had been educational and of course my time spendet in school. heh.
Casul readers consider it trange chemicals but they are easy to optain if you have internet and little bit of english language knowledge and a paypal or bank transfer possibility. If you have a credit card you are the lucky ones who had a access to anything or almost anything.  Just research, so i self to do.

I think the coconut is good stuff to use if you cant get the real products.  2,4-D "is usually used stuff" but it can form mutations so that is´n good. I look one thread and it´s about mutations. I haven´t see 2,4-D in there. I just a glance it might be there? All your thought are good but there are hardship to extract or execute.

I never seen any chart of effective, only what i have read. So i don´t know.

How tolerant are the plants. That is what i can´t even guess. I had only experience with IBA and i don´t remember how much it was(300-1000PPM) Between i think. I left the salvias cuttings for IBA-water solution for about 5 hours and then put in a regular water and the next day they are dead. Sixs beatyfull salvia cuttings died. byyh,hyy :'( . It was an accident. Usually dipped in solution only 15-60min. Something came up and I left them in a too long time.
But i think it can do a media where are 50ml per liter or over the growth regulators. I gues?

Book on this topic:
Plants from Test Tubes: An Introduction to Micropropagation
http://ifile.it/5ylibo3/0881923613.rar

This is pretty awesome, can you rehydrate a dried piece of plant and make it a viable tissue culture say a stem for instance that has been dry for a month?

Considering the nutrient medium needs to have vitamins, minerals, amino acids, and carbohydrates for everything to grow properly you either are going to need to make it, or more likely since it will involve so many chemicals, just buy a pre-made mixture of the plant growing medium. This is probably and always will be the best and cheapest way.
However, if you are like myself and hate paying for shipping, and spending money in general - you'll desperately be looking for an alternative right about now if you are considering doing any micropropagation.

Well I've had an idea, and i think it will work - but this is based on nothing at all.

In an attempt to clear up a potato infusion I made, since I am going to be making PDA soon - I froze it, believing it would cause the un-dissolved potato suspension to precipitate, since the water wants to crystallize in a pure form, all of the contaminants are generally pushed together - I thought that would allow them to interact and than fall out of solution more easily upon thawing.  -- It did, there is more flakes at the bottom, and the main solution seems slightly clearer.

Well thinking about this made me wonder, what if I did it to V8 Juice? This might cause all of the solid particles to come together and fall out of solution upon thawing and leave you with a clear solution after. I than was reminded of this instructables  article: http://www.instructables.com/id/How-To-GelatinAgar-Filtration/

The guy adds agar, freezes the gelled V8 and allows it to thaw over a coffee filter, this leaves a clear yellow solution, which I believe has vitamins, minerals, and the soluble carbohydrates that are needed for growing a plant -- After all, that is where it originated from - being mostly tomato and celery juice, along with a few other plant based juices.

I am currently freezing, with out the use of agar,  11.5 oz of V8 juice to see if I can precipitate the compounds and hopefully be left with a clear solution - I think the formation of the crystals will help. I also added about 1/2 oz of boiling water with 1/4 or 1/8? of a teaspoon of gelatin dissolved in it - this is because gelatin is a flocculation and helps emulsions/suspensions fall out of solution - No idea if that is enough to affect it, but I think there is a chance it could help it out a bit. (was kind of just a last minute thought)
 

I will update on this later too see how it goes.

Anyways - the great thing about this, if it works (I'll have to try to test it) is that V8 juice is easy to get, and I believe so loaded with nutrients that it will probably need to be diluted in order for the plants to effectively grow in the gelled medium.

Any input on why this will or will not work, or references of someone doing similar would be interesting.
I have found a few sources on V8 medium - they use this:

Receipt for 1L Medium:
• 200mL V8 Vegetable Juice
• 3g
CaCO3
• 20g
Bacto Agar

Apparently since V8 is acidic, the purpose of the CaCO3 is to neutralize it. However another place reports that it doesn't matter -- this is because they fear the hydrolysis of the agar.  Often it is centrifuged to make it a clear solution, this obviously makes the material sink, and than it is decanted.

11.5 oz is 340ml -- so a single V8 can is able to produce about 1.7 liters of growth medium. The down side to what I have read about the V8 medium is that it is used mostly for bacteria. I have not read anything about it being able to support plant life - however, I believe it should be able too. According to the can it contains 12 grams of sugars, 3 grams of protien, 200mg of sodium, and 1180mg of potassium. as well as having vitamin A, Vitamin C, Calcium, and Iron - But obviously it is going to contain many other compounds.

The V8 medium I copy and pasted would have about 7 grams of sugars per liter, assuming the fiber was removed which makes up about 3 grams of the 11.5 (340ml) V8 drink I am using.



Also since instructables  makes you sign up in order to download the PDF I have uploaded it below this post.

Edit: Also here are more instructions on micropropagation - http://www.biotech.iastate.edu/lab_protocols/AV_Micropropagation.html I hope that is useful.

Also this is pretty awesome: http://www.shroomery.org/forums/showflat.php/Number/1932583#Post1932583
Getting a lot of cacti from a few little ones..

UPDATE:
Freezing V8 and allowing to thaw does not allow for it to separate - i am now doing it over with 2 packets of gelatin in it.
The V8 will not provide enough nitrogen, I do not believe, so ammonium nitrate will be added to the mixture and I will try my hand at micropropagation, in at least getting a plant to grow and stay alive. I will not mess with the hormones yet as i do not have any currently.
Right now I am just waiting for the V8 jello to freeze.

what sounds better to get for an generic medium?

   Murashige & Skoog Modified with Adenine, Inositol, and Thiamine
With macro- and micronutrients, 80mg/L adenine sulfate, 100mg/L myo-inositol, and 0.40mg/L thiamine HCl

OR

Murashige & Skoog with Gamborg's Vitamins
With macro- and micronutrients and Gamborg's vitamins

That seems to be the generic ones...
Not sure...

Any suggestions?