# Cloning Plants By Tissue Culture



## search for animal chin (Dec 15, 2008)

*CLONING PLANTS BY TISSUE CULTURE*

*by Michael H. Renfroe, Ph.D.*






Many plants are cloned by tisssue culture techniques and sold commercially. Some of the ferns such as Boston fern and staghorn fern are propagated through tissue culture. Also, many varieties of African violet are propagated asexually by tissue culture. 





We can take a leaf from a plant like the plant below.





The leaf is then cleaned of contaminating microorganisms, fungal spores, small insects or whoever might be on board.






Continue to Part 2
*CLONING PLANTS BY TISSUE CULTURE*

*by Michael H. Renfroe*

*Part 2* 
The leaf is then cut into small pieces in a laminar flow hood that provides a clean working surface. The small pieces of plant tissue that are cut out of the leaf are called explants. Below you can see what they look like.





The explants are then placed on a chemical medium that provides nutrients for the plant tissues to grow and usually some plant hormones to encourage development of new organs from the plant tissue. Below is an explant that has been placed on a chemical medium inside a test tube.





If you look at an explant with a scanning electron microscope, it would look like this.





From this explant, new shoots would start to develop. Before they were obvious to you, as they just started to develop, they would look like this with the scanning electron microscope.






Continue to Part 3
Back to Part 1
*CLONING PLANTS BY TISSUE CULTURE*

*by Michael H. Renfroe*

*Part 3* 
After six to eight weeks, the explant will develop new shoots, as below.





These shoots may be cut free from the explant, and placed in a larger container on a new medium that will help roots to develop.





The rooted plant can then be transferred to soil. At this stage, the humidity must be kept high until the plant can adjust to the new surroundings. This process of adjustment is called acclimatization, and involves the growth of new leaves that will function in the less humid room air.





The cover is slowly opened more and more over a two week period so that the plant can gradually adjust. Then the cover can be removed completely and you have a new African violet plant. 





From one original violet, you may produce hundreds of genetically identical plants.





Because the plants are genetically identical, and are of similar developmental age, they tend to produce flowers at the same time. This is very important to someone who is growing the plants and wants to get them to market just as they start to flower.





Many flowering plants are propagated this way. I hope you enjoyed learning more about how plants may be cloned using tissue culture. 
If you want a more detailed explanation, please visit my Getting Started in Tissue Culture web page. 
Back to Part 2
Go to beginning of Cloning Plants by Tissue Culture.


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## diemdepyro (Dec 15, 2008)

where can you buy a kit.


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## search for animal chin (Dec 15, 2008)

yes i have done this, it works very easy and it took 4 -5 weeks to get roots, HOWEVER, it works great for that "o shit" i wish i had a clone of this plant but its gonna be cut down in 2 -3 weeks situation. it will be the way we all do it in a few years, my child hood friend works for a produce lab and they are doing tissue culture and getting roots in 2 weeks, of course a "lab" is way differant than my grow room, however i'm getting my room close to lab status.


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## Sheckster (Dec 15, 2008)

Seems like a long time to wait... 
What do you see as the biggest advantage besides getting a lot of clones for a small amount of plant material?


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## search for animal chin (Dec 15, 2008)

well one advantage would be; you grow that one in a million, only shit, i've been blessed plant, thats perfect for your growing room or climate, its a month or a day away from being chopped down. cut 5 leaves off and in 4-8 weeks you could have 500 babies all the same plant, sounds like the perfect indoor (strain)plant to me...? If i had a special plant that would be perfect for my grow room, it would take me a week or 2 to clone it, then a month or 2 to be able to take about 60-80 clones, but my room will hold 500 plus plants well thats well over 4 months and a chit load of work and time & materials. now i use tissue culture and within 8 weeks i could have well over 500 plants all the same, same everything to the day....makes it nice ....and as my friend says it will be easy to do within 4 weeks max within a year, just something new and interseting....?


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## dat130ysmurf (Dec 15, 2008)

Here's a html document on affordable plant tissue culture that can be done at home. To view the document download the .zip file, extract the file, open the file....

This plant tissue culture looks similar to growing psilocybe mushrooms using a liquid culture technique..... To see info on growing shrooms using liquid culture check out http://www.shroomery.org/ ...

If anyone tries the plant tissue culture let me know how it goes.


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## tinyTURTLE (Dec 15, 2008)

http://www.percival-scientific.com/products.php?cat_id=5

culture kits here. no prices listed, so pretty expensive.


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## jhershner11 (Dec 15, 2008)

seems to be alot of work and materials. Also needs to be exact or risk complete faliure. Strerilizing to that extent requires a lot of chemicals and clean workspace i.e (lab). But still very interesting


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## scrxbandit (Dec 16, 2008)

I'm actually a good friend of Bill gram, the creator of tissue kit. Ive done the process in bio lab, completely analogous to bills kit. Bill has a bio history, and is making it available for every body with some level of competence. keep every strain youve ever dealt with in a test tube. for years. he tells me about finding tissue cultures that fell out of his pocket and just proceeded to multiply under his seat. bargain for 200 bucks. His partner mike at hydro solutionz is the inventor of the smart light controller for running double lights on single ballasts. great company


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## OregonMeds (Dec 21, 2008)

That smart lighting controller seems gimmicky. Is it just me?
Why push ballasts 24/7 when you can buy 10 more cheap (or not so cheap) 1kw ballasts and timers for less than that thing.

Neat idea, but seems as practical as tissue culture is over cloning in most cases.


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## 1kooguy (Dec 21, 2008)

Keep it simple.


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## slackjack (Dec 21, 2008)

Rumor has it that all of some apple variety (golden... somethin) come from a single branch from one tree, and that branch continuously had the premium apples. It would be great to be able to do this with cannabis.

or 

that'd be fucking awesome to do away with moms and just take a few leaves off the heaviest budded plant from each harvest.
simplicity is for the meek growers, which will inherit our harvests. Bwahahahaaha!


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## Titan4jah (Dec 22, 2008)

can you really do this in your own home ?? sounds like you need a room all to its own for this to be a reliable source of propagation, how long does the tissue last?


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## cackpircings (Dec 22, 2008)

OregonMeds said:


> That smart lighting controller seems gimmicky. Is it just me?
> Why push ballasts 24/7 when you can buy 10 more cheap (or not so cheap) 1kw ballasts and timers for less than that thing.
> 
> Neat idea, but seems as practical as tissue culture is over cloning in most cases.


 
Just got a kit I will show you when I get it...


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## Titan4jah (Dec 22, 2008)

cackpircings said:


> Just got a kit I will show you when I get it...


 
hell yah you should post the process so i can follow along....lol wher did you get the kit , did i miss something lol


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## pharmacoping (Jan 16, 2012)

I do this very well, and assemble kits for sale too /prepared solutions/pre filled sterile containers/proper nutrients, and even genetic specimens, for observation purposes. Some samples include Thyme, Rosemary, Tahoe OG Kush,PlushBerry,Kandy Kush, and hundreds of other non plant dna samples. The study of these samples will can prepare you for the art of tissue cultivation/cloning. From a single spec thousands of master programmed clones can be taken monthly ! Imagine no more mother plants or clones using your plant spaces? How important is that to you? These are stored/divided as masses of tissue or unusable roots. Only in the last few days do they become a "plant". You can easily have roses with thc, or mj without leaves, or even glow in the dark, really, I've seen it ! It's not the future, it's now ! Patents are already being granted for different mj types grown this way. suicide genes,inability to clone/seed..are some of the programing in these super corpopharma plants. Instructions are available online, but the right mixes are almost impossible to find and invaluable to growing your own rare herbs successfully, and storing their dna forever. With these supplies you'll learn how to master this fast because the work is done !! Plan on spending a couple hundred bucks or less, depending on the equipment you have at home...jars/mixer/pressure cooker,etc.

I'll answer some q's here, but private message me for instructions for above. 
pics coming soon, soon as I figure that thing out !

peace


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## Guile (Jan 16, 2012)

I tend to have some ultra simplistic perspectives on things.... though they are generally not far from the truth...

I have not looked that closely into it yet, but my impression biased on other research is that you can simply take the node section (or growing tip) from a plant you harvest and place it into a gel with low concentrations of nutrients (200ppm or less) and hormones (Cytokinins & Auxins) with the result being a clone. Most the places I see it done the plant material is processed like in a blander but I feel this might be unnecessary if only limited clones are necessary.. Keep in mind that cleanliness and environmental controls will likely be key concerns.

Hormone ratios/concentrations I have found while researching plant tissue culture experiments are incredibly low, if you obtain your hormones in nearly pure form you would need a swimming pool full of distilled water to get the concentrations correct (assuming you have a scale that is accurate to the milligram to start with)


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## pharmacoping (Jan 17, 2012)

Hormone and nutrient ratios are plant specific. I use most parts of the plant to perform this, depending on what type of growth,structure, etc I desire, leaf,node,root,stem,flower,pistil, etc. more than clean is needed, sterile is key. 

peace


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## ch1ll (Jan 19, 2012)

Don't you get problems with too much stress on the plant, causing it to become a herma?


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## pharmacoping (Jan 20, 2012)

no hermie issues caused by tc, yet. Only hermie issue in three years was the Tahoe OG Kush pack of gear I got last year. I treated the choice one with DM Reverse, at a tenth of rec dose, twice, and her traditional clone, once, and have taken hundreds of traditional and culture clones from her since. she is gone now, but her prodigy live on in various in vitro grows. never saw a sex issue again. For traditonal cutting/cloning, I have never used anything as effective and fast as the DM Replicator. very fast.fyi


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## wyteboi (Jan 20, 2012)

OregonMeds said:


> That smart lighting controller seems gimmicky. Is it just me?
> Why push ballasts 24/7 when you can buy 10 more cheap (or not so cheap) 1kw ballasts and timers for less than that thing.
> 
> Neat idea, but seems as practical as tissue culture is over cloning in most cases.


no its not just you , its an idea stolen from a standard weed nerd an turned into an "invention" that cost probably way too much. we have been makin "flip flop" controllers for years around here. (with just a few cheap parts)

in real life the controller is way cheaper then a ballast so it helps if you dont wanna buy another one. 





i love the tissue thing , it wont be efficient in my opinion , but its something to play with. i love science. 


i have a leaf in my fish tank that developed roots and i dont know what to do with it ? its a underwater plant for the tanks. will that leaf grow another plant , if not on its own , can i help it ?




thanks soil


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## pharmacoping (Jan 20, 2012)

your roots will grow fine, given you've got good 02 saturation, and a stable system. ot may continue to grow many different ways under water. you wont be able to manipulate it though, with fish. It should be cool to watch though. 
tissue culture isnt't practical for most, you're right, with the ease of cloning. but one fine example of practicality is availability. imagine instead of seeds you could purchase verified pure rootballs ready to sprout up in a couple days, and the advantage to the grower to have fine genetics delivered overnite, with golf ball size roots, before the thing has even sprouted. It's an incredible jump start that allows me to keep a full time perpetual grow successful, with many more pants flowering than without. This system is already in use by most greenhouse/flower suppliers right now, and they will tell you how very efficient and practical it is, and they dont even have legal plant limits to consider like we do

peace


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## wyteboi (Jan 22, 2012)

pharmacoping said:


> your roots will grow fine, given you've got good 02 saturation, and a stable system. ot may continue to grow many different ways under water. you wont be able to manipulate it though, with fish. It should be cool to watch though.
> tissue culture isnt't practical for most, you're right, with the ease of cloning. but one fine example of practicality is availability. imagine instead of seeds you could purchase verified pure rootballs ready to sprout up in a couple days, and the advantage to the grower to have fine genetics delivered overnite, with golf ball size roots, before the thing has even sprouted. It's an incredible jump start that allows me to keep a full time perpetual grow successful, with many more pants flowering than without. This system is already in use by most greenhouse/flower suppliers right now, and they will tell you how very efficient and practical it is, and they dont even have legal plant limits to consider like we do
> 
> peace


i was wrong ...... with little to no knowledge it would be in-efficient. if done right it could be way more efficient.




soil


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## pharmacoping (Jan 22, 2012)

I love honest people. thank you I've heard if an honest person who is mistaken is given the truth, one of two things happen, either they will no longer be misunderstood, or no longer an honest person. once a shelf is filled with "unusable roots" not using plant counts,of your favorite herd you continue the dividing/rooting, while selecting (biweekly for me) the "unusable roots" of the strain you want to run, allow it to shoot up, now taking up plant counts, and put in the veg room to cycle.exactly 45 days later they go to flower,for roughly 60 days, and then to harvest.(weekly here) it could take a month or two to get a stocked shelf of these roots, but after that, control is yours. I dont manipulate any more than we all do when we use "rooting gel" to start clones. same hormones too, in a sealed jar, little light, agar,sugar, until I need their service. frankensteins are not my goal, and are destroyed each time. I have used colchi**** in vitro, and did produce a fine(techy name) plant with twice the normal sets of chromosomes, pictured here. PlushBerry, from TGA Subcool, attitude seeds. 

On the original note, I have one of those flip switches PowerBox, nice, works, if your ballasts are identical brand, and no issues with hot fire bulbs exist. digital ballast come with an internal fuse that can, and does trip occasionally(high temps, short,expired bulb,???) and these cause it to happen also, in my experience, on one product, used only by me, brand new. I used it some time ago to light two flower rooms at opposite times(12/12 to 12/12) and it did work mostly, but one discovered lighting failure is unnacceptable, so it sits on my shelf 'o toys. not sorry for the purchase, but was a waste of time/thought. If you get another ballast/timer, you won't regret it. plus, I think these digital ballasts like a break daily, as opposed to running 24/7, imo.

peace


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## Guile (Jan 22, 2012)

You know I never considered cloning a plant from the roots up... 

My understanding of the law and its definition of a plant was: containing both root and foliage.. Having dealt with conventional cloning methods exclusively for so long that I condensed my personal definition to "has roots" and I think that has gotten in the way of me thinking about cloning plants this way..

I like your idea about mailing a root ball that could be induced to sprout by the recipient.. Many times I've wanted to share clones with someone across the country and there didn't not seem to be a good way to do it without breaking the law.. However in my understanding a cutting (assuming it is either/only root or foliage) should not be illegal to ship?

How long could you keep one of those root balls dormant and stable for shipping before they start becoming less viable? Could you priority ship them (instead of overnight)?


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## pharmacoping (Jan 22, 2012)

mailing the foliage is illegal. even seeds are illegal here. however, an invitro callus or rootball is not recognizable by any means. cept dna. if you could regulate sugars and protect the rootball from attacks, theoritically you could raise roots in your system. a piece of a live root can be told to sprout up, or keep growing roots/dividing. In vitro culture allows all to be sterile, with some luck of course. as the culture matures it can fight off many attacks, and you can re rinse/transplant again, if needed. I have callus over two years old, and also rootballs the same age(older,maybe?). I dont push it, but pretty sure they survive in the fridge for weeks, and in a fair temp package for days and days. packaging can be manipulated easily also, heated/etc. as long as they are not dried up, are transferred every couple months or so and not moldy/dead, their viability is limitless, I believe. been sharing this way for a long time. some top seed breeders are approachable, and they tissue culture. Instead of years to an F1, these guys can do it in months, in vitro. some will gladly share callus for test grows, instead of seeds, because they are pure, and sterile, and exact. with a punnets square they can purify a strain quickly, its amazing what they're doing. cannabis breeders bible explains much of it.


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## Guile (Jan 23, 2012)

I thought as long as a plant were not complete it was basically seeds/stems, the dry weight of a cutting could never be considered more than personal use, and even thats a bit of a stretch (the law like everything else is subject to compromises as long as you have a reasonable position to argue). A decent part of standing up against the law is, well.... standing up against it... Help set reasonable legal precedents (isn't that more or less how laws get changed, when you don't have the money to successfully lobby).

I should probably apologize in advance, I'm sure I have not covered all the suggested reading material at this point..

"in-vitro callus" Would mean something like cultured scar tissue? If so is it at all specific to where the plant is scared to start with (I was under the impression that "growing tips" were most manipulable)? Are there specific ways to obtain scaring or can I do something like "burn" a plant with say, peroxide?

Could I use say.... the remnants left over after a harvest (roots and main stem)? They tend to want to live, there must be an incredible amount of life/energy left to them (can that be put to good use?)...

What if the culture were cross contaminated with the cells from another closely related plant, say its seed sister or clone sister (the mess of roots you might get from a hydro table after harvest for instance)?


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## scroglodyte (Jan 23, 2012)

very cool.....thanks


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## pharmacoping (Jan 23, 2012)

seeds, stems..you are correct, but we are taliking about shipping, not posession. ship one leaf and you have broken multiple federal laws. a stem stuck into rockwool becomes a plant count, with or without roots attached/formed.

callus is not a scar tissue, caused by knicking or damage to a plant, they are a grouping of disorganized undeveloped cells proliferating. when you tissue culture mj you'll see explants, callus, mold, roots, leaves, and some mutants. callus wil look like a puffy chunk of cauliflower. This is the basis fo rstorage of my genetics, cutting off of them is very successful. meristem or tip culture is the easiest, but these plants are totipotent and any piece will work, albeit with different patterns and success with each. The tips are the best for beginning as they are most likely without virus, and have the most hormones within. 

you will notice some swelling in tissue on a stalk. this area is full of mutated genes/virus' that the plant has overcome. Taking material from this area can produce the frankensteins you might be looking for. 

one cell wont rub off into another, in a tray or in vitro, otherwise we could breed via grafting root stock.... seedless breeding can take place invitro,(embryogenesis?) but I dont do this, however seed companies do. 
the bacteria for these types of manipulations are available online, and are responsible for some wild creations in the plant word.

those roots are usable, in vitro and even out(regeneration). If you leave a couple leaves on the stalk after harvest you can put it back into veg and regenerate the plant with success, if desired, but you know that I think. I have rooted leaves, but it proves to be a much longer process to bring it to harvest. I use new shoots, trimmed of all foliage, scraped, and sterilized in bleach/alcohol before beginning. 
many are hung up on moldy failures. I get some, sure, but 100% of the time its brought in with the plant piece, not dirty equip, or non sterile equip. I know this because you can easily see it under a scope, ////eyelash,dirt, etc. when I mix agar I've never seen mold form, unless there was a plant in it. maybe I'm an awesome sterilizer, or just fol
low directions without fear of the unknown.

(grafting hint..no agar used. wax, elmers glue, rooting hormone. easiest way is to fill a hollow new stem with a scraped stem of a new lateral tip.)


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## wyteboi (Jan 23, 2012)

Guile said:


> I thought as long as a plant were not complete it was basically seeds/stems, the dry weight of a cutting could never be considered more than personal use, and even thats a bit of a stretch (the law like everything else is subject to compromises as long as you have a reasonable position to argue). A decent part of standing up against the law is, well.... standing up against it... Help set reasonable legal precedents (isn't that more or less how laws get changed, when you don't have the money to successfully lobby).


well seeds can be sold for the purpose of helping someone do something illeagal. -----conspiracy /federal 
the stems contain a horrible schedule 1 narcotic. THC ------ can be charged as possession and/or dealing / can be ugly, but the judge would have to be a dick to give out a lot of time for that.

this is dead on. 


Guile said:


> A decent part of standing up against the law is, well.... standing up against it...


now our lawyers argument is gonna be "what the fuck , your honor , sticks an marbles! i demand this garbage be thrown out"...... thats a decent lawyer. your everyday cheap lawyer is gonna get the dealing dropped if you sign guilty to the possession......... and now i got a felony for weed over stems and seeds , nothing more. and then automatically my next joint was a felony just because i already had a felony for pot. 
so two drug felony's for seeds an stems. 

bottom line.... keep a high paid lawyer around when fucking with pot and charges will stay minimum. 

i dont really think they can be too rough with this plant matter shit because of loopholes. plus like pharma said , they would have to run a DNA test just to see what it is.
its all illeagal but consequences are determined by who you know and where your at.








if your going to ship stuff (legally of course) just make sure its airtight and it will make it to its location. if it dont , the box busted open an the postman is running out to get a 400 right now. 




soil


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## wyteboi (Jan 23, 2012)

pharmacoping said:


> I love honest people. thank you I've heard if an honest person who is mistaken is given the truth, one of two things happen, either they will no longer be misunderstood, or no longer an honest person.


well facts are facts , i cant argue with facts.



pharmacoping said:


> On the original note, I have one of those flip switches PowerBox, nice, works, if your ballasts are identical brand, and no issues with hot fire bulbs exist. digital ballast come with an internal fuse that can, and does trip occasionally(high temps, short,expired bulb,???) and these cause it to happen also, in my experience, on one product, used only by me, brand new. I used it some time ago to light two flower rooms at opposite times(12/12 to 12/12) and it did work mostly, but one discovered lighting failure is unnacceptable, so it sits on my shelf 'o toys. not sorry for the purchase, but was a waste of time/thought. If you get another ballast/timer, you won't regret it. plus, I think these digital ballasts like a break daily, as opposed to running 24/7, imo.


i dont like digital ballast for that reason. more money and more problems. ive got magnetic ballasts from the 80's that still work just fine. 
lighting failure is not an option for me or you. 
the flip flop switches we build dont really have the option of fucking up , all they do is switch contacts. so as long as the timer is working correctly then there is no probs. the magnetic ballast dont need a break.

now with that being said i would still recommend buying the extra ballast and timer , *specially* if your using digital ballast. the 12 hour break will help the ballast to last forever plus you wont fuck up 2 rooms if one single ballast goes out. 



soil


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## pharmacoping (Jan 23, 2012)

I almost miss the humm of my old ballasts !! really. you're right, they just hardly ever fail, and usually gave an audible warning while dying, enough to fix it for 30 bucks!. the digis do cut out for various reasons, failures suck, but several more are sparked at the same time, so tomorrow, fixed. your ballasts are a better deal too. I still like the idea of switching for heating, (i do with timers now) but instead of a 2000 watt baseboard heater cranking, 2- 1k lights are lit, and the heat is recirc to the opposite rooms. it works great.

some bud porn..cheers !

p


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## wyteboi (Jan 24, 2012)

pharmacoping said:


> I almost miss the humm of my old ballasts !! really. you're right, they just hardly ever fail, and usually gave an audible warning while dying, enough to fix it for 30 bucks!. the digis do cut out for various reasons, failures suck, but several more are sparked at the same time, so tomorrow, fixed. your ballasts are a better deal too. I still like the idea of switching for heating, (i do with timers now) but instead of a 2000 watt baseboard heater cranking, 2- 1k lights are lit, and the heat is recirc to the opposite rooms. it works great.
> 
> some bud porn..cheers !
> 
> p


ah...  so what your sayin , is when room #2 is sleeping , room #1 is used to heat #2? thats a beautiful idea. i never even considered that. 
i dont use two bloom rooms , but the more i think about it , my cheap ass is considering the savings. 

hmmm a 1500w heater for 12 hours a day or a divider in the room. easy decision.  


is that pink an green bud the same ?


good knowledgeable posts p ! 




soil


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## mellokitty (Jan 24, 2012)

this is waaaay too much good information before coffee...... subbed up and will read properly later...... <3


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## pharmacoping (Jan 24, 2012)

california orange bud(real hashy favor, good stone) and Plushberry(top 10 medicators)
(seperated by a light trap curtain) one side vegging, other in flower currently. veg is 18hrs on, all nite, the 6 off is during high noon. flower room is lit 8-8 during the day. each has its own heater(elec baseboard,digi thermo, hardwired240), air is circed full time between the two, including c02, 8-8. very efficient for me. all the lights are 240volt, even the t-5's(Badass brand) and the 8 light controller is hardwired in, so timing is a breeze. still costs several hundred dollars monthly in electricity for ac,dehumid,heat,lites,pumps, etc. but worth it.

peace


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## Bonkleesha (Jan 24, 2012)

you would almost have to make your own micropropagation media for cannabis. that can be a science in itself.


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## pharmacoping (Jan 24, 2012)

you are correct Bonkleesha, but the base mediums readily available work well. It's the rest of the mix that took forever to figure out. The kits sold and info available multiplies some flowers well, but if multiplying was needed, cloning would be the key. Different formulas are needed for each stage of tc. first one is callus, then dividing, then rooting, then sprouting...each requiring a special set of circumstances inside their womb. I"m in the tissue culture tent daily, transfers, inspections, disposals, etc. I'm 46 in 2 days, and remember being in the first marijuana growroom when I was 4yrs old. my parents had several, before you could buy all the toys and right nutes/lites online. they had lots and lots of hps streetlamps and some strange experiments, but a pretty successful garden biz for lots of years. was hooked then, nothings changed, cept technology, legality, and the price paid for quality herb !


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## Guile (Jan 25, 2012)

Sorry to interrupt the continuity of the conversation.

So the callus is not a response to plant damage?

While looking around at information on callus culture One of the impressions I got was that you are probably working with predominantly "undifferentiated" cells.. "Parenchyma" (living cells from the core of roots/stems).. 
"Meristematic" (found in zones of the plant where growth can take place). To form the callus 

Is there an easy way to isolate these cells? If left with the "differentiated" cells will they have an influence on callus formation?

Is damage to the differentiated cells related to undifferentiated cell formation? (or just consequential to obtaining it?) Are we manipulating part of a repair response (or just completely manipulation the situation)? 

What I was after with my foreign plant material question was more aimed on weather of not it could be mixed without causing the callus material to attempt to isolate/reject it? Would the entire experiment fail because everything is rejecting each other? Would things sort themselves out by creating 2 genetically different callus formations? 

Could you use UV lighting (or an ozone generator) in your incubation aria to keep molds/bacteria at bay while still allowing the callus formation? Both can be relatively "harsh" but _if_ callus is a response to damage it almost seems that it could work in the favor of a crude experimenter.


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## pharmacoping (Jan 25, 2012)

no apology necessary. 
callus " a proliferating mass of disorganized, mostly undifferentiated or undeveloped cells" (Plant From Test Tubes).
this mass can resemble a chunk of cauliflower partially submerged in medium. I use this stage to multiply and divide, as well as to store genetic moms. I cut off off these and induce rooting, for genetic storage, although more resources are necessary than storing callus culture, or to be transferred to a "shooting" vessel to begin the plant stage.

Osmosis will not transfer dna from one plant to another, without certain circumstances. electricity, polypropyene glycol,bacteria/virus,mechanical dna injection, radioactivity, uv light,radiation all are being used currently(NOT BY ME) to do that. although worm learning comes to mind.....
hormones are available already cut for ease of use. most are unnecessary for keeping culture alive and flourishing for experiments, storage. if you can imagine a grain of salt dissolved in 5 gallons of water, and 1/2ml drop will make another gallon of hormoned water, and a drop of this makes a handy litre of medium mix..not scientifically accurate, but shows how dilute these are at the end. uv light would most likely cause genetic disruptions, failures/freaks. and ozone is unknown in vitro to me. I stay clean, works, mostly. I 

my incubation area is a grow tent no longer needed. vents taped shut, no air movement. lysol everytime in and out, wash,clean clothes. minimal peaking. tent was sprayed with alcohol in the beginning. your issues will not be the area, or your sterile medium. It will be in transfer from one jar to another, and most will come from your plant material. there is a fine balance between sterilizing live (or dead) plant material, and disrupting the dna and ending its life. when they die, they can mold. strong established plants mold seldom, even when exposed to air. I no longer use a positive hepa air in the transfer chamber either, so there....I believe this caused more issue than not. 1 year without, and I have way better results, maybe it was failing in design, not sure, but not necessary here. best frankenstein experiments come from the first knot on your stalk of a mature plant. this is where a virus attacked, changed dna,restabalized and the plant continued/survived. this area is full of strange cells, ready to be cultured. 

I apologise for hijacking this thread.
please visit my thread for great updates https://www.rollitup.org/grow-journals/506026-pharmacoping-tamisium-5000perpetual-grow-extracts.html#post6994167
thanks
pc


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## Guile (Jan 25, 2012)

Sorry again if its messed up the continuity of things. I have been editing my post for a while not realizing you had responded to it in the meanwhile..

I keep seeing things that look more like what I would call "micro-propagation" than cell culture.. Maybe My impression of what "cell culture" might be is wrong... If you start with a seed or seedling it seems that you are only stretching the seed or clone..

I somehow figured we were looking to "clone" a mature plant using parts that would not otherwise be (growing tips or other conventional clone/propagation material). More or less stems and/or roots..
I was also under the impression that we were exploiting a survival/repair response from the plant to achieve this..

An ultra simplistic perspective would be if you were to be able to say quarter a plants stem length wise to expose the Parenchyma, (assuming its undifferentiated cells inside) then influence it to spring up a column of miniature clones (or atleast callus formations that could be used to make them)? Otherwise extract the undifferentiated cells and provoke them to make the callus that can be manipulated in that direction.
If the Parenchyma of cannabis contains the undifferentiated cells you are after, could the differentiated cells be dissolved or mechanically stripped away so that it were predominantly the undifferentiated cells being incubated? Would this provoke more successful callus formations?


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## pharmacoping (Jan 25, 2012)

https://www.rollitup.org/grow-journals/506026-pharmacoping-tamisium-5000perpetual-grow-extracts.html#post6994167

response


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## wyteboi (Jan 26, 2012)

pharmacoping said:


> I apologise for hijacking this thread.
> please visit my thread for great updates https://www.rollitup.org/grow-journals/506026-pharmacoping-tamisium-5000perpetual-grow-extracts.html#post6994167
> thanks
> pc


well this is your thread now, the op seemed to dissapear an guile an myself are open ears. please maintain this thread too. 

you know your tissue , an we need ya around. your a great help. thanks.


same with you guile , keep this thread up an going. 

thanks guys!

soil


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## pharmacoping (Jan 26, 2012)

thanks for the kind words wyteboi ! I'm mixing up some custom mj rooting compound as we speak. it goes into vessels, sterilized,gelled, and shipped. the little mag stirrer is getting a workout today. I couldnt stand and mix for hours without my mind xploding !


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## pharmacoping (Jan 27, 2012)

_"I keep seeing things that look more like what I would call "micro-propagation" than cell culture.. Maybe My impression of what "cell culture" might be is wrong... If you start with a seed or seedling it seems that you are only stretching the seed..I somehow figured we were looking to "clone" a mature plant using parts that would not otherwise be, like growing tips or other conventional clone/propagation material. More or less stems and/or roots..I was also under the impression that we were exploiting a survival/repair response from the plant to achieve this..An ultra simplistic perspective would be if you were to be able to say quarter a plants stem length wise to expose the Parenchyma, assuming its undifferentiated cells inside then influence it to spring up a column of miniature clones? Otherwise extract the undifferentiated cells and provoke them to make the callus that can be manipulated in that direction.If the Parenchyma of cannabis contains the undifferentiated cells cells you are after could the differentiated cells be dissolved or mechanically stripped away so that it were predominantly the undifferentiated cells being incubated? Would this provoke more successful callus formations?

_
You're way ahead of me there buddy ! I dont experiment much, not a mad scientist either.Short answer is....I have no idea,but heres what I do I take a cutting spec from a new tip, midway down. It grows callus, gets bigger. then I cut a piece off,transfer,and it grows roots. I transfer the roots, and they shoot up a bonzai tree quickly. I harden, then put them in the veg system. thats the extent of what I do today. I keep jars of "unusable roots" waiting for my order. anything done over the years was just figuring out what not to do, then directions came out to do this. I followed directions. on the cheap. with success. only issue is sterilizing plant material . the rest is second nature now.Only taking up a plant count number at the last moment when told to shoot, this allows me to have rooting genetics on standby, negating the seedling/cloning phase. these "seedlings" have a ball of roots that rival a month old plant....all before they even sprout ! 
Once a farm is established of chosen root ball genetics, and regular transfers take place, it really is a no brainer. lots of back ups in case of failures, common sense, years on the internet, or a week in a book. they're only super plants for a couple weeks, then look quite normal, except for the excessive lateral branching, ie lots of roots in the beginning.

many strains will grow from seed directly into flower(seed from 12/12) with minimal harvest weight loss. if i experiment any, it will be with moving a bonzai from a jar and putting directly in the flower room. other than that, I have no plans to exploit the technology any further than my own applications. lots of success can be achieved in vitro with a good rooting gel, on the cheap, if needed. varying amounts do really wild things in culture.

I culture plant material in different stages, so that I can micropropagate them to fulfill my needs. I hope that helps​


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## Doer (Jan 27, 2012)

OregonMeds said:


> That smart lighting controller seems gimmicky. Is it just me?
> Why push ballasts 24/7 when you can buy 10 more cheap (or not so cheap) 1kw ballasts and timers for less than that thing.
> 
> Neat idea, but seems as practical as tissue culture is over cloning in most cases.


Well, heat control is the reason you run 2 rooms and 2 lights, one ballast. And ballast can last longer if it's not cycled.

I've tried 8 weeks of cloning the traditional way and got Zero. And I have one of the WW in bloom that is a wonder grower compared to the other 4.
I'm contemplating a cloner. Invest $300. Surely this is more reliable and less expensive. Test tube environment is small and controlled.


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## pharmacoping (Jan 27, 2012)

I woud not suggest buying a cloner, if you're expecting different results. Rockwool, Replicator, Sharp Razor, angle cut mid section lateral shoot branch, scrape a little, dip, and place in the wet rockwool. follow directions on the rockwool. clones amore difficult to maintain if taken during a flowering cycle, but very possible. You can also harvest when ready, and leave some leafy small growth at the bottom attached, put it back in veg cycle, and you can try again.

tissue cloning will cost you more than 3 bills to begin. and it has to remain sterile. It cant replace traditional cloning for small grower, but is the only way to avoid using up clones as part of your legal plant limit.


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## Guile (Jan 27, 2012)

I probably should have read more before making speculations to start with. Alot of how I learn is to use my intuition (or incites based on other research/experiments) to speculate an outcome then pick up enough vocabulary and individual appreciation to move on and experiment. 

I wouldn't go as far as to say that I'm a mad scientist, just learn best from first hand experiences.. Formulate a hypothesis, conduct experiments, record data (if you have the time, rinse and repeat). Then formulate a better educated theory/hypothesis and start again. At some point you will gain a true understanding of how many aspects of things conspire to obtain a particular outcome (now that's an education, understanding the Why as well as the how). Honestly its how I always learned, my mother tells a story of me disassembling spring loaded cupboard hinges (child safety hinges) to see how they worked back when I was 3.

Knowledge is the product of Knowing, how can you know if you never did? Otherwise you are just accepting other peoples individual appreciations (which were likely guided by a paper written by someone else, subject to their own individual appreciations). as absolute fact. (where I accept my own bias, I sure as hell am not going to exclude anyone else's).

I do hope you realize how much I appreciate the information and incites you share. I've just been listening and reading a little bit more lately because at this point I already have alot to process before moving much further forward (otherwise I might lack fundamental understandings required to build from)..


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## pharmacoping (Jan 27, 2012)

Hey Guile,

Thank you for the kindness share. I have to assume that you spelled "incites" rather than "insights" purposely. now thats wit !


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## Guile (Jan 28, 2012)

No problem... 
If you scrutinize my writing enough you would likely find many spelling errors. I'm a phonetic speller for the most part, though I use spell check to help identify where communication issues might arise (the web browser plugin I use has its limitations). Despite having been enrolled in multiple typing/keyboarding classes I'm not much of a typist either.

I use to have a hard time dealing with my lack of spelling prowess, particularly in elementary school (the level of education that they seem to focus on that sort of thing). I was actually labeled as "learning disabled" in 1st grade due to my lack of intuition when it comes to spelling. Ironically or perhaps more serendipitously I also ended up in the "gifted & talented" programs as well. My pear group growing up was a product of the polar ends of the educational standard. It was a bit of a culture shock when I went away to collage at 16 (I had received a full academic scholarship when I was 15 however the law required I become 16 before leaving high school). As I high school dorp-out (do to never legitimately passing an English course) I made my collage eligible for some kind of government program because I was a "minority"..

To be honest I'm socially awkward too... People rarely understand me well, the way people seem to react to my words leaves me to believe that many times there is a complete mistranslation of my intent or appreciations. Much like everything I do, I use words intuitively, biased on the "feeling" I get (gut instinct so to speak).

I never realty thought/cared much for my mind (and the way it worked) until I suffered a traumatic brain injury a few years ago.. Early on during my lengthy stay in rehab I underwent a bit of mental testing. After the first battery of tests the doctor told me that I could easily be considered "normal" given my background and education (I've gone to a couple different schools).. And that I had retained at least 95% of my mental capacity. Considering how "stupid" I had been up to that point I decided to take whats left of my mind more seriously and not waste it.. (if you don't use it you loose it).


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## pharmacoping (Jan 28, 2012)

a kinetic personality for sure, is the most intense, and misunderstood, in my experience. I hope we have many years to share.

peace


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## nitro20% (Jan 31, 2012)

and the there is this. this i found last night and sums up tc for mj for dummies like me.
http://www.pakbs.org/pjbot/PDFs/41(2)/PJB41(2)603.pdf


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## pharmacoping (Jan 31, 2012)

nitro my man ! gee i'm still wondering why an expensive lab would waste time culturing marijuana !!!!!


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## nitro20% (Jan 31, 2012)

MONEY MONEY MONEY, this is "THE CASH CROP".yeah i wonder if the funding was payed in full by results of EXCELLENT CANNABIS??? k so heres another chemistry question, if i would have listened in school i probably would know this, or at the very lest at 36 yrs. old,,,forgot it.......ok so here it is,,,if i have a concentration of iba 1.0% & 0.5% naa,,,,to get to 0.1 iba & .05 naa mg./l??????
today is the day,, up until 1:00 a.m. cleaning and building.oh and thinkin.got to stop that.
my friend that has been doin this turn around on the mj tree $$$$ thing has mite and white mold. his mothers (king kush, grand daddy purps,and of course my poor blue dream). getting hard to clone and easily pics up root rot etc.... perfect, that is one of the reasons to do this,, ill fix em'!!!!!!!!


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## Guile (Jan 31, 2012)

nitro20% said:


> MONEY MONEY MONEY, this is "THE CASH CROP".yeah i wonder if the funding was payed in full by results of EXCELLENT CANNABIS??? k so heres another chemistry question, if i would have listened in school i probably would know this, or at the very lest at 36 yrs. old,,,forgot it.......ok so here it is,,,if i have a concentration of iba 1.0% & 0.5% naa,,,,to get to 0.1 iba & .05 naa mg./l??????
> today is the day,, up until 1:00 a.m. cleaning and building.oh and thinkin.got to stop that.
> my friend that has been doin this turn around on the mj tree $$$$ thing has mite and white mold. his mothers (king kush, grand daddy purps,and of course my poor blue dream). getting hard to clone and easily pics up root rot etc.... perfect, that is one of the reasons to do this,, ill fix em'!!!!!!!!



If you simply want to dilute the concentration 10:1 then you just want to add 9 volumes distilled water to 1 part concentrate..

If you are trying to obtain 0.1 ppm from a solution of 1% Iba the easiest way would be to try to find something that mentions PPM in its dilution ratios (then just scale accordingly).

Otherwise whats it's specific mass/density? (the figure listed on its chemical, MSDS, or CAS description) also is it 1% by mass or 1% by volume? (to establish a ratio biased no mass/density). If your base solution has a different specific gravity than water you may also have to take that into consideration (depending on how much information you can come up with and how you want to skin that cat). 
its cool what you can figure out with a scale, graduated measure, hydrometer and a calculator (if your resourceful you might get away with half that stuff.) Math is beautiful dude and its a language that's easy to respect (not alot of special rules or contradictions).


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## nitro20% (Jan 31, 2012)

Guile said:


> If you simply want to dilute the concentration 10:1 then you just want to add 9 volumes distilled water to 1 part concentrate..
> 
> If you are trying to obtain 0.1 ppm from a solution of 1% Iba the easiest way would be to try to find something that mentions PPM in its dilution ratios (then just scale accordingly).
> 
> ...





man i love this. yeah i got the conversions and 1 more page of notes. but i dont have an equation to solve, ur right,,,so help skin this cat(hate cats) dip n grow is a alcohol base liquid and it is 1.0%iba & .5%naa by wieght,,98.5% other ingredients.smells 100% alcohol. so, with that i want to get it to .1mg/L iba..it comes with a concentration cup,,1 tsp. is the X line 5x = 5 tsp. i guess that was obvious. the naa will be what its is.Im shooting for the #2test result of this study:
*http://www.pakbs.org/pjbot/PDFs/41(2)/PJB41(2)603.pdf*


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## Guile (Jan 31, 2012)

nitro20% said:


> man i love this. yeah i got the conversions and 1 more page of notes.
> 
> 
> 
> ...


98.5% other ingredients does pose a bit of a problem... Can you get a breakdown of the other ingredients and ratios they are used in?

I would guess that you might want to find out the specific gravity of the solution (with a hydrometer) then the specific mass, derive the mass of the 2 active ingredients (1.5% @ the appropriate ratios and quantity biased on weight). From their chemical descriptions you should be able to determine their mass per volume (specific density) now I think you can figure out the ppm biased on mass, specific density, and molecular weight. I'm sure you can find a calculator for it online unfortunately I don't know the formula. 

I'm also high and despite rewriting this like 3 times it still doesn't sound right to me, but you are only figuring out how many parts are in a volume biased on weight (specific mass) and applying it to the ratios established on the label (also biased on weight) to determine the number of parts in the mass of hormones used (for the given volume of concentrate).

The molecular weight/specific density (mass per volume) of the hormones are likely the key considerations...

Here is a simple one :One ppm is equivalent to 1 milligram of something per liter of water (mg/l) or 1 milligram of something per kilogram soil (mg/kg).
So essentially, 1ppm = 1mg/l *Source(s):*

http://groups.molbiosci.northwestern.edu/holmgren/Glossary/Definitions/Def-P/parts_per_million.html

It doesn't take into consideration the unknown specific gravity of your base solvent but I doubt you would be off by much..

1 liter of water would weigh 1000grams 1% of which would be 10 grams, if 1mg/L = 1ppm, then 10000 milligrams should equal 10,000ppm (that seems like alot to me) what ratios do you mix this stuff for say foliage feed or long soak application?


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## nitro20% (Jan 31, 2012)

Guile said:


> 98.5% other ingredients does pose a bit of a problem... Can you get a breakdown of the other ingredients and ratios they are used in?
> 
> I would guess that you might want to find out the specific gravity of the solution (with a hydrometer) then the specific mass, derive the mass of the 2 active ingredients (1.5% @ the appropriate ratios and quantity biased on weight). From their chemical descriptions you should be able to determine their mass per volume (specific density) now I think you can figure out the ppm biased on mass, specific density, and molecular weight. I'm sure you can find a calculator for it online unfortunately I don't know the formula.
> 
> ...



yep.,,,math and numbers ,,theres somthing great about it all..i guess it would be the oldest universal language know to animals with a propose.(man kind)k,,,so this is what i needed to get to a ppm??http://www.dipngrow.com/wp-content/uploads/2010/08/Parts-Per-Million-PPM.pdfwell here it is now i got it. but im still not done solveing the last equation....so weight by mass of iba=A weight solution (dip and grow) =X i think its divide A into X = W there it is.i think. then convert W to mg/L. To think i would pay 1000.00s to sit and be graded on this weeks learning disability.this is way funner. 
OK TISSUE CULTURE DUDES u can have ur site back, now its time for results WITH PICTURES i know.


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## Guile (Jan 31, 2012)

nitro20% said:


> yep.,,,math and numbers ,,theres somthing great about it all..i guess it would be the oldest universal language know to animals with a propose.(man kind)k,,,so this is what i needed to get to a ppm??http://www.dipngrow.com/wp-content/uploads/2010/08/Parts-Per-Million-PPM.pdfwell here it is now i got it. but im still not done solveing the last equation....so weight by mass of iba=A weight solution (dip and grow) =X i think its divide A into X = W there it is.i think. then convert W to mg/L. To think i would pay 1000.00s to sit and be graded on this weeks learning disability.this is way funner.
> OK TISSUE CULTURE DUDES u can have ur site back, now its time for results WITH PICTURES i know.



It looks like your dip n grow would be proximately 10,000 ppm IBA and apx 5,000ppm NAA. Assuming Dip n Grow isn't flammable at room temperature, its base solvent is predominantly water and those figures should be within 10% accurate. 
so if you wanted a concentration of 0.1ppm you would use a ratio of 1:100,000 or 1ml to 100L.. 
I would probably go about it by putting 1ml Dip n Grow into a liter container and top off with water to get 10ppm then take 10ml of that solution and dilute into another liter of water to get 0.1ppm.

I'm a bit more sober now and it still feels wrong but I'm not seeing where I'm messing up yet...

Don't worry too much about my earlier ramblings, I was high and over complicating things. this equation for finding ppm biased on mass and the density of water should be within 10 percent (or so) assuming that Dip'n Grow is under 100 proof..


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## pharmacoping (Feb 1, 2012)

whoaaa, you a smarty pants guile ! thanks man, good to see a serious mind here.
I buy it already diluted, because I never learned how to drive a calculator


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## nitro20% (Feb 1, 2012)

Guile said:


> It looks like your dip n grow would be proximately 10,000 ppm IBA and apx 5,000ppm NAA. Assuming Dip n Grow isn't flammable at room temperature, its base solvent is predominantly water and those figures should be within 10% accurate.
> so if you wanted a concentration of 0.1ppm you would use a ratio of 1:100,000 or 1ml to 100L..
> I would probably go about it by putting 1ml Dip n Grow into a liter container and top off with water to get 10ppm then take 10ml of that solution and dilute into another liter of water to get 0.1ppm.
> 
> ...


yep should have went with a less concentrate. newbie's!!! think that we can do cheaper/better.no you are right with that one. Thats how i was thinkin it was broke down.. but listen to this one: NxE=JUSTBUYTHEKIT with a remainder of PRESURECOOKER!!!ha aha ha haha. Newbie x Experience= always a realization.but back to work (with pictures i know)i think i have to re-size them cause that firewall thing..connection..etc. would let this old ass Dell through.lights closet,hepa running now,bleach n clean. to ace for plastic(mini lab)powered gloves no good. to my buddy's ,oshit things to do, cant rush this.LATER!!!oh goin with @1000ml-1 to 1.5 ppm iba, which will make ppm naa @ .5 lower,,perfect!!!


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## Guile (Feb 1, 2012)

pharmacoping said:


> whoaaa, you a smarty pants guile ! thanks man, good to see a serious mind here.
> I buy it already diluted, because I never learned how to drive a calculator


I buy mine in nearly pure/powdered form so I don't have to deal with unknowns. 
I didn't go digging for a calculator either (if I messed up that's why) all the math here was a factor of 10, all I had to do is move the decimal point (the kind of thing you feel comfortable doing in your head when you are stoned). Its partly why i didnt want to get into figuring out everything's specific mass to use volume and density to sort it out... Especially since it felt like I was going to have to do all the looking to get the figures I needed... and it would have required that I payed attention to my math.... 

but with water having a specific gravity of "1" (meaning a liter weighs a kilogram) it just made all the math simple (a factor of 10, so I only had to move the decimal point). The only other consideration is that ethanol has a specific gravity of about .780 (meaning a liter of it weighs about 780 grams).. Its my understanding that alcohol (drinking alcohol) is not flammable at room temperature in concentrations under 50% (100 proof).. meaning that the base solvent couldn't be more than half ethanol. Half the difference in specific gravity between water and ethanol is 11% so your margin of error dealing with the unknown base solvent would be less than that (10.something at the most).. this would translate to similar accuracy in your final solution.. Besides you are only accurate as you can prove, anything beyond that is strictly speculation...


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## Guile (Feb 1, 2012)

nitro20% said:


> yep should have went with a less concentrate. newbie's!!! think that we can do cheaper/better.no you are right with that one. Thats how i was thinkin it was broke down.. but listen to this one: NxE=JUSTBUYTHEKIT with a remainder of PRESURECOOKER!!!ha aha ha haha. Newbie x Experience= always a realization.but back to work (with pictures i know)i think i have to re-size them cause that firewall thing..connection..etc. would let this old ass Dell through.lights closet,hepa running now,bleach n clean. to ace for plastic(mini lab)powered gloves no good. to my buddy's ,oshit things to do, cant rush this.LATER!!!oh goin with @1000ml-1 to 1.5 ppm iba, which will make ppm naa @ .5 lower,,perfect!!!


If you haven't figured that one out yet its just 3 drops per Liter

The concentrations you describe seem low to me, unless the cells you want to influence are in like constant direct contact with it... Are you putting this stuff into your culturing gel? or using it in an aeronautic or DWC type cloner?

It seems that mimicking the culturing gels they use here (to promote rooting) in a liquid solution (simply omit the gelling agent) should make about the best cloning machine solution ever..


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## nitro20% (Feb 1, 2012)

Guile said:


> If you haven't figured that one out yet its just 0.75ml/L, apx: 3ml per gallon or Tablespoon per 5gal jug..
> 
> The concentrations you describe seem low to me, unless the cells you want to influence are in like constant direct contact with it... Are you putting this stuff into your culturing gel? or using it in an aeronautic or DWC type cloner?
> 
> It seems that mimicking the culturing gels they use here (to promote rooting) in a liquid solution (simply omit the gelling agent) should make about the best cloning machine solution ever..


yeah i know they seem low,,but for all i read on tissue cultureing for the harder woods like/and mj(theres alot of studies on tissue culturing on mj) they all seemed to use a .5 to a 2.0 ppm of iba for the first to weeks or so, and naa run about .5 ppm less. NOW,, im with that other guy, every thing i say is a lie QUOTE.ive had no/to nill guide (except for u guys) so what ever i made up is the truth so far,,with no results...but its easier then that.....DDDDUUUUUU,,,1 part dip n grow to 19 parts water=50 ppm!!!!so easy its [email protected]#[email protected]!#$ SSSOOO @ 1ml (concentrate) per 1200ml water= 1.5625 ppm. YYYYEEEEEAAAA i did it.im confident with that! to my buddys house,, theres some sick plants to cure!


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## Guile (Feb 1, 2012)

nitro20% said:


> yeah i know they seem low,,but for all i read on tissue cultureing for the harder woods like/and mj(theres alot of studies on tissue culturing on mj) they all seemed to use a .5 to a 2.0 ppm of iba for the first to weeks or so, and naa run about .5 ppm less. NOW,, im with that other guy, every thing i say is a lie QUOTE.ive had no/to nill guide (except for u guys) so what ever i made up is the truth so far,,with no results...but its easier then that.....DDDDUUUUUU,,,1 part dip n grow to 19 parts water=50 ppm!!!!so easy its [email protected]#[email protected]!#$ SSSOOO @ 1ml (concentrate) per 1200ml water= 1.5625 ppm. YYYYEEEEEAAAA i did it.im confident with that! to my buddys house,, theres some sick plants to cure!


One of us is off by atleast decimal point (I'm willing to accept if its me, but please point out where).. I get 20:1 making 500ppm and 1ml to 1200ml water to get 8.3ppm

1ml to 6.5L (about 7 quarts, or 1 and 3/4 gallons) should give you about 1.5ppm (IBA and 0.5ppm NAA) if my math is right..

If there are 20,000 drops in a liter (20 props per ml) , and your concentrate is 10,000ppm then you will want to use 3 drops per liter to achieve 1.5ppm 

Brings to mind how they say to only use one drop per gallon super-thrive, most everything I read seems to suggest the ideal concentration of vitamin B to be quite low sometimes around 0.5ppm. 
The concentration of "B vitamins" in "Superthrive" must be similar to the hormone concentrations in "Dip n Grow"


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## nitro20% (Feb 1, 2012)

thats funny cause last night right at bed time 3:00 am i came up with that same answer. and this morning with the final confident answer it was about 3-4 drops in a liter @ 1.5 ppm iba.which is what im doing right now!!! (WITH PICS I KNOW) gettin my mini plastic lubor-u-tory' ready. cooked the tools and got king kush, diesel, blue dream, head band,grand daddy purps,and my own afi-gui-snow roturallas. told u it was a universal language. i did it probably wrong but got the same result,,,thats scary!!!this is what i did, just took and made the dip n grow scale into ml per parts water. then stared with 1ml concentrate @ 199ml. parts water still equals 50 ppm.then to the calculator divided by 2 a few times,, 1ml concentrate to 398 parts water = 25ppm iba. 1ml to 796= 12.5ppms iba.etc etc...?yeah?no? the reciepe is 3 drops of concentrate to 1l , hopefully reach 1.5 or so.cant be to picky,i picked this cat.


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## Guile (Feb 1, 2012)

nitro20% said:


> thats funny cause last night right at bed time 3:00 am i came up with that same answer. and this morning with the final confident answer it was about 3-4 drops in a liter @ 1.5 ppm iba.which is what im doing right now!!! (WITH PICS I KNOW) gettin my mini plastic lubor-u-tory' ready. cooked the tools and got king kush, diesel, blue dream, head band,grand daddy purps,and my own afi-gui-snow roturallas. told u it was a universal language. i did it probably wrong but got the same result,,,thats scary!!!this is what i did, just took and made the dip n grow scale into ml per parts water. then stared with 1ml concentrate @ 199ml. parts water still equals 50 ppm.then to the calculator divided by 2 a few times,, 1ml concentrate to 398 parts water = 25ppm iba. 1ml to 796= 12.5ppms iba.etc etc...?yeah?no? the reciepe is 3 drops of concentrate to 1l , hopefully reach 1.5 or so.cant be to picky,i picked this cat.


Go with what is recommended by the manufacturer. my margin for accuracy can't match theirs (they know whats in it).

Though if you had those figures yesterday we might have gotten here sooner..

If you give me the mixing instructions from the label (or package insert) there should be enough information to figure most any concentration out..


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## nitro20% (Feb 1, 2012)

its that web site. ppm of dip n grow. thats what i went off of.its say hhhh nothing, no box, but it did come with a 1 to 1 dilution cup. @ tsp per. concentrate line IS 1tsp. im trying not to 2nd guess myself but man if my little break it down by 2s equation seemed pretty accurate. 1 ml per 1200=1.5625ppm.every time.and thats 15 to 20 drops per1200ml.broke that down 6 times so 9 to 14 drops per l.but the thing is,, ratio of 3 drops per L, will not hurt the first 10 day stage, to weak.i will have some time to "get it together" so what do u think. well i guess thats what u been doing.


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## wyteboi (Feb 2, 2012)

i take it , the chart on their website wont help you none nitro ? 

i dont speak tissue culture, but i will .... gotta LOT of reading to do.


can you guys lay out some real simple info for me ?

are these pieces of plant sitting in a hormone or solution , in an airtight environment ? or is this stuff open in a sterile "lab" (or box)





soil


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## Guile (Feb 2, 2012)

I just found this PDF file... _PARTS-PER-MILLION_ MATRIX - _Dip_'N Grow (I can't believe you got me to do your homework) This has all the info you need on it and would have made it pretty simple to figure out any concentration you would need based on whats listed.

Considering that the undiluted strength is 10,000ppm I have a feeling we figured things out pretty well yesterday.. 

3 to 17 gives you 1500ppm (thats using 3 parts concentrate out of 20 parts total) given that it is 1000 times as strong as you want you would apply a 1000 to 1 ratio making 20,000 parts only 3 of which are concentrate. 3 parts out of 20,000 with 20,000 drops in a liter means 3 drops per liter..

If you use 1:199 (1 out of 200) = 50ppm which is 33.3333 times as strong as you want. so you multiply the water used accordingly (to get the correct disillusion) you end up with 1:6633 (6.63L assuming you used 1ml concentrate) otherwise 1 drop to 331.65ml (332 plus 1 equals 333ml or 1/3 of a liter, or 3 drops per liter)

If I'm not mistaken my math from yesterday showed that 1ml to 7 quarts or 6.62L (10ml, less than a tablespoon of water from the above formula) would give you "about" 1.5ppm 
6620ml divided by 20 (the number of drops in a ml) equals 331ml per drop or 3 drops per liter (give or take a drop or 2 of water).

I would be inclined to use the figures biased on 1:199 as it is the closest to the scale you are trying to obtain (smaller margin of accuracy). Or my figures from yesterday, they seem to be more accurate than I had hoped.


About the 1:1200 thing... 
Starting with 1 drop @ 10,000ppm, adding 1000 (999) drops of water will get you to 10ppm (this is way more than 20% from where you want to be) adding the other 200 drops (for a total of 1200) will give you 8.3ppm (like 5 and a half times as high as you want to be)

*


*


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## Boyz N Da Hood (Feb 2, 2012)

This is a very interesting read! Just read the beginning and I'm intrigued! Gonna roll up a blunt and read up a lil more... Quick question does this also apply to weed plants? I don't see why not but you never know..


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## wyteboi (Feb 2, 2012)

Boyz N Da Hood said:


> Quick question does this also apply to weed plants? I don't see why not but you never know..


yes , any plant. i think. im sure some are a lot easier then others. i dont even speak the language yet , but its very interesting.





soil


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## Guile (Feb 2, 2012)

The way I have it figured you will probably need a pressure cooker (the kind they use for canning meat and low acid foods) and some wide mouth canning jars (1 cup jobie's if you can find them, I have some 12oz ones and they seem a little tall). 

You can probably make your incubator out of a couple plastic (Sterilite) totes nested together to make a warm water bath using chlorinated water and fish tank heaters for temperature control. I would probably cover the rim of the upper bin with weather strip (make as air tight as possible) and mount a UV light inside the lid and just leave it running because the metal lids to your jars will keep most the light off your culture so it should be relatively unaffected however it might cut down on contamination in the bin (I have an ozone generator idea too that could maintain darkness but it might be a little harsh).

Other than that alot of sterilization (in your pressure cooker or using alcohol). I have seen a few recopies for making the gel, just add that mixture to your jars before pressure cooking them. Allow jars to cool to room temperature before adding the plant material you prepared with sterile tools/instruments, and procedures (if you wanted to introduce a virus you would do so at this point). Wash your hands, tools, jars, and working surface with alcohol before each opening/or use of the tool, incubator, or jar. (lint free alcohol wipes might be handy).

It seems doable... I have a feeling the "don't open the door until its done cooking" rule might apply.. I would probably prepare several jars checking each with a diminishing degree of frequency. check the same one every day, another every other, the following every 4th, so on.. As fungus and other contaminates claim victims just move the others up in queue. The odds are the air in your room is contaminated, so it might be a struggle (though I guess you could make a "clean room" out of plastic sheathing, wear a respirator/mask, and build a bigger ozone generator, but seems like too much hassle for some clones).

This 1.5ppm IBA 0.5ppm NAA ratio we worked out getting from Dip'n Grow should work for rooting (I personally considered using it in a DWC cloner), I assume you would also add some nutrients (I've even seen sugar) and of curse the gelling agent before pouring into your Mason jars and pressure cooking (per instructions for canning meat). This should work on a pretty small growing tip (like micro propagation might imply, it would just be a very small cutting). 
If you want to create a new growing tip from cells harvested elsewhere it seems like it could be a bit more involved..


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## pharmacoping (Feb 2, 2012)

no fish tank heaters, or chlorine baths necessary. absolutely NO UV lights,or ozone, other than that, pretty close.

I naturally check my baby's often, as transfers happen daily sometimes. a small 100 watt 4 lite T5 fixture hangs over the totes on a timer. storing/growing a clone is exactly the same protocol as culturing a new growing tip , cept for the recipe. dont open any containers unless doing another sterile transfer. a closet works great. mites are your enemy dust mites love the recipe, they carry mold. keep the area sprayed with lysol, and no traffic. really is no big deal. you'll see. sugar is a must in culture, they are not photosynthesizing to make carbs, so sugar is tube fed. pressure cooker, aluminum foil, paper towels, scalpel, tweezer, baby food jars/lids, bunson burner, denatured alcohol. no respirator, tyvek suits, or sterilized rooms here. I use a simple grow tent, spray lysol upon entry/exit, inside and outside the tent. your mold will come from your explants, and sterilizing those pieces is really the only difficult part. next to figuring out recipes


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## pharmacoping (Feb 2, 2012)

small bits of plant material are either suspended or laying on, or partially submerged in an in vitro specialized mix gelled, and then sterilized in a pressure cooker, while being sealed within their incubator, a baby food jar/lid is awesome for rootballs. the collection of jars is kept in a still, clean area with some light. transfers of these cultures to new vessels, or dividing them to make multiple "clones" is done in a tipped rubbermaid, wiped with alcohol, and quickly done. one hand will open the jar while the other is tweezing bits into it, and closed quickly. the jars are not "canned" with tight fitting snapped lids after a transfer, but will preserve the gel for a long time if sealed and dark. temp should be 70-80 f, lite on 14-16 hrs a day. roots get none. hormones(cytokins and auxins) sugar,agar,water,correct basal salts,plant preservative are in the mix at varying rates. 
I stock hundreds of rootballs of specified strains, until needed to sprout. they are transferred to a sprouting/shooting mix, covered, and then go to the veg room in cycle. roots are divided daily to replenish stock. 

hope this helps


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## nitro20% (Feb 2, 2012)

yep you are 100% right....i came up with that number (2-3 drops per l.) before i found that site. sorry man i thought i put the page up once(about the ppm).and my math was all messed up in the last entries ,,the funny thing is,,,is rhat i 2nd quessed our stoner math and change it...not goin to say what i did,,but i swear i saw the tiny little plantet pieces go from a kinda limp to straight up, kinda like when the wind blows up the hole of my pants,,,booing! so either it will work or not.if not it was good practice for sanitation. its hareder then u think. at the last bit of mix i started to notice hairs and things.i thought i was keepin everthing covered well.i wonder if there is such contaminates,, if the pressure cooking takes care of that to.i have agar comeing,, the health stores prices are 11$ for 6 tbls.holy shit..so if this turns into hot jars,,which is very likeley,,ill do it again @ 2-3 drops per 1000l.

im a first batch, heard about tissue culture decided it was cool 3 day ago PROFESSIONAL,,, beeeiiiiiitttcchhhheesss!!! JUST KIDDIN, K this is what i know,,no results,,,but the first cutting, the actual top from the mother,,goes in to a mostly glucose based and a form of plant preservative material (PPM)or(nadcc)and gel agent with VERY WEAK!!! iba & naa.these planlets are like premature babies basically with no nerve stucture,,just a tissue with parts or a cell to start from,,from here the goal is to reach CULLIS state. which is when the plant cells know exactly what to do and were to go..by then if all things are a go, the plantlet u started 40 days ago can be split into 3 to 10 plantlets a jar.some for more micro-production and the ones for soil treat like barely rooted clones for 7 days,,till strong enough to GO FOR IT!! fuck,,,its fun though..SUGGESTION: do not buy or get any thing till u are SERIOUS BOUT THIS,,,CLEAN ROOM,PRESSURE COOKER, A PLACE WITH NONE TO NO MOVEMENT,HEPA FILTERS,E.T.C.THIS IS NOT ANYTHING LIKE CLONNING OR ROOTING A PLANT,THIS IS A WAY TO WEEN OUT A PLANTS DEFECTS DUE TO AGE,PEST,E.T.C. OR IF YOU REALLY HAVE NO LIFE TO LIFE AND NEED SOMETHING TO DO (ME)THE KITS ONLY GIVE PART OF THE THINGS NEEDED FOR 90 % RESULTS AT BEST (I THINK)I BOUGHT EVERY THING I NEEDED AND IM IN IT FOR 300 +$(no kit). I HAD AQUARIUMS,HEPA FILTER MACHINE (200.00), LIGHTS, VENTING, ITS GOES ON AND ON.. THATS JUST THE BASICIS,,I THINK..



I might be really high, but this is my experience,,please for the overly confident kit makers,,no hard feelings,,but every ones sanitary view might not be enough for the home they live in.cats,dogs,birds e.t.c. In the 3 years ive lived in this house,,my shoes have NEVER been past the sweep of my doors. my dog gets bath once a week, i am to say the least CLEAN.
yep,,you are right, i have NO life. not by choice though,,lets just say,,if u are deciding to take up a meth hobby,,,use clean needles!! it might come bite u right in the LIVER 12 yrs. later.....


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## nitro20% (Feb 2, 2012)

oh and to make 1,000,000,000 plants in 1 year from 1 planlet, that Is the main goal in micro-reprodution by tissue culture..
damn dip and grow could turn into dipnGO GO GO GONE!!! move a bond from here to there and you can get some drastic effects, especially if proteins can break apart the rings at the changed points and the change of the bond location in the pyrrol ring worries me a bit). The long butryic acid chain seems like something that could be a candidate for replacement to produce illegal tryptamines or their analogs. My question is, "Given that tryptamine is banned in this paranoid age, have we found a new widely available precursor chemical?"quote to a study

oh and sorry to ALL of us that had to listen/read that illiterate excuse for math i was 100% responsible for.note: when breaking down ppms by 2s, must take that new volume and double, but not the parts per million in question.please feel free to pass warning,,i deserve it.


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## nitro20% (Feb 2, 2012)

pharmacoping said:


> no fish tank heaters, or chlorine baths necessary. absolutely NO UV lights,or ozone, other than that, pretty close.
> 
> I naturally check my baby's often, as transfers happen daily sometimes. a small 100 watt 4 lite T5 fixture hangs over the totes on a timer. storing/growing a clone is exactly the same protocol as culturing a new growing tip , cept for the recipe. dont open any containers unless doing another sterile transfer. a closet works great. mites are your enemy dust mites love the recipe, they carry mold. keep the area sprayed with lysol, and no traffic. really is no big deal. you'll see. sugar is a must in culture, they are not photosynthesizing to make carbs, so sugar is tube fed. pressure cooker, aluminum foil, paper towels, scalpel, tweezer, baby food jars/lids, bunson burner, denatured alcohol. no respirator, tyvek suits, or sterilized rooms here. I use a simple grow tent, spray lysol upon entry/exit, inside and outside the tent. your mold will come from your explants, and sterilizing those pieces is really the only difficult part. next to figuring out recipes



you make it sound simple, man i was having a rough time thinkin i was not being clean enough.like i said there was hair in the mix at the end,but it got in there as i filled the jars, probably. have a 30 gal aquarium that i turn on end with 2 layers plastic,1st is THE COVER. then there is the one that i put [+ +][ +] holes in the two openings.so is it ok to take the pressure cooked med. and put them in my tank (91% alcohol & gloves every step of the way) to cool and so i might cook more or cuttins ready? how long is SAFE for an unsealed lid protected from the particles of death that fall @ 1 ft. per min.??ha ha. man, they have been cultured for about 9 hours, 1st look, i must be high as fuuuukkkk, could they be,,, should i say it?? are they GROWING? could it be that fast??????if i "HOT JARED THEM" (ppm) would i see signs of the to?


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## pharmacoping (Feb 3, 2012)

I leave the jars in the pressure cooker overnight to cool, while hot they draw air into themselves, ...........unless transferring or hardening off, the lid should stay affixed to the tops of your containers. some tape or wrap the containers. I've considered keeping the containers inside zip locks for more protection, but has not been necessary in yrs since I moved into the tent. 

yep, you see growth really fast. i didnt cut arm holes in my latest sterile tub transfer area, no issues . arms,gloves,outside of jars, are all sprayed with alcohol/lysol, seems good here


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## Guile (Feb 3, 2012)

pharmacoping said:


> no fish tank heaters, or chlorine baths necessary. absolutely NO UV lights,or ozone, other than that, pretty close.


I find that my cuttings seem to respond well to warmth.. I keep my place pretty cool in the winter months 60-65F (many basement "project arias" never go much above that without adding heat somehow) I have noticed an improvement in rooting times when I bring cuttings up in temperature to around 70-75F (actually I have gone as far as 85F without running into problems). 

I know this might sound silly but I believe a plants bimetallism is pretty closely related to heat.. 
The only reason I said to use chlorine in the water bath is for when you take out the second (upper) bin (that you keep your jars in) an set it on something else to top off the water bath you don't transfer contaminant. 
I chose that method for temperature control because I always have fish tank heaters around (I use them to culture other things in water) and I find heating mats/pads to be less accurate..

I haven't used UV light or Ozone around small cuttings before so I have no idea how harsh you can be before damaging your plants, I figure UV defuses pretty quickly over distance so keeping it near the lid (where the air will get in) and with the metal lids on the jars it might be a good way to manage that micro environment. 
I have used homemade ozone generators to "sterilize" my grow room. A healthy dose of ozone after lights out can help keep large/dense buds from rotting as they mature (you just put it on a timer like you would your lights or pumps). A large enough dosage will even take care of a mite problem (though you would have to let it go for hours).

I just figured biased on the amount of failure I have heard about that keeping the cleanest (and most regulated) environment should yield the best results..


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## pharmacoping (Feb 3, 2012)

I agree, cleanliness is key, but I wouldnt keep any water anywhere near the growing cultures, even bleach water. 
Ozone will steal your terpine profile in a grow room, not a good idea except for maybe exhaust, if you dont have a sealed grow.

the t5 lamp inside the tent keeps temps around 70-80, esily maintained in a tent, in a closet, in my lab room, which is climate controlled, and also where traditional cloning was taking place. 

lowering humidity and increasing air flow is what will stop bud rot in its tracks, very effectively. 

My opinion, 99% of my mold failures in vitro are due to infected explant material, not the pressure cooker, or room, or tools. everything is flamed off before coming in contact with another specimen. the jars, once taped up, have been impenetrable thusfar to any mites or mold, here, since moving inside tent a year ago. more failures were seen when the grow chamber was on a shelf in a room, but not one jar has gone moldy (in the tent) if they've made it for 5 days without. dirty explants cause me mold within the first few days of transfer, occasionally. and I have killed explants with bleach, alcohol trying to achieve this issue, until last year when I got good at it. I used to transfer inside a sterile zip lock, with the vessel and tools inside ! that worked very well for me, as my head could see the view from the top, very helpful with timy bits.

I like the "mote" with the two bins, but believe its overkill. 10,000 ppm(bottle of C02) for a couple hours will kill every living bug in your room, and please your plants. repeated with the breeding cycle of mites(or any pest) will rid the garden for good....then, dont take in stranger clones, unless tissue cultured, of course !!


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## Guile (Feb 3, 2012)

pharmacoping said:


> I agree, cleanliness is key, but I wouldnt keep any water anywhere near the growing cultures, even bleach water.
> Ozone will steal your terpine profile in a grow room, not a good idea except for maybe exhaust, if you dont have a sealed grow.
> 
> the t5 lamp inside the tent keeps temps around 70-80, esily maintained in a tent, in a closet, in my lab room, which is climate controlled, and also where traditional cloning was taking place.
> ...


Yes ozone kills the smell, and probably affect's the flavor (I've never compared side by side). I probably should look into Co2 again, I've encountered some interesting sounding experiments recently involving Co2 (that could make other applications more feasible).
In the meanwhile Ozone seems like a decent way to keep a "clean room" "sterile" (or atleast as clean as possible). Besides I think Co2 is heavy and might escape under the sides of your plastic tent before reaching lethal concentrations at the highest surface in the room (or atleast take alot of bottled gas to do it) 

I use a 500cfm exhaust fan and multiple low lying inlets (both inside and out, for temp control reasons). I manage heat the best I can without phase change cooling systems. I think my biggest problem is humidity and of coarse I have been putting off getting a phase change dehumidifier. 
This is probably going to sound cheap (even coming from me) but I can't stand running big power through my grow room.. I hate the kind of power my lights eat up and I run relatively low light density by many peoples standards. I'm looking forward to building a greenhouse this spring, (damn near makes me horny thinking about my ladies laying out in the sun)..

I like the idea of having a dedicated micro environment to work in, (but I also like working with my bare hands in plane view) I could defiantly see the advantages to making a box like you describe. You could probably use some kind of chemical (or cleaning) glove band clamped to a ring (drain pipe?) epoxied around an arm hole on the inside. If you used Polly-carb panels and the right epoxy you could tailor build the box to your own specifications (though I would probably build 4-5 of the 6 sides out of plywood coated with a thick coat of an oil based enamel paint (like Rustoleum) and alot of silicone calking at the seams (despite copious amounts of epoxy). the top (only opening part) could be as simple as a sheet of poly-carb, a piano hinge, some weather stripping, and some kind of locking mechanism (or weight to hold it shut).. 

I could also see building it from a $30 stock tank.. I use these for reservoirs and they are well built.. If you buy 2, you could nest them with water heaters in-between so you can better control temperatures (make the cords come out through the arm holes and things should fit tighter together).

Not even sure "mote" is the best description, that seems to imply that the liquid is exposed.. I'm more of less trying to use water to vacuum pack 2 totes together, if it could be sealed well enough I'm sure you would never have to worry about water loss, but where the wires from the fish tank heaters will prevent a seal I'm willing to speculate that you could need a topping off eventually. I've used a similar method to warm my cloneing/propagation flats..


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## pharmacoping (Feb 3, 2012)

I ozone 1 time a year, after the room wash in the fall, for a day, with plants in room. then back on the shelf. I've had ozoned weed totally devoid of detectable terpines and it scared the shit out of me, compared notes, sure enough true. these are exact replicated plants that stink to the highest order every day ! only happened once, while running the ozonator. my grow room is as sterile as the other side of the wall, outside, except one day a year, its really clean that day !
my tissue area is very clean, but surely not sterile, no moving air, breath held until capped/taped. when I transfer inside a flipped tote, the air is filled with lysol, and the inside is alcohol wiped with sterile paper towels, outside jars wiped also. tent door/zipper and interior is sprayed with lysol upon entry. no fancy stuff here. just the ability to flower more plants, instead of cloning more plants. 

aha, the c02 does not escape a secret jardin grow tent before 10000ppm are reached in seconds, with a small bottle(20lb$20tank) of c02. not experimental at all, its standard for every seed breeder I know of, thats where I learned it
. 
I only tc in a tent like that. I have a 400 sqft sealed room with a c02 gen on the wall. I can set the ppm's higher, and death occurs within an hour in both curtain divided rooms. never had mites, but they need to breathe o2 also, so they'll die too. it takes minutes to get the room to that concentration with a propane burner a quarter the size of a bbq grill. trimming in close qtrs will raise your immediate concentration near the plant to 1500ppm in seconds for comparison. also-your 3 foot plant will consume 100% of a garbage bag of 1500ppm c02 in less than an hour, depending upon temps, etc. c02 is a great solution to a hotter running room, as the metabolism increases they bum out because their c02/water/food is limiting them(liebligs law) so adjusting these factor will always result in increased weight, rate of growth, and over all health. a garden without additional c02 is limited....ambient c02 is bout 400ppm, used by your plant in a minute(one importance of moving air) instead of 1500ppm as can be consumed. outdoors is a non issue as the air immediately surrounding each leaf is exchanged infinitely, unlike an enclosed room. it was probably the most important "toy" I've purchased, and in weight/flower times, it paid for itself in the first 60 days of use.

Imagine only being allowed to breathe a fourth of the oxygen you are able to, all while being stressed out, enclosed, with artificial lighting and ......we would be much less productive breathing 1/4 of our available breath.


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## Guile (Feb 3, 2012)

I run a propane heater for temperature control this time of year (but have no means of testing Co2 concentrations).. 
I've read about enriching the air in a grow room with up to 1500ppm Co2 and running average temperatures up to 85 degrees. I just never really went that way because gas, system automation and temperature management costs were all expensive considering factors.. I have often fermented beer/wine in my grow room, and recently began using a propane heater (for its emissions, though I believe it might contribute to my humidity problems).

The other experiments I meant had to do with super-critical Co2 extraction techniques. (a couple different phase change systems I'm contemplating).


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## pharmacoping (Feb 3, 2012)

secret jardin tents are all but sealed, negligible leakage, 100 bucks for a small one 2x2. 

what is a phase change dehumidifier? I got mine at lowes, digital, rocks, flawless automated in the grow room, $150 3 yrs later. electric heat in the house(tc) is dry so i dont need one in there. 

if you get a chance to checkout c02 next to butane extraction, you'll see why the home guy goes for butane, those pressures are scarier than a handful of tane in a stainless sealed tank, run back and forth with no leaks. 
c02 can target thc better, probably, but, when I "clarify" oil with heat/cooling, filtration, to remove (cbd/cbn/waxes) I end up with a product that scared montel williams and is unusable by most. it comes back from the lab sometimes at 88% and is ridiculously potent. only one patient consumes this so I do it for him. would put a girly in the er though. It's so very different than the oil containing the other compounds like terpines and cbd and cbn, which balance the psycho effects of thca.

my box has no glove hands, or cut outs, only flipped on its side, on top of a small table., in the tent. I reach inside to flame tools, transfer, and then out. no other special precautions have afforded me any better results than I have now. it cost 11 dollars at lowes, I removed the hood, set it on a small table, and turned it on its side to protect things falling from above in the 1/2 second I'm inside a jar. works great. I kneel in front of the opened tent and reach into it. only in there for a minute to do 25 transfers/incisions, inside the open end of the rubbermaid, thats opaque, letting light in. roots are sheltered from lights in a different area in a dark tub.

why not put your mini tc area in a less traveled, dry warm environment, where cords and heaters and waterbaths arent necessary? unless you're building a pro lab, then thats awesome ! I've found that a grow room is the absolute worst place to conduct tc . I could gain more success in a shed, on a work bench, next to the vice. the garden atmosphere is conducive to all kind of airborns that wont, or do(botyritus(sic)-budrot) affect ganja growing, but will def affect in vitro activity, for sure, been there.


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## pharmacoping (Feb 3, 2012)

it does add humidity. mine is a 20$ propane tank monthly,and liquid cooled by a seperate reservoir(in the cold) filled with rv antifreeze. no heat issue at all, luckily. I run the lamps and electric baseboard heat with 240volt in the growroom, but the ac and the rest of it is 120v. I am running at exactly half capacity, as planned when built. maybe wife becomes a cg also??
I pull five gallons weekly water from the dehumidifier, at a cost of 14$ monthly. barely runs daytime lites on and ac runs every 60 minutes for about 10 minutes, in the winter, longer more in the summer, average cooling cost is 68$ monthly, but at night digi thermos drop temps a bit, naturally, and it tries to get more humid. 
When I grew in 6 tents I also put my jugs in there to ferment, and measured it. but its vented out without control, unless you got a controller. in the sealed room its very efficient.


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## pharmacoping (Feb 3, 2012)

Guile, I knew you would appreciate this pic, this buds for you, and ...Cheers ! 
thats my 10 day bad ass hooch from a store bought organic juice. use a pinch of champagne yeast instead of normal, and add a few teaspoons sugar
and its done in a week for girls, and killer in 10 days for boys.


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## nitro20% (Feb 3, 2012)

ALL NONE BELIEVERS ,, I HAVE RESULTS/WITH PICTURES!!! in under 24 hr. of culture i have kept myself from entering the (area).i have have entered twice (91% alcohol all the way)once at about 9 hours after culture,then at 9:00am today.i saw that there was a definite change in the size of some cultures, at least 1/16 of new leafy material was observed (my afis.) on two of 6 jars.fault notes:the seal tape used (ace poly sealing tape for windows) was pealing off the jars i stretched the tape around.one taped with no stretching of tape were fine...NOT GOOD. i think i might have seen nonuniformed bump/bumps in the smoothness of the medium.(contamination???) My puter is putering so if anyone would like to post these pic for me or just for personal i could email them,my puter likes email. 
gulie,,,i went and did the math last night, there are already made up equations for this math (of course) broke it down like a shot gun,,,and you don't want to know the first answer. its probably NOT right, and you are probably tired of hearing bout it,, but it was fun retrieving all the atomic weights and conversions.....the WRONG answer was:21.37g/l dip n grow...ha ha ha ha 2 hours and ?.i felt smart for a second.ha ha!!! SMART?? ah shit,, short term memory loss had me again. wish i had more jars,,, but i think for the first timer a MOCK UP for experience, to see and learn the hows and whens..i could see were i might be takeing my bed to the dump and doing the tc conversion on my last room.
as for the BETTER RESULTS off of a work bench thing,,funny deal,,my spear room is a NITRO/eletric boat and truck RC hobby room.shoved every thing to one side of the 18 ft. work counter.
Its the dustiest room in the house.this is where my clone/mother cabinet is,and now the closet with upon entry, 1st shower curtain,then closet doors then a plastic that goes from the floor to the shelf above.making a clean area within the closet.the hepa filter draws air 1/2 from inside of the clean/plastic-ed area and half from inside the shower curtain and the closet doors but out side the plastic.its winter so that room stays very cool,only heat source is the pioneer Jr grow light in the cabinet/and some morning sun.culture area is 68 -70 deg. humidity is what it is.no control.


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## Guile (Feb 3, 2012)

pharmacoping said:


> secret jardin tents are all but sealed, negligible leakage, 100 bucks for a small one 2x2.
> 
> what is a phase change dehumidifier? I got mine at lowes, digital, rocks, flawless automated in the grow room, $150 3 yrs later. electric heat in the house(tc) is dry so i dont need one in there.
> 
> ...


 Phase change just means liquid to gas (or gas to liquid) same idea that most compressor driven refrigeration systems use.. The dehumidifiers I am most familiar with are very much like an air conditioner (infact many air conditioners have a dehumidifier setting I believe).

Perhaps I am overestimating the contamination risk. During the summer I have a feeling that the air in my place gets pretty thick with spores. Natural yeasts are common enough, it only takes a couple days to get a sour dough bread starter going (in the summer when the windows are open) I live in a pretty damp aria and I think it contributes to some pretty lively air...

If you only saw my house... I have every nook and cranny stuffed with something to make room for some project or another. Partly the reason I'm planing to build a greenhouse this spring is to move mother plants outside for a while and free up some space (not to mention saving on electricity). I have more projects than space most the time. I've even thought about buying a shipping container to turn into another project aria..

I don't honestly see myself doing much micro propagation unless I read/hear something really special dealing with genetic manipulation through carrier virus like you had mentioned earlier. (like adding the gene for DMT, Salvinorin A or an interesting Tropane alkaloid) If I go that far I would probably take it to the extreme with environmental control to protect my fragile ego (I hate repetitive failure). 
Just speculating the kind of resources that might be necessary to achieve a reasonably high level of success, If I have a good idea it might help someone else (or benefit me in the future, if I do explore this further at that time).

I have considered using a small fire on demand hot water heater (like the ones for camp sites) with some sort of Co2 switch to trigger a circulating pump that would basically connect to a 50 gallon drum or radiator outside (low heat Co2). 

I'm still contemplating other heat exchanger options too, like burying a couple 50 gallon drums under ground with evaporative cooling towers and fan inlets above. I figure with a couple big fans, a pond pump and some recycled radiators (or heater cores) mounted in a bezel (on either side of my air inlet) should help with temperature control without costing the kind of money AC would.

Could you draw me up a basic schematic of our Butane extraction device, a simple theory of operation would be wicked kick but too.... How to you remove the waxes and other impurity's?

By the way, I like how you think... You seem pretty resourceful...


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## Guile (Feb 3, 2012)

nitro20% said:


> ALL NONE BELIEVERS ,, I HAVE RESULTS/WITH PICTURES!!! in under 24 hr. of culture i have kept myself from entering the (area).i have have entered twice (91% alcohol all the way)once at about 9 hours after culture,then at 9:00am today.i saw that there was a definite change in the size of some cultures, at least 1/16 of new leafy material was observed (my afis.) on two of 6 jars.fault notes:the seal tape used (ace poly sealing tape for windows) was pealing off the jars i stretched the tape around.one taped with no stretching of tape were fine...NOT GOOD. i think i might have seen nonuniformed bump/bumps in the smoothness of the medium.(contamination???) My puter is putering so if anyone would like to post these pic for me or just for personal i could email them,my puter likes email.
> gulie,,,i went and did the math last night, there are already made up equations for this math (of course) broke it down like a shot gun,,,and you don't want to know the first answer. its probably NOT right, and you are probably tired of hearing bout it,, but it was fun retrieving all the atomic weights and conversions.....the WRONG answer was:21.37g/l dip n grow...ha ha ha ha 2 hours and ?.i felt smart for a second.ha ha!!! SMART?? ah shit,, short term memory loss had me again. wish i had more jars,,, but i think for the first timer a MOCK UP for experience, to see and learn the hows and whens..i could see were i might be takeing my bed to the dump and doing the tc conversion on my last room.
> as for the BETTER RESULTS off of a work bench thing,,funny deal,,my spear room is a NITRO/eletric boat and truck RC hobby room.shoved every thing to one side of the 18 ft. work counter.
> Its the dustiest room in the house.this is where my clone/mother cabinet is,and now the closet with upon entry, 1st shower curtain,then closet doors then a plastic that goes from the floor to the shelf above.making a clean area within the closet.the hepa filter draws air 1/2 from inside of the clean/plastic-ed area and half from inside the shower curtain and the closet doors but out side the plastic.its winter so that room stays very cool,only heat source is the pioneer Jr grow light in the cabinet/and some morning sun.culture area is 68 -70 deg. humidity is what it is.no control.


I'm a little unclear about the Dip'n Grow mix ratio... what have you determined to be the correct dilution rate to acheive the 1.5ppm IBA you were after?

Nitro toys are always fun, I useto do airplanes and wanted to try boats (I've seen some fast ones). I have played with RC cars and helicopters too but strictly electric on them (so I could run them indoors).


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## pharmacoping (Feb 3, 2012)

my c02 generator is exactly what you describe with a bunch of ul listed safety switches, no gas, no 02,too hot, no water shut off, etc. however it is an obvious retrofit by a reputable company(advanced nutrients) and was well worth the hassle avoidance for 399 delivered. no fail for three yrs now. I cant draw, but google has the tamisium.
three parts pictured above, top is butane, middle is herb, bottom is holding tank. liquid butane gas is manipulated via heat/cold thus moved at a controlled rate from one tank, through the herb, to the other tank, then evap'ed back to the butane tank, leaving the extract in the holding tank, and the butane for reuse, and never exposed to the air around you.


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## pharmacoping (Feb 3, 2012)

waxes and impurities can be removed with acids, or the way most of us do it, temperature manipulation, filtration. wax freezes, oil doesnt...that should get you going, trichomes and other impurities are removed with activated carbon or even coffee filters. I cut mine with 99%alcohol, mix it in a magnetic stirrer for a hour with a pouch of carbon, then evap the few mls of alcohol until its original weight, then comes amber glass/shatter,and some other treats. the perfect temperatures manipulate without adversely affecting the extract. experience will teach faster than a manual for sure. its an expensive hobby, so we tend to pay attention and learn quickly. try to preserve your terpines by keeping the heat gentle, after decarboxylation. when you smell the right end, you'll never forget it and it'll set the bar for your future extracting. be careful, it is uber potent, google the er visits from extracts.
Matt Rize has some awesome information available free, here too !. 
I got to get around to telling him I dug out the bags for the first time, gonna give ice a chance, and he inspired me.winkwink


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## nitro20% (Feb 3, 2012)

Guile said:


> I'm a little unclear about the Dip'n Grow mix ratio... what have you determined to be the correct dilution rate to acheive the 1.5ppm IBA you were after?
> 
> Nitro toys are always fun, I useto do airplanes and wanted to try boats (I've seen some fast ones). I have played with RC cars and helicopters too but strictly electric on them (so I could run them indoors).


yeah me to,,it dont break dont by 2s? unclear on ppms and how they split to make 1 more part a million.but this is the basic formula i used. i just added the naa atomic mass (1263000g/ml) to the solution its dilute in,in this case its 3ch80 witch is .7854 g/ml....then iba @ 127000g/ml.,,,total contains @2 fl.oz or 60ml by weight/mass.1%iba .5naa 95.5 other/3ch80.THEN:*Example #9:* The density of an aqueous solution of nitric acid is 1.430 g/mL. If this solution contained 36.00% nitric acid by mass, how many mL of the solution would be needed to supply 150.20 grams of nitric acid?*Solution path #1:*
1) 1.000 mL of solution weight 1.430 g, of which 36.00% is HNO[SUB]3[/SUB]
2) 1.430 x 0.3600 tells you the grams of HNO[SUB]3[/SUB] in each mL of solution.
3) 150.20 g divided by the grams of HNO[SUB]3[/SUB] per one mL of solution.
*Solution path #2:*
1) Determine how many mL of the solution you need:
150.20 g nitric acid x (1 g solution/0.3600 g nitric acid) = 417.2 g solution.​2) Determine the volume of solution that weighs 417.2 g:
417.2 g solution x (1 mL/1.430 g solution)

MY ANSWER 21.37g/l or 21370.572 mg/l,its seems like alot. I added mmmmmmm not going to say.BUT THEIR GROWING.​


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## nitro20% (Feb 3, 2012)

hey i got a bunch of real baby food jars,but the lids arnt what i thought. i dont know if u seem them... u probably have,, no good or can they cook and not melt the rubbery part and then push down hard to seal and into a zip lock?i answered it i guess,,just get a box of jars...its about $$$$ right now..should watch myself. spending is fun to.


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## Guile (Feb 3, 2012)

nitro20% said:


> yeah me to,,it dont break dont by 2s? unclear on ppms and how they split to make 1 more part a million.but this is the basic formula i used. i just added the naa atomic mass (1263000g/ml) to the solution its dilute in,in this case its 3ch80 witch is .7854 g/ml....then iba @ 127000g/ml.,,,total contains @2 fl.oz or 60ml by weight/mass.1%iba .5naa 95.5 other/3ch80.THEN:*Example #9:* The density of an aqueous solution of nitric acid is 1.430 g/mL. If this solution contained 36.00% nitric acid by mass, how many mL of the solution would be needed to supply 150.20 grams of nitric acid?*Solution path #1:*
> 1) 1.000 mL of solution weight 1.430 g, of which 36.00% is HNO[SUB]3[/SUB]
> 2) 1.430 x 0.3600 tells you the grams of HNO[SUB]3[/SUB] in each mL of solution.
> 3) 150.20 g divided by the grams of HNO[SUB]3[/SUB] per one mL of solution.
> ...


Dude, you are taking it further than it needs to go (over complicating it like I did the other day)..
You can see by the picture below that the undiluted concentrate is 10,000ppm IBA (that's all that you will glean from the math, I only went there because we had no idea how many PPM the concentrate was to start with, however assuming the base solvent had the specific gravity of water (1.0 or 1 kilogram per liter) and figuring the weight of the IBA biased on 1% of that got us dead on accurate according to the picture below)



All you need to do is use the scale for 1,500ppm IBA and dilute it 1000:1 (so you have 1.5ppm) 
Thats 3 parts Dip'n Grow to 17 parts water for a total of 20 parts, like the number of drops in a milliliter... So if you made this using drops you would have 1ml of solution at a concentration of 1,500ppm (IBA).. Unfortunately that is 1,000 times too strong but if we add 999ml water we will have diluted it to a thousandth of its concentration making it 1.5ppm... in other words 3 drops per liter.... or 1ml Dip'n Grow to 7 quarts water...


All low acid food cans and jars should hold up to the heat of a pressure cooker (that's how they were canned the first time around) I haven't seen a baby food jar in a while i guess, last I noticed they still had metal lids.. 
If you want to make a bunch in advance and save them for latter I believe you will want to leave the lids a little loose until after they have been cooked for their prescribed time then run cold water over the pressure cooker until the pressure equalizes and immediately remove its lid then go about tightening the jar lids (the first few might still be boiling in the jar while you tighten the lids). the tops should pop in on their own as everything cools, if any don't use them first..


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## jkahndb0 (Feb 3, 2012)

[video=youtube_share;BvPQaMG_S-c]http://youtu.be/BvPQaMG_S-c[/video]
[video=youtube_share;NnKrOVNtyxY]http://youtu.be/NnKrOVNtyxY[/video]
[video=youtube_share;caAB8jrcCGE]http://youtu.be/caAB8jrcCGE[/video]
[video=youtube_share;neOEWTcJ-NA]http://youtu.be/neOEWTcJ-NA[/video]
[video=youtube_share;_6u4QSAC218]http://youtu.be/_6u4QSAC218[/video]
[video=youtube_share;JIWPJ4DzvjU]http://youtu.be/JIWPJ4DzvjU[/video]
[video=youtube_share;O87lRW_0ecE]http://youtu.be/O87lRW_0ecE[/video]
[video=youtube_share;0q_XRHnn9pI]http://youtu.be/0q_XRHnn9pI[/video]

http://www.kitchenculturekit.com/index.htm

http://www.planttc.com


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## Guile (Feb 3, 2012)

jkahndb0, Thanks.
This series is pretty cool (brings things into perspective).

That PPM seems pretty interesting... The MSDS says its active ingredient is proprietary... 
Looks like miconazole, copper sulfate, potassium sorbate, and cycloheximide can be used to similar affect and you can know whats in them. If you preserve meat (dry/smoke) or make wine you probably have some potassium sorbate around (it looks like it is commonly used @ 100-300ppm to inhibit mold/yeast)

The big question for me is, if you mix up that growing medium minus the gelling agent and substitute Dip'n Grow for the Super Nova would it be the ideal solution for an aeronautic or DWC cloner?


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## nitro20% (Feb 3, 2012)

ok this fuk me up more:http://abacus.bates.edu/~ganderso/biology/resources/dilutions.html
dilute a X solution by 900 every time and get 1/2? isnt that what this site is saying.im not argueing at all cause i have that same (3 drops litre) idea to....being not smarted' in this stuff,i question myself.But heres the thing, not going to say how much i put in, but if i added way way too much would i know by now? that is the $340 question..plus 22$ at ace for 8 oz. small mouth jars and plastic lids an 1hr ago.
note:metal lid caps fit into the plastic lids of ball jars,,might be a way to double seal..

like i said i got pic if anyone needs or wants ideas or just to stare at some more jars and chlorine n stuff ha ha. THIS 1st TIMER  has results in progress with results as of bout 5 hours ago!!!


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## Guile (Feb 3, 2012)

nitro20% said:


> ok this fuk me up more:http://abacus.bates.edu/~ganderso/biology/resources/dilutions.html
> dilute a X solution by 900 every time and get 1/2? isnt that what this site is saying.im not argueing at all cause i have that same (3 drops litre) idea to....being not smarted' in this stuff,i question myself.But heres the thing, not going to say how much i put in, but if i added way way too much would i know by now? that is the $340 question..plus 22$ at ace for 8 oz. small mouth jars and plastic lids an 1hr ago.
> note:metal lid caps fit into the plastic lids of ball jars,,might be a way to double seal..
> 
> like i said i got pic if anyone needs or wants ideas or just to stare at some more jars and chlorine n stuff ha ha. THIS 1st TIMER  has results in progress with results as of bout 5 hours ago!!!


The link you provided states it right off...
*1. Simple Dilution (Dilution Factor Method based on ratios)*

A _simple dilution_ is one in which a _unit volume_ of a liquid material of interest is combined with an appropriate volume of a _solvent_ liquid to achieve the desired concentration. The _dilution factor_ is the total number of unit volumes in which your material will be dissolved. The diluted material must then be thoroughly mixed to achieve the true dilution. For example, a 1:5 dilution (verbalize as "1 to 5" dilution) entails combining 1 unit volume of *solute* (the material to be diluted) + 4 unit volumes of the *solvent* medium (hence, 1 + 4 = 5 = dilution factor). The dilution factor is frequently expressed using exponents: 1:5 would be 5e-1; 1:100 would be 10e-2, and so on.

I think you could use concentrations up to and perhaps above 10ppm IBA and 5ppm NAA. I get IBA in powdered form and my insert says I can use it at 50-150ppm for the "Basal Long Soak Method" submerging 1" of the cutting for 12-24hrs... At 1/5 that concentration I doubt you would see any immediate problems... You might not even see long term ones... I was thinking about going around 5-10 ppm in my cloner the next time around.. Though I will probably go lower now, like 1.5 or 3 (atleast I wont have to do the math again)... I'm thinking about foliage feeding the plants with the same solution in the cloner too...


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## nitro20% (Feb 3, 2012)

oh to that other dude with the tub video,,my friend has a degrees in botany and she has tc experience, she seems to think that the mix may be a little strong.that video caught my eye too.i have an IQ of 1 , but this is a GGOOODDD ONE.
http://www.pakbs.org/pjbot/PDFs/41(2)/PJB41(2)603.pdf
that is the exact opposite of the videos mix,,mmmmmmmm???????


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## nitro20% (Feb 3, 2012)

yeah im not agrueing,, AT ALL,,,,I like the 3 drop thing,wish i had a ppm meter...right,would that be helping me...
so i wonder when the contamiates will show up,,,tonight,tomorrow,,beginners luck @ 100%


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## Guile (Feb 4, 2012)

nitro20% said:


> yeah im not agrueing,, AT ALL,,,,I like the 3 drop thing,wish i had a ppm meter...right,would that be helping me...
> so i wonder when the contamiates will show up,,,tonight,tomorrow,,beginners luck @ 100%


Hope you don't think I'm stressing, I like these sorts of things... its interesting to look back and see where I make my mistakes. I just hope your not getting too frustrated or feeling talked down upon, there is no intent for that here (but I know how it can feel when its hard to read the language)... 

A PPM (TDS) meter might confuse things further at this point... They work by converting the electric conductivity into a ppm (or Total Dissolved Solids) reading. The odds are that your Dip'n Grow will not read 15,000ppm (10,000 IBA plus 5,000NAA) and the water you use to dilute it is unlikely to be 0...

We are just working with weight and volume here.. We assumed that the Dip'n Grow had a specific gravity around that of pure water 1.0 (thats 1 kilogram per liter). for comparison sake 100 proof alcohol has a specific gravity around 0.9 and "Kool aid" has a specific gravity around 1.1 (think about how much sugar you use to make that crap)..

Assuming the Dip'n Grow (concentrate) had a specific gravity between that of 100 proof vodka and Kool aid (say the specific gravity of water, 1.0) we speculated that 1% of the 1 kilogram that a liter of Dip'n Grow weighs should be IBA weighing about 10 grams (within a 10% margin of accuracy). 

Latter we were validated when the mixing instructions were brought to light.. They clearly state that there is in fact 10,000ppm IBA in the undiluted Dip'n Grow concentrate.. This means that it has a specific gravity of 1.0, that there is in fact 10,000 miligrams (or 10 grams) of IBA in a liter of Dip'n Grow weighing 1 kilogram (1,000 grams).

Now its just a matter of figuring out how many parts containing 10,000ppm (10,000mg/L) we need to dissolve in a larger volume of water to obtain 1.5mg IBA per liter (1.5ppm).

We know that a "drop" is considered to be 1/20th of a milliliter, and a milliliter is considered to be 1,000th of a liter (There are 20,000 drops in a liter.).

If we wanted say 1ppm (1mg/L) IBA and our concentrate is 10,000ppm (10g/L) we would simply dilute it to 10,000 times its volume with water (our solvent of choice). 
So 1 drop of Dip'n Grow to 9,999 drops water (to make 10,000 parts total) would give us 1ppm (1mg/L) IBA. We know there are 20,000 drops in a liter so 10,000 drops would be 1/2 liter (or 500ml) 

But we want 1.5ppm (1.5mg/L) thats 50% more than 1.0 so if we doubled the water but added triple the concentrate we would get a 50% stronger solution making 1.5ppm (1.5mg/L).. 

Thats 3 drops per liter... 

I think I might keep confusing the issue by giving you the 1ml per 7 quart thing but what I am really saying is 1ml per 6.666 liters. The thing is that 6.666 liters just seems like an odd measurement to work with (I can see someone using more than one measuring device in an attempt to be accurate) 7 quarts are equal to 6.624 liters which is only 42 milliliters off (that's better than 1% accuracy) If you realy wanted to be a stickler you would just add a teaspoon extra water to each quart, and another one for good measure (but now you are using a second measuring device again, which kinda defeats the purpose. Besides do you really need to be under 1/2 if 1% accurate?)


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## nitro20% (Feb 4, 2012)

I have GOOD news from the tc thread>>>>as of 72 hours of culture, i see growth of 1/8 to 1/4 inch on all 15 cultures....and no signs of contamination,,,mediums look smooth with a tiny bit of liquid on top,tiny bit.one has a "rough" look,, but it shows no signs of contamination as of yet,,culture is growing...every thing is going BETTER then i expected..ive only entered the area 5 times in 72 hrs..alcohol misting all the way..the tape i used isnt the best..got to get REAL seal tape i guess..planning to culture more when agar arrives. (today)HHEEELLLLL yah!

i see the same thing as you do,,,i saw two ways to look at it,,,your way i understand and is correct. but all the math i did and every thing i thought i read,other then the matrix break down,says 1part con. is diluted by say,,, 4 ml. water, to repeat the exact doubling process i must and 4ml. more water witch gives me 8 ml,,, to the now to break that down double again,,, i would just add,,, i thought,,, 4ml. more water,,,,,not 8ml as i would have from the last...now you say,,take the 8ml. and add 8ml more,gives me 16ml.,,,then add 16ml. to double the original consentrate @ 32ml to the one part we started with,,,, witch intern breaks the PPMs down to what we are after...add that is what the dilution equation is not saying on your behalf,,, as my 1 IQ understands it m1v1=m2v2... maybe im doing the math wrong,,but u tube has step by step instructions on chemistry solution dilution math..in dummy terms,,haha maybe i need STUPID DUMB DUMMY terms.


but thats it though... 1.5 mg/l of 1% iba,, in a solution that is 57.7 g (57700ml/g) total v/m,,, to 1000ml. parts /water ,,,u will need 2.5 to 3 drops of said consentrate....to achieve 1.5 mg/l or 1.5ppm iba...

oh I GOT PICS IF ANY ONE WANTS TO SEE THEM ..I MUST E-MAIL THEM THOUGH .


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## pharmacoping (Feb 4, 2012)

sweeeet !!! so happy to hear that marijuana tissue culture is possible at home, for more people everyday !! imagine the progress in a few years, now that this mystery is being solved by us stoner alchemists. Pretty sure they try real hard to make this look near impossible, as indicated by many comments made here. I wonder what corporate benefits they may have by keeping this tech out of our hands? govern-mental process


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## Guile (Feb 4, 2012)

pharmacoping said:


> sweeeet !!! so happy to hear that marijuana tissue culture is possible at home, for more people everyday !! imagine the progress in a few years, now that this mystery is being solved by us stoner alchemists. Pretty sure they try real hard to make this look near impossible, as indicated by many comments made here. I wonder what corporate benefits they may have by keeping this tech out of our hands? govern-mental process


What kind of mixtures have you used to make your gel medium for cannabis cuttings? what were your experiences with them?


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## nitro20% (Feb 4, 2012)

HEY EVERYONE!!!HERE IS ANOTHER UP-DATE FROM TISSUE CULTURE 101 (tc101inprogress) 
THE BEGINNER!!! i guess thats what we're doin.. for all of YOU that skip over the entries to find the latest(me to),,,heres the deal...I decided to study this, for the knowledge, 3 weeks ago...as of 80 hrs ago, I TCed my first ever cultures..I cut all green leafy material from 19 pieces of branch/nods....i have witnessed the growth of 1/16 to as much as 3/8 inch on all cultures..no signs of contamination as of 1 hr. ago one culture seems to be darkening a little, but i still see growth..still not sure when contamination shows its ugly head?..someone here does though,,im sure? i am not a beginner farmer,,as it may seem,,the last few weeks made me wonder how those big ass pumpkins even lived or those Fresno peppers,,those colorful fukrs are HOT/good taste too..
i believe that TC may have a huge affect,as it has for the SEASONAL PLANTS to be sold yearly,to the FOOD GROWER or one who like farmers market where one/I might be able to supply a whole bunch of "pure" vegetable plants or such..no more GOV. genetically modified shit that goes through our nation one way or the other....given one might have to find seeds from natural/organic food store to ensure of not getting a modified seed to start with,,but after that one study finds it possible to make 1,000,000,000 plants from 1 in 1 year...even at half that a number of people "COULD" start a whole new place or places to get pure 100% FOOD (that shit sounds like a movie)...personally i eat ONLY food that i can read,,(i miss restaurants)but MY OPINION.and if you read back a little,,,dont look like im surrounded by a hole bunch off IQ's,,damn there goes the last ONE!


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## nitro20% (Feb 4, 2012)

i've try and built everything on the market, seriously i have,,,,except for all the 1000000000000000 new cloning gels and things. i even had a version of that cloner every body talks about,,7-8 yrs. ago...and i even updated it to what these new ones look like,,but the look of it didnt help...
i have my best results,,to were i only have to take what i need to fill one of those kiddy pools.(they make great plant base).and i use some sort of pellet,clean not jiffy or something from pay-less.work better i think.. then snip,dip into Olivia's cloning gel and with the pellets soaked in a (im not saying it) a SOLUTION that contains 1 gal. water and about mmmmmmm bout 1 1/2 tsp of liquid karma.i might put less if its dead winter due to to cold weather,, i find that just a pioneer jr. in a oak cabinet no pad, and 10 cfms.(fan modded)they want to purple up on me,,u know what that means...me it means "clones" plus "veg./flowering" divided by "JUST for personal"= not worth my time/energy.
oh and i use bleach on ALL things use to cut or store clones every time around.i hate being so clean some time, it make pot taste like bleach after.wish i knew cleanness 15 yrs ago.


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## nitro20% (Feb 5, 2012)

so i was om the web dddduuuuhhhhh,,,, and typed in dip n grow ppm,,know were it took me HHHEEERREEE!! WOW!!! dip n grow is getting more of a name for itself. so lets help,,"i like to help" (fedX)ha ha ha remember that comedy with steve martin,,,
Having results, like this, may need to be heard,,there's not some GRAND BREAKTHROUGH that will make u a $1,000,000,000 in 10 to 14 weeks indoors HAHAHAHA.(thats the newbie goal,,,, right,RIGHT) but for some it might be a new thing to smoke on and maybe play with...with that in mind,, if u search this thread u may find allot of "man, what do they think they are changing" ora,,or math problems (ignore all that). i heard about TISSUE CULTURE 14 yrs ago,and the guy made GOOD acid ,,,and the shrooms,,,wow! I heard what noise came out, but never listened,,,,,80 hrs ago i started the process of "tissue culture" by way of Micro-Reproduction,(just as a,,,, lets see if i could, project) I did not by a kit,and with a little research found that i could BUILD MY OWN KIT (sorry kit and gel makers,,no hard feelins) all of the things can be bought at one huge all in one store..i try to stay out of "THE WALMART" kind of stores!!!!we have to stand up for something...hears a list of what i used and the medium recipe..again,,sorry kit and gel makers, Any how, after research kits was not the choice for me.

-canning jars/plastic lids, and a few extras, pressure cooker, all the items to cut hold plantlets and seal jars properly,all types of ways,, tote or a fish tank/plastic works youll need it for the clean 
GROW area.i dont know but a hepa filter will help CLEAN air and better the odds. just think about what u cant see that make u sick,im sure that you've already done some research at this point..So you know bout cleanness and lighting and cleanness & cleaning supplies,,get the hint.
this is basically a med. that,,, with no experience,,, came up with and as 82 hours ago their all growing, with no signs of contamination:

all per ml. and stuff,, is dedicated to guile!!!HAHA even though I went with my last second thoughts.

-mix pool sanitizer (NADCC/plant preserve) @ 1/8 teaspoon per 750ml,,this will be your rinsing and cleaning solution for ur cuttings.unlike bleach,this will not have to be rinsed off before you transfer,although i mixed very little consentrate to get the cuttings to the place of culture.
dont think this has storage life,chuck it when ALL done.
-fill 1000ml to 500ml
-add 30 ml + of your NADCC/ppm note:i added 30ml but will try 5ml/L. more next time.
-add 15 drops dip n grow,,, this will be your iba/naa nutrients for shoot production.
I might have added more then intended,1 to 3 drops was to verdict
-2 tablespoons sugar
-add 500ml water to make 1 litre.
-and your agar agar,, i used flakes at the lables instructions.
in that order
-this is a part were things may fall in ur mix,,so cover it, open the edge dummy,,,and microwave on high.get to boil then try to a just so you dont burn it. i think my first batch burnt,i chuck it...
you can tell when you've cooked it enough,,,common sense,,ull need that.
at this point fill your dishes TRY NOT TO REACH OVER THINGS!shave you arms haha.lids on and to the pressure cooker,,lids lose,tools and things too...

well if you dont know what to do after you pressure cook then "stop" you wasted money, u must study WHY this is preformed before before YOU PREFORM it.or whats the point. Keeping a clean story short,cut nods,some to all "leafy material",3/8 to 2 inch pieces and plant in your chlorine jars,,i saw things happen within 10 hours!SERIOUS.
i have pics but my puter is putering and that aint happen.ill email them though! iaintalier', its true, this is highly possible in your own home/kitchen...my man, pharmacoping,the starter of this high jacked thread, knew it all along, and if "I" can do it, HEAINTALIEIN' either!!!!!!!!!!!!
heres what im basing my info from:http://www.pakbs.org/pjbot/PDFs/41(2)/PJB41(2)603.pdf


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## pharmacoping (Feb 5, 2012)

guile,

I've used all the above that nitro used, with many different results. over the counter carrot callus recipe works to get you started. when ready for a change, cytokins and auxins are added at tiny varying amounts, until your desired results are achieved. I never fussed with the math much, and didnt use pool sanitizer(awesome nitro) as I always had acess to the culture wares in the last5 yrs. but before that.....
I've used store gelatin, crushed vitamins, antibiotics, colchicine,bleach,different types of sugar,crushed and sterilized foreign pant material, crokus bulb extracts, banana peels,cloning gels,bushmaster type additives,pk's,salts, etc. lots of successes, and many freaks wanting to break out of the jar, some grew tall, some bonzai, some had barely any leaves,some with dusted calyx clusters(think dr grinspoon). I dont want to manipulate any other way(for now) and just keep producing pure callus material for the mj community, the ones in the know.
I'm not doing it the right way, just my way. we can see several applications in the thread already. I grow callus and rootballs,purify strains, dr tissue cultures perfected clones...and nitro is going to show us "roses with thc" soon !!! lol


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## Guile (Feb 5, 2012)

Do either of you guys use any kind of nutrients or B vitamins?

I like the *NADCC *tip too.. Next time I'm in town I'm going to stop by the pool place and pick some up.. Have you tried using it in the place of peroxide (or enzymes) in your reservoir?

You have better experiences with Olivia's gel, peat pellets, and liquid karma than anything else? Do you use peroxide, enzymes, or that NADCC.. Who about B vitamins? 

I have to agree about the kiddy pool, I useto do that back in the day.. Best damn flood tables/reservoirs you could buy for $5-10.oo apiece. And they get you decent light distribution with homemade aluminum flashing parabolic reflectors too (took the stitching out of an umbrella once to use as a pattern because I didn't want to figure out the math). To use with the reclaimed oil cooled vertical HID bay lighting out of factory's and such (that was before I started building my own switchable ballasts using sodium rebuild kits and switches).

Do you fill yours with aggregate, like crushed brick or screened gravel? I remember once using 1gal pots to try and get about 25 plant per square meter spacing, (putting clones directly into flowering as soon as the roots broke through the peat pellets)..

You ever make 5 gallon buckets of wine to place in the corners of your white washed paneling "gorw room" around your swimming pools (for Co2 enrichment)? Every 2 weeks I would start a new one. Usually apple, grape, lemon, and tea.. Bummed me out when they started marketing "hard lemonade" and "twisted tea" that could have been me (mine weren't carbonated though). After the first couple weeks fermentation would slow down and by 8 weeks things were usually ready to bottle/drink..  I had a pretty ghetto 1 gal still too, made a couple glass of brandy at a time though .. To be young and creative... I've gotten so square...


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## nitro20% (Feb 6, 2012)

SO it been 4 days and i see that 2 jars containing 3 plantlets (2/1 and 1/1) might be not doing well,,not concerned YET no sign of contamination...note: NADCC only will have its benefits for about 72+ hours..plant preserve material has a wider scope for TISSUE CULTURE .. growth IS noticeable, essentially in the widening of the stem,,jars look good.....
*-----important message------(error0425)-----important message------*
AFTER CAREFUL CALCULATIONS AND A KICK TO MY ESTEEM,,,THE RIGHT DILUTION IS 5/16ML PER LITRE OR 99.8% SAY 20 DROPS IS A ML.,,SO AT THAT IS...3.2 DROPS OF DIP N GROW PER LITRE IS 1.56 PPM..
----------------------------------------------------------------------------------------
Im not seeing the growth that i dreamed i read about,,but i know from every thing i read,the harder wood plants that are in lower ppm of iba do much better.
1-2.5 ppm iba with the naa at .5 lower.(dip n grow 1v/w iba and .5v/w naa) but at 13to15 drops,,there still growing..
yep,, auxins and cytokinins,, haven't found a source that i could measure haha. but ive looked into willow tree water (got that) and coconut water,but if you know something better/easier id love to here it. that,,, i will need to add in a few weeks. `
I dont know bout thc roses,,,im sure stranger things will happen,,like,, 60 miles south,I-5 corridor,,its snowed once,ski park,, no go,,some wonder if its cause they complained enough,,the gov. finally let it snow on em, ONCE!! couple feet.mmmmmmmmm 

Nope i thought about using willow tree water,,but i never knew what was in it..now i think that i will use dip n grow to soak my peets,, all the hubub about the cloning gel/powders that's all that's in them is auxins and cytokinins.
i put 1 gal pots in there transplanted from clone cab. then 1 1/2-2 wk.veg/then flower,,same thing..nothing huge,,but it supplies my demand for bout $100 in electric/month.i smoke ALOT!!!!AAALLLOT!!!!!!!one of those raptor hoods would over it be sweet the 1000 hortiluxes (hps/mh) would love it.. maybe soon,, local dude,,, $200.00 for the, made in the U.S.A. one. 
As for any thing beyond basic n/p/k with high Ps at bloom,, nope,, consistency defines ALL failure..Im not saying that there is no benefit to all the (tech stuff) there is some excellent mj/and things...hormones are auxins and cytokinins,,and the b1 and thingslike "super thrive" i thought correct me though.please!
sure seems like alot of people high as fuk for there to be 1 right way, pharmacoping? I glad theres more than one way...


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## pharmacoping (Feb 6, 2012)

Guile,
I dont add vitamin b or superthrive or over the counter nute mixes. Eco has pure basal salt nutrients, with msds, since the seventies. I've used nothing but Eco nutes for......ever, until 3yrs ago and swithced to dutch master gold line, nver looked back either.Nothing wrong with pure salts, but..DM makes life and exposure much easier.
These eco salts contain every major and minor ingredient necessary, without additives or hormones. I use a tsp of these salts in my litre mix of agar. My nutrient ratio has not changed in vitro since I switched from a crushed vitamin to Eco salts.
s
My largest reservoir(100 gallon) is a big round plastic kiddie pool too !! every tote I've used breaks down (from the nutes, I guess, as there is no uv exposure under trays) and the pools work great for 20 bucks !

I used to use willow water for cloning. it's salycilic acid and the branches were chewed historically for ions, for pain management. if discovered today this acid has so many uses it would be a miracle acid. Replicator(DM) roots clones and makes a killer foliar for seedlings too, faster than any willow stick I've soaked. I use the Rep now exclusively when I clone traditionally


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## nitro20% (Feb 6, 2012)

Ive had my pool for 3 years and i cant tell you how many cycles have been through there.....cleaning with bleach every time,,that thing is still like new..way cheaper then 24 plant bottoms...

cool,,but do use rep.for ur auxins and cytokinin? or is that ur secret...what are vitamins compared to n/p/ks..or is that the deal ,, salts and organics.Are the p/ks the ones im looking for to achieve my goal for the nexted medium?


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## pharmacoping (Feb 6, 2012)

I buy the diluted hormones as necessary from various online suppliers. I used to use cloning dips for tc, but not in awhile, or with the level of success I was looking for, only a training exercise for me. check out the pure salts at Eco online, they're cheap too. mixed and ready to go, and pure. 
You're looking for a weak version of exactly what you would use for tender seedlings, all the way through tc, only hormones change. sugar is constant also, as there is very little photosynthesis happening for carb production. you really should have a 30$ hanna ppm dip checker and start with RO water. otherwise months of trial and error could be yours.


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## Guile (Feb 6, 2012)

nitro20% said:


> SO it been 4 days and i see that 2 jars containing 3 plantlets (2/1 and 1/1) might be not doing well,,not concerned YET no sign of contamination...note: NADCC only will have its benefits for about 72+ hours..plant preserve material has a wider scope for TISSUE CULTURE .. growth IS noticeable, essentially in the widening of the stem,,jars look good.....
> *-----important message------(error0425)-----important message------*
> AFTER CAREFUL CALCULATIONS AND A KICK TO MY ESTEEM,,,THE RIGHT DILUTION IS 5/16ML PER LITRE OR 99.8% SAY 20 DROPS IS A ML.,,SO AT THAT IS...3.2 DROPS OF DIP N GROW PER LITRE IS 1.56 PPM..
> ----------------------------------------------------------------------------------------
> ...


I don't think that many people realize that to be successful (not just at growing either) you only need to be resourceful, otherwise spend a bunch of money... The only thing better than seeing "made in the USA stamped on something is a complete lack of a stamp (because you made it yourself). If you can do it out of recycled/repurposed stuff you are saving the environment to boot. Biulding your own stuff is environmentally friendly in alot of ways, think of all the logistics that have circumvented (transportation = poor air quality and depleted natural resources). Every time you reuse/repurpose something you have cut out at-least 3 round trips (some of which might be to the other side of the world). Not to mention the damage to the economy caused by shipping your money over there... Think locally for the sake of the earth globally...

What nutrients do you use? I useto use Shultz or Peters "complete" fertilizers and add Epsom salt to for extra magnesium that pot plants need... I switched to the GH "flora series" using only the "micro" and "bloom" but have considered changing back to powdered nutrients again as I am spending nearly $1 a day on nutrients (despite buying in bulk and recycling as much as possible). From what I understand anything that works for tomato's (like the GH series) should have high enough magnesium so you don't need to add Epsom salts. I haven't been lately, but I've been using KoolBloom which has magnesium sulphate (Epsom salts) in it.

By the way I am incredibly unclear on what goes into most rooting gels/powders other than the auxins IBA and or NAA. I have been trying to sort out the best possible rooting solution I can come up with, and I am quite certain it will contain a bit more.. I think these culturing mixtures are an excellent place to start... as you are doing essentially the same thing, just on a different scale. None the less I plan to add Superthrive (B vitamins) to my mixture and maybe potassium sorbate (@ 150ppm or so) to my mix (I have a bit of it around for preserving meat and wine) otherwise something else to keep yeasts and molds down...

If you are trying to promote "shoot development" its my impression that you want to be working with both Cytokinins and Auxins, not just Auxins... If I were to guess it would probably be something like BAP and NAA... From what I understand NAA alone will not aide in rooting as much as IBA alone However NAA is more active in other plant development, making it more favorable for shoot development.. I have often wondered why they are not commonly used as a trio, and I'm still a bit unclear (perhaps getting a cutting to do more than one thing at a time increases the likelihood of failure).

It seems that when trying to provoke a plant to develop both new shoots and roots its often done in 2 stages... First shoots, using predominantly Cytokinins and Auxins then transplant to new medium for developing Roots using Auxins (and sometimes Gibberellins).


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## nitro20% (Feb 6, 2012)

Yep Im seein that one little drop more/less may per litre changes ALOT of the values of the medium. they started out like things were goin to happen,,but maybe a few are not growing so much at a time....I had this feeling it was to much iba,,,but im goin to make up a new batch here soon and with the right amount this time,and transfer 4 plantlets to new medium and do some other cultures,,(veggie seeds)...so a drop wouldnt, hurt of this eco or of a o/1//1 hydro nutrient additive...
AND my agar powder says 2 tbls per 1 pint about a half litre right? would you use this amount in your mediums,,,cause my readings say about half that in TC.. your opinion...

Had a thought on a new KIT... ALL is in there. tent,lights,cooker,tots,etc,, just like hydro did when it went public....I was 17 in my first appt. (with a 19 yr.old chick)22 years ago,,put a seed on my fish tank and it sprouted,,the next issue of high time had a article on HYDRO,,kidda like what we're doing right now...but im not just going to think about it this time...objectives/goals=SHOT ASS LIFE!!!


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## nitro20% (Feb 6, 2012)

yep,for some reason i just cant stand the wastefulness i was teached my whole life!! fukin schools/intsitutions...shit head lie'in gov..its not my fault that the world hates us,,,i just figured it out...theres nobody really helping the USA thats not in it to GAIN SOMEHTHING PERSONAL.Its not their fault either,they got lied to, too!

I use the GH flora series,with liqiud karma,,i use to use all organic (to slow indoors/$),,this is way faster,, and easier,,with the temp change here(day/night),you got to watch what soil/nut. u use..fungus nats are my little freinds..,fly tape tape helps me warn the others,,,ha ha ha..I save the $ for organics for the outdoor crop... im glad that i studied this, cause there is alot of interesting things that makes cloning alot easier,,,this is one of those things that Will always be LEARNIN' me.


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## Guile (Feb 6, 2012)

nitro20% said:


> Yep Im seein that one little drop more/less may per litre changes ALOT of the values of the medium. they started out like things were goin to happen,,but maybe a few are not growing so much at a time....I had this feeling it was to much iba,,,but im goin to make up a new batch here soon and with the right amount this time,and transfer 4 plantlets to new medium and do some other cultures,,(veggie seeds)...so a drop wouldnt, hurt of this eco or of a o/1//1 hydro nutrient additive...
> AND my agar powder says 2 tbls per 1 pint about a half litre right? would you use this amount in your mediums,,,cause my readings say about half that in TC.. your opinion...
> 
> Had a thought on a new KIT... ALL is in there. tent,lights,cooker,tots,etc,, just like hydro did when it went public....I was 17 in my first appt. (with a 19 yr.old chick)22 years ago,,put a seed on my fish tank and it sprouted,,the next issue of high time had a article on HYDRO,,kidda like what we're doing right now...but im not just going to think about it this time...objectives/goals=SHOT ASS LIFE!!!


I way over did it on my first hormone experiment, still suffering the fallout. I still have 3 tables exposed to my "bad" mix in rotation and atleast 1 of them looks like it will almost be a complete loss...

If you want to get technical a pint is 473 milliliters but I think 500ml (1/2 liter) would be close enough for a gelling agent...

If you want to be really accurate just weigh 2 tablespoons of powder and do the math 

By volume package instructions give you what looks like 1cc per 15.76ml or 1 teaspoon to every 78.83ml 
Where as 4 tablespoons per liter (or 2 tablespoons per 500ml) would give you more like 1cc per 16.66ml (or 60cc's per liter)
If you have a 1/3 teaspoon measure and use it in conjunction with your 2 tablespoons it would (more or less) account for the extra volume (water) in a half liter. Hell 1/4 teaspoons are fairly common and will get you closer to the label concentrations than adding nothing extra at all (just make it a "heaping" 1/4 teaspoon).

Did you look into the "SuperNova" or "Nitrozyme" products the guy in the video mentioned? Atleast one of them is a seaweed extract (whatever that means anymore, I know that is used as a base to some "miracle" additives, that they manage to sneak other "proprietary" stuff into).


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## pharmacoping (Feb 6, 2012)

that chinese paper lays it all out specifically whith correct ratios for desired affects. ppm, sugar,eco salts,water are the same for me in every culture. iba naa and bap are in every culture vessel I have, just at varying amounts, two drops of this to root, with one drop of that, and this many to divide, etc. no sense on me reinventing the wheel here, scientists have already done it. I never use gibberellins. eco is mixed according to directions to attain a under 200ppm mix and works here consistantly, with no other nutrients, besides sugar. I dont even care why it works, now that I know it does !! The public carrot callus recipe is successful for callus production and division also. a necessary starting point for tissue culture.
Three stages, three different vessels, only three hormones, used in conjunction, at varying rates, depending upon desired effects. the difference between my callus and rooting gel is two drops more of one hormone.

I'll be honest, I built every system I ever had in 20 yrs or so, and used off the shelf stuff constantly. but when i got legal all of that stuff went into the barn, and I wrote a check for pro systems, equipment, and supplies that was paid for in a short time, and have nevr looked back. It was fun, even getting wet, or stinky, or having strange results.....but not fiscally responsible. now I get no surprises ever,no leaks,no experiments or additives, and consistant results...which I never got from home depot supplies. cant say i regret either way, but should have wrote a check yrs ago,instead of trying to reinvent this damn wheel. from lighting to nutrients to c02 to temps and humidity, I started to feel like a retired old dude farting around tinkering, now I feel like a retired old dude farting around and tinkering, but getting consistant results and cashing in from them.

peace


Callus Production/division

Rooting


Shooting

hardening off


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## Guile (Feb 6, 2012)

pharmacoping said:


> that chinese paper lays it all out specifically whith correct ratios for desired affects. ppm, sugar,eco salts,water are the same for me in every culture. iba naa and bap are in every culture vessel I have, just at varying amounts, two drops of this to root, with one drop of that, and this many to divide, etc. no sense on me reinventing the wheel here, scientists have already done it. I never use gibberellins. eco is mixed according to directions to attain a under 200ppm mix and works here consistantly, with no other nutrients, besides sugar. I dont even care why it works, now that I know it does !! The public carrot callus recipe is successful for callus production and division also. a necessary starting point for tissue culture.
> Three stages, three different vessels, only three hormones, used in conjunction, at varying rates, depending upon desired effects. the difference between my callus and rooting gel is two drops more of one hormone.
> 
> I'll be honest, I built every system I ever had in 20 yrs or so, and used off the shelf stuff constantly. but when i got legal all of that stuff went into the barn, and I wrote a check for pro systems, equipment, and supplies that was paid for in a short time, and have nevr looked back. It was fun, even getting wet, or stinky, or having strange results.....but not fiscally responsible. now I get no surprises ever,no leaks,no experiments or additives, and consistant results...which I never got from home depot supplies. cant say i regret either way, but should have wrote a check yrs ago,instead of trying to reinvent this damn wheel. from lighting to nutrients to c02 to temps and humidity, I started to feel like a retired old dude farting around tinkering, now I feel like a retired old dude farting around and tinkering, but getting consistant results and cashing in from them.
> ...


Which was the "Chinese" one? What plant variety/s did they base their information on (Isn't pot one of those things that gets you killed in China). I have read things about tropical fruit that just doesn't seem like it would yield the same results in marijuana.. 
I have seen similar experiments conducted: one with cannabis the other an ornamental flower, cereal crop, vegetable or fruiting plant and the hormone concentrations/ratios were completely different...

I can relate with what you are saying to some extent... Since I became legal I have done 2 complete grow room redesigns using all the hydro shop stuff (once for just me, and again when I picked up a partner, to take advantage of "shared growing space"). To be honest with you I have been disappointed many times with the limitations of the hydro store stuff... 
My Batinacare 3' tables are more like 42 inches and don't properly fit in the only 3 foot stands I could find (clearly meant for like 38") .. A 6.5' grow tent I bought was more like 6'2" or 6'4" (don't remember now because its been given away.. Hydro shop pumps are expensive for what they are, $150-200.oo reflectors that don't distribute light any better than home made ones... 
My grow room looks impressive to everyone who has seen it (been offered store credit if I slap a bumper sticker on my system and take a picture of it for advertisement), yet it rarely fails to piss me off more than most the setups I built from scratch... There are some hydro store parts I have evicted from my garden... The overflows on my flood tables for instance got replaced with pluming parts from Lows because the hydro shop stuff has no adjustment finer than about 1.5-2" But nobody can see that so I still look high-tech.... Not to mention finding flood table covers with any spacing other than 25 plants per square meter (obviously I still make my own)..

To be honest I'm pretty well fed up with the entire hydro shop industry... Literally nothing it has to offer lives up to the expectations they imply, so you will buy more crap to reach your otherwise failed expectations, then they sell you something else so you can be as efficient as you should have been considering your previous upgrades. Its rediculis, and the impressions they place people under... Have you looked around here and seen what some people are spending on nutrients (not even fully knowing whats in them). I read an article recently about Dutch Masters Superbud... I'm sure its old news now but it ilistrates my point perfectly..

I deal with greenhouse supply companies now, they will tell me whats in almost anything I buy (so there are no mysteries) and everything is sold in bulk making it convenient and cost efficient. And most the freight truck drivers have no issues putting things where you want them... Beats the hell out of struggling with 5-6 gallon jugs in/out of the shop, your car, and your house. When I built my latest grow room I made sure it had an outside door and small porch (makeshift loading dock) to make deliveries easier (room construction would have been a nightmare without it).


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## pharmacoping (Feb 6, 2012)

http://www.pakbs.org/pjbot/PDFs/41(2)/PJB41(2)603.pdf
thanks to nitro ! for this one. It's almost the same ratios I use in vitro. i wish he showed this to me twenty yrs ago !!


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## pharmacoping (Feb 6, 2012)

was just thinkin....if I had a friend with gout, I might trade my medicine for his ? just sayin..


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## Guile (Feb 6, 2012)

pharmacoping said:


> http://www.pakbs.org/pjbot/PDFs/41(2)/PJB41(2)603.pdf
> thanks to nitro ! for this one. It's almost the same ratios I use in vitro. i wish he showed this to me twenty yrs ago !!


You know how many times I've looked at that? I have it saved to my desktop and I never once looked at it close enough to notice the "Hong Kong University of science and technology" (I usually skip right to the body of it)...



pharmacoping said:


> was just thinkin....if I had a friend with gout, I might trade my medicine for his ? just sayin..


Do you mean colloidal silver?


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## pharmacoping (Feb 6, 2012)

no, I think my buddy would have to be taking colchicine, for gout.


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## Guile (Feb 6, 2012)

For creating a polyploid?

Acros Organics - 227125000 - Colchicine 97% 500mg, Ea

Tell them you are a small self named business and you use your Social Security number as your EIN.
You are currently consulting as an independent/outside researcher and need to conduct control studies in house. In other words your physical address is now your business address (assuming you don't live in an apartment complex). 

If that doesn't do it I think you can still incorporate on legal zoom for around $40.oo (this is how alot of "stuff" gets done). 
Once you are incorporated I don't think the government can even deny you a permit if you otherwise meet the rules provided by the regulating body. Just outsource everything you cant provide in house, as we all know corporate responsibility boils down to "Well, it worked on paper".. This is a free country and we all technically can play by the same rules (assuming you are a property owner anyway)..


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## pharmacoping (Feb 6, 2012)

http://www.phytotechlab.com/Detail.aspx?ID=761

I found colchicine ! 

Guile- I was watching the whole dutch master thing with that product when it came out, shame on them. but know that all the big players have the same product line now, and many then also, without ingredients always listed. I hate those additives,(paclobuzatrol?? wtf) and required full disclosure from DM and got it. I even called them once on the phone, for hours these guys are genius in my book, and transparent, and apologetic about their shameful "first product" . I forgave them, and their line of cheap nutes are paid for monthly with one branch, of one plant, one time, monthly. very inexpensive to me, and the best I've ever used. they should actually give me gifts if they're reading this !! for the pr.


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## pharmacoping (Feb 6, 2012)

polyploidy's are very kind, I was privy some time ago. you are correct. its very successful in vitro, and even sold as such.


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## nitro20% (Feb 6, 2012)

yep,, my bad everything adds up,,,,i got to learn my conversions!!!! I feel confident about the china -mans report too..just got to get there. i have that problem of seeing new things and thinkin i can reproduce the same effect...but as we all come to realize,,you end up with a KIT of some sort or u spend so much money u give up and then its money wasted...I know that i could sum this up with a little money spent in the right places,,BUT fuk it feels like im so close or like in my writings "STOP you wasted your money"!!! i dont have money to waste per say but these are the things i do with it to keep me alive,,so its all how you define WASTE..this aint that hard,,i know it. The cultures are doing well..maybe im just expecting to much...but clones dont just start growing from the time i cut them with in 24 hours,,so im doing well i hope. just no experience in this town,,no one to ask..so what about a o/1/1 hydro nut. additive,,will this have the similar effect towards our goal of auxins and things ..how fast do ur grow on the average,,it seems like i might be able to split in the next 7-8 days....


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## pharmacoping (Feb 6, 2012)

I dont know anyting about a 0/1/1 hydro nute, but do know they need 100% of the nutrient requirements of a plant, just alot less of them. I am able to divide in the first week of callus production, or roots,for that matter. It willl take a month minimum to get to a herd of root balls ready to sprout. be patient. the china man is spot on


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## Guile (Feb 6, 2012)

nitro20% said:


> yep,, my bad everything adds up,,,,i got to learn my conversions!!!! I feel confident about the china -mans report too..just got to get there. i have that problem of seeing new things and thinkin i can reproduce the same effect...but as we all come to realize,,you end up with a KIT of some sort or u spend so much money u give up and then its money wasted...I know that i could sum this up with a little money spent in the right places,,BUT fuk it feels like im so close or like in my writings "STOP you wasted your money"!!! i dont have money to waste per say but these are the things i do with it to keep me alive,,so its all how you define WASTE..this aint that hard,,i know it. The cultures are doing well..maybe im just expecting to much...but clones dont just start growing from the time i cut them with in 24 hours,,so im doing well i hope. just no experience in this town,,no one to ask..so what about a o/1/1 hydro nut. additive,,will this have the similar effect towards our goal of auxins and things ..how fast do ur grow on the average,,it seems like i might be able to split in the next 7-8 days....



Hey man, don't give up... its guys like you doing this stuff in real world/practical conditions that make it possible for the rest of us to follow... We are gaining insights from your experiences too.. When you become successful imagine how many people will follow... Lead the way


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## pharmacoping (Feb 6, 2012)

wanted--one special interested mature person with proven web abilities in design, marketing, and counting money. pm me with your state of residence.nitro,dude, this is your town, ask anything from any of us. I'm not hiding anything. the nutes dont have hormones in them, so your desired effect cannot be gained by nutes alone. get some auxins, 12 dollars online, diluted, my bottle lasted me 1.3 yrs ! and a few graduated plastic pipettes for a buck. this will save you tons of time and money, and will get you on the road fast. start with the right stuff first to prove your results, then work your way back on the cheap.


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## GrnMn (Feb 6, 2012)

Great read, thanks!


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## nitro20% (Feb 6, 2012)

You want to know whats funny is that all the TC thread anywhere really never show the end result or the pic are of like a clone just put in a jar???never do u see the after..the best results ive seen is your pics,,thats tc 101 in progress..more pic!MORE PICS!!!it just looks cool/scientific,,love it..man there growin,,,i took them down to there stalk,,i see growth on all but 2 but even they have LITTLE "things" starting to stick out..so its a good sign..no black or fussy or moldy or contamination,,so thats GOOD..so far 100%. just got to be patient,,ur right...this is like cloning,just more delicate.Im not given up on nothin man....this just like anything ,,we're not talkin money,,the mor u put in the more comes out,,bottom line "EFFORT".
I got the new batch in the aquarium now its cool for sure..bought a shoe box sterilite tote..thoes will come in handy..so jared,toted,aquaiumed,alcoholed,,i think there safe like that to cool,,i let them sit,cooker/cold water for 15 min. that took all the heat off them.. GOT TO GO CULTURE>>tryin a ornamental house plat from a botanical garden..(AWARD WINNING)


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## pharmacoping (Feb 7, 2012)

I have better results when I let the pressure cooker cool down naturally on the stove top, sometimes over night, as opposed to cooling down quicker. my agar lasts much longer, less damage to containers/lids. I dont "can" the jars, meaning suck the sealing lid closed, they are loose until I open the pressure cooker, then tightened and taped for storage until used. 
when you cool it down quick I believe the negative air pressure inside is quikly replaced with non sterile air sucking into the cooker, possible contamination too? I'm no gynecologist, so its just a guess here. but better results, are, ah better.


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## nitro20% (Feb 7, 2012)

Nice... thanks..so u say u can tighten the lid and tape for later use? how much later? 1day 1hr? those pis didnt have any tape..why?those jar lids are the thread type im looking for..ddduuuu hence the name, BABY FOOD JAR LIDS.
So i was on the web...YEP! and got *CARROT CALLUS **INITIATION** MEDIUM FROM PITH TISSUE.*is this it? your right bout the "gout" thing,of course.....i looked up all the ingredients,,and the* cupric sulphate pentanydrate *is for cattle.. goat and sheep hoof root,,which is a form of gout ,,right..but the others are a 13.0.44 potassium nitrate..and things to keep the hole thing from braking down..and a dye....pretty basic..(if i had a LAB)..nope this one i will buy..the research has been done..no time to make a square wheel roll..THANK i think that this is the missing link to phase two..RIGHT..

wasnt to impressed last night,,,the lids on a few jars came unscrewed,,but still on top..not got to the sanitation thing...i see what ur sayin about the hot/cold air thing..i am not arguing with experience,,but i want to think that the air in the aquarium/alcoholed might be safer then open air/port hole on the cooker and a dog running for 5 hours or over night...could have used the over night thing last night....i defiantly need some longer tools,,ha ha ha ha (TISSUE CULTURES HAVE LONGER FORCEPS) ...I had a hard time with barely opening the lid to transfer,,jars were deeper this time,,,i got some s/s tweezers and these clampy thingys with the clamp dremeled off..the medium was tougher this time..but it says that h/w plants want a harder medium. 

got to browse the web for a non/harmful free picture managing download...​


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## pharmacoping (Feb 7, 2012)

nitro,

I tape the jars with shitty lids, like stock bf jars, the lids dont tighten back down, but do close, so I tape them, sometimes if loose, because I'm cheap on things that really matter I guess. Those plastic lids are awesome, but the pressure cooker will ruin them if not careful. gotta put them on loose or cocked, then sterlize, then let cool down naturally. I have two dogs, raise chicken,rabbits, fish, marijuana, vegetables, on a farm, with muddy boots often, grow outdoors, stove is inside, cooling overnite, forever. no issues man. you'll see its your eylash, or mostly the dirty plant material. ppm will solve some of this issue though. honestly, mold is not on my worry list, for yrs. its no biggy, i got lots of cultures. In that last batch of about 25 transfers over a week ago , I have one container, with two transferred calluss to root that went moldy, one of those is dead, and I'll probably re wash and bleach the other live one for transfer again, which mostly works with older growth.

the pic is blurry, but a close up of the first two lateral branches on an explant, with no roots, happens sometimes, should catch up on next transfer,, or I'll divide it and root it seperately.

I've kept the jars dark in my transfer tent(covered the jars with aluminum foil) last for several weeks. mine start to get watery, but have never molded(never !)

cooling faster than natural will cause condensation buildup in the jars, drown explants, and water down your agar. dont do it grass hopper! please, for the love of tissue


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## pharmacoping (Feb 7, 2012)

first two lateral branches on a Ace of Spades culture, with no roots. happens sometimes, she'll catch up ! she is screaming for vengeance !!


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## pharmacoping (Feb 7, 2012)

carrot culture medium is the gateway to what we are trying to achieve. its a no brainer, and I urge you to get some ppm,and the four hormones you'll want, and spend as much as you can afford on more ppm,agar,and other supplies while there. you can experiment with different pre mixes, or create your own, or duplicate one. i suggest buying hormones in small dilute amounts for ease of use. they'll last a year at least, before you run out. dont forget pure white cane sugar on the grocery list.


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## Guile (Feb 7, 2012)

Hey dude I was looking at that PDF file you brought to light. I assume you are looking to mimic the hormone concentrations used in that paper... The thing is they are using 0.1ppm IBA and 0.05ppm NAA.. If I'm not mistaken I believe that would be 1 drop to 5 liters. (about 5 quarts 1 cup).

Also, do you guys add your Cytokinins to the same medium that your Auxins are in?


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## pharmacoping (Feb 7, 2012)

dude prolly nitro, but i got some 2 cents?I have mimicked that mix, and it works great. adjustments are made per your expression demands. I've never set out to murder a culture, but I also never killed one due to high hormone concentrates. The hormones will cause expressions, some more, some less,some desired, some undesired. Most of these traits should be grown out to make sure the desired results are exactly that. then you've found you're mix. I'm not even sure that too much or too little hormones will kill a plant, maybe stunt it, or suspend its growth, but maybe not kill. that china paper will get a culture going fast, with viable results. I use a mixture of three hormones of the four I own in every culture. I have been know to post treat also, for more roots, or branching, etc. in veg


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## Guile (Feb 7, 2012)

pharmacoping said:


> dude prolly nitro, but i got some 2 cents?I have mimicked that mix, and it works great. adjustments are made per your expression demands. I've never set out to murder a culture, but I also never killed one due to high hormone concentrates. The hormones will cause expressions, some more, some less,some desired, some undesired. Most of these traits should be grown out to make sure the desired results are exactly that. then you've found you're mix. I'm not even sure that too much or too little hormones will kill a plant, maybe stunt it, or suspend its growth, but maybe not kill. that china paper will get a culture going fast, with viable results. I use a mixture of three hormones of the four I own in every culture. I have been know to post treat also, for more roots, or branching, etc. in veg


My experiences is about the same with established plants.. I haven't killed one yet but it can sure mess up yields when done wrong...


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## nitro20% (Feb 7, 2012)

YEAH i know ITS CAUSE of the mg/l-10 (1.0mg/l-10) I KNOW!! ITS fun though right!! tried to figure it out without bothering the thread to much with math....but it came to be that hard wood & mj were requiring 1.0 to 2.5 ppm iba...
SO thats what we went with this time or close..tiny drops,,, defiantly 20 drops per ml,,,3-4 drops..im good...witch reminds me,,how are they doing..done at 100am...


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## pharmacoping (Feb 7, 2012)

i do use cyto and auxi at the same time, most of the time, unless something is needing tweaking


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## nitro20% (Feb 7, 2012)

this time i treated with dip n grow 12 hrs.....my 1st. medium batch i put in 15 drops dip n grow and there all growin some-what..so to much will be the determining growing factor here or to little... eye lashes,,,not a problem..my transfer area is NEWBIE SAFE!! I HOPE..


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## Guile (Feb 7, 2012)

pharmacoping said:


> i do use cyto and auxi at the same time, most of the time, unless something is needing tweaking


Do you use TDZ? and you recommend around 200ppm complete nutrient? Whats your position on the B vitamins?


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## pharmacoping (Feb 7, 2012)

Guile,
My experiences is about the same with established plants.. I haven't killed one yet but it can sure mess up yields when done wrong... ,
---------------------------------------------------------------------------------------------------------------------------------

Unless outside, my results were never ever consistant, until I built the right room/combined with proper genetics , with the right controls, since then(3yrs) I have had no surprises, no issues, no variances, no failures or deaths or late flowers, and almost total automation.. its totally about the environment we provide, once stable and constant and correct, they flourish every time. I have no regrets on spending the money to do it once/right, after decades of home depot growing with varying results


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## pharmacoping (Feb 7, 2012)

i have no experience with tdz. I use 150ppm when growing or storing callus material, 200ppm for rooting/shooting. never higher than 200ppm for me in vitro.yrs ago I used crushed vitamins, so sure B was there. But in recent yrs I use Eco salts and they contain every needed major and minor vit/min necessary for plant perfection, however, will affect ph, which should be adjusted before sterilizing. I have never personally added thrive alive to anything on a regular basis.I used it once in a free trial kit from technoflora, but that was it. I have no position on them in tissue culture. But like in any hydro application, if your nutrient is lacking, and additives are needed,why not use a nutrient that is complete?


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## Guile (Feb 7, 2012)

pharmacoping said:


> i have no experience with tdz. I use 150ppm when growing or storing callus material, 200ppm for rooting/shooting. never higher than 200ppm for me in vitro.yrs ago I used crushed vitamins, so sure B was there. But in recent yrs I use Eco salts and they contain every needed major and minor vit/min necessary for plant perfection, however, will affect ph, which should be adjusted before sterilizing. I have never personally added thrive alive to anything on a regular basis.I used it once in a free trial kit from technoflora, but that was it. I have no position on them in tissue culture. But like in any hydro application, if your nutrient is lacking, and additives are needed,why not use a nutrient that is complete?


Just picking your mind... Its nice to be able to hold a conversation with and informational resource..
hat Cytokinins do you use? What concentrations? What are Eco salts?


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## pharmacoping (Feb 7, 2012)

bap,naa,kinetin,iba,ppm,1 mg/ml concentration somewhere in this thread I listed the four hormones, and their concentration I purchase them dilute. I cant post "my personal" recipes, but will say that the china man is spot on for most application, albeit a little slow. I dont know how much is in the diute mix, I use drops per litre when mixing, with pipettes showing measurements. until the last transfer before going to veg, there are no hormones or sugar, just agar and nutrient and ppmgoogle ECO hydroponics and you'll find his website. he sells nutrients in pure basal salt formulations.


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## nitro20% (Feb 7, 2012)

K so there all showing signs of growth (1st batch) 3 of them are bout the same...I dont know,,, but,,,, i think i have a GOOD SIGN..The ones that i did last night seem to NOT have the liquid ("dont do it grasshopper") that the last ones have...i think that i see the signs of the break down of the agar,,,but this is about the time that the NADCC totally wears off,,so transfer is soon....this is my 2 week transfer to new medium with the aux/ coyt. or a small amout of eco salts+the same iba and such from the first medium.>>>>I THINK....
I know about ppm...and i plan on getting it....my specimens are mostly clean other then some normal white mold...AGAIN I THINK.....
*I WILL HAVE RESULTS WITH THIS, THE WILL TO SUCCEED IS OUT MATCHED BY ODDS OF FAILURE
*
Or seriously we need to get some internet cops so that some people wont have to wade threw all the BS to find one honest sight that gives it to you like it is...sellin thangs' is not making the economy better,,,it may seem that way, but man oh man,,, at the end of the year when u make that dump run,,, it sure is * hard throwin** all that money away** HUH*??? the thing is,, id be willin to spend *ALOT of money* at a place that would NOT try to sell me that ONE product that is SHIT,,and they know it....or the same as the other guy but with a cooler label... u know what i mean RIGHT..i have a high shopping/money spending moral code.....ONCE and its on the company and NEVER will i give them MY money EVER AGAIN....BUT THATS NORE HERE OR THERE...


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## nitro20% (Feb 7, 2012)

No sugar????? When u transfer to new medium do u wash them again..i guess u would right..


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## pharmacoping (Feb 7, 2012)

white mold aint normal, should be no cloudy,fuzzy, color bleeding, red ooze, or discoloration of any kind on or near the tissue sample. it should look like clear agar, and an explant in it. If not, discard, start again. young ones wil not survive fungi. plus fungi is now in your sterile culture environment. remove it quickly. older cultures have a better chance. you'll need to clean your explants better. almost there dude !!


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## nitro20% (Feb 7, 2012)

NO NO NO not in the jars NO NO!!!! just a leaf or two on the original potted mother... NO NO were good there..I washed the shit out of them in the nadcc and in diluted alcohol...i was very clean and and titty... if i would have had any signs like that HOLY SHIT......NO NOT in the jars.


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## pharmacoping (Feb 7, 2012)

AWESOME ! good to hear, I was scared for a minute. so you got the powdery mildew... i got a brand new sulphur vaporizer in my rooms with a nite time only plug on my room controller that I've never used, and sulphur pellets, for the just in case...whew. they do make a simple spray to cure it though i never used it either. good to get rid of asap, before it infiltrates flowers, and slow veg growth

peace


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## nitro20% (Feb 7, 2012)

That mold was on my friends plants not mine,,,,but thats WHAT DID HAPPEN!!got some stuff for it cause of the clones and bam,,dead, not the mold either,, my pore clones,, most of them..Thats what i mean,,,SELL me anything cause they got to pay the bills,,,im not payin bills,, im play around the bills,,theres a huge difference,,,If if did this for cash,,,,or to pay the bills the guy would have fukd me...thing is hes the only game here so if i needed something fast say a bulb,,well then i cant cross his store out and take my business else where..oh well.. 

gonna make a new batch now but ive done some research on this additive i have for my rooms and WELL...aux. cyot.. YYYYEEEEEYYYY.......im startin to think about all the things i read from ur posts and im getten it...couldnt say it all at once though... almost ur hole recipe ,,except for,, how much of,, is all right in this thread... mabye 1 special thing u might add thats ur thing,,but its here,,some where..huh.... sssshhhhh... dont tell people how fukin cool this is..next is ppm and more lighting/storage,,cause those thangs are growin,,perky tips and weird growth happening, little leaves..mind you that i cut ALL the greenery off most and now i see little growing thangs and bigs ones to.....one of them,, i do see some spots ,,, they are in the medium kinda in a group of 4to 6 but the culture is one of the healthiest.. its time to trasfer to new medium,,,the ppm wont be here in time so the nadcc will give me 4-7 days longer..that was the CHEAP part about that nadcc was 4-7 days....then im back to spendin money making mediums more often $$$$(that was alot of Ms).
so i guess i would wash the cultures off AGAIN when i re-jar them? in a solution or sterile water?


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## nitro20% (Feb 8, 2012)

hell yah,,,thats me,,,not about TC but a custom built from the GNRs' creations work bench..60+mph 30%+ nitro rigger...soon to come 1/10 scale v-drive drag boats,,, 137ft. track 75+mph..


the cultures got growth out of the tops the cut ends..this i did not see this morning,,their little leaves...but i definitely see black spots appearing in the medium by but not touching the stem,,
i know its time to get them in the NEW medium batch,,,added some stuff,,less sugar and 2 drops of?????with the dip n grow..well see!! A MUST HAVE FOR THIS IS PLANT PRESERVE,,,nadcc will have me makin medium ever 4-5 day for the sane TCs,,SAME money spent,,,,bottom line all you TC grasshoppers plant preserve is a must!!!!
*and for all you who just tuned in im doing this without a kit,,,BUT if this works then think about a dude that KNOWS HIS SHIT-- WITH PICTURE RESULTS who offered a deal like that....mmmmmm

*shit that guy might be around HERE!!


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## nitro20% (Feb 8, 2012)




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## nitro20% (Feb 8, 2012)

there they are!!
test run
pretty good hepa filter 2stage with my fiter at top..


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## nitro20% (Feb 8, 2012)

*
not bad for the first time grasshopper,,,not much but they were stems 8 days ago!!!got a new mix and their all alive and going in there tomorrow..I PUT THE METAL LID UNDER THE PLASTIC THEN I PUT THEM IN A ZIP LOCK AND THEN TAPED THE ZIP PART,,THATS THE WAY GRASSHOPPER!!!**
*
*this ones for u gulie or who ever 
*


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## nitro20% (Feb 8, 2012)

*this is the thread were there might be a thang happening or two..so come all tissue culture dudes and lets talk some shit ,but dont come with no ammo (pics) and defiantly dont tell the others that this can be done and how freakin cool it is!!!!!! *


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## Bonkleesha (Feb 8, 2012)

a note on cleaning the explants: we did rose micropropagation 2 terms in a row, keeping the explants in a 20% bleach solution for each. the ONLY difference was that the irst time, we left our explants in it or 10 minutes, and the second, we did 20 minutes. about 50% of the irst ones got contaminated. NONE! 0%! of the second ones showed any signs o contamination. cool, huh?

note* we did it in an enclosed HEPA hood... not the gloves one, but a hood, still.


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## nitro20% (Feb 8, 2012)

YEAH Thats what im talkin about..lets have a thread....a real thread that will do good and help a muthrfuk out in this hush hush subject...come all newbies/and vet.s allike lets talk culture!!


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## pharmacoping (Feb 8, 2012)

Soooooo cool nitro !!! my man !!!! I wish I was there to celebrate !! great photos !! 
20% bleach for a0 minutes will kill the incised mj explant. 10% 10 minutes, rinsed twice,then 70% alcohol, couple minutes, rinsed again twice in sterile water. they dont die. they dont mold much. experiments in your environment, with plants cut from a different room than mine, may show different contaminations at different times I imagine. so probably personalized for each app. dirty plants? outside? inside? humid?dry? pets?pests? surely make huge differences in results all the way through. 

Bonkleesha, do you have some ros pics in vitro, would be so cool here. most serious mj gardeners love flowers too !!


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## nitro20% (Feb 8, 2012)

heres some more info..

"When the plants start slowing in growth or the jar gets full of tissue, you'll need to "replate" or "subculture" (propagate) that tissue. Prepare your desired media and, in your sterile work area, remove the tissue from the jar it's growing in. If you simply need to replate (transfer the tissue to a new jar with fresh media without dividing), then just pull it out of the old jar and place it in the new media jar. If you need to divide and "subculture" the tissue, then you'll have to have a sterile work area to set it on so it can be divided or cut into pieces."

and again,,, there is so many different ways!!!EXPERIENCE rules


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## pharmacoping (Feb 8, 2012)

correct, but did it slow in growth? mine slow in growth 3 months later...just sayin, never a week. it will fill the jar with a ball of foliage(pics coming)


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## nitro20% (Feb 8, 2012)

*SO FOR ALL THE NEWBIES ** **LIKE ME, THAT THINK YOU CAN DO IT YOUR SELF, YOU CAN,,, BUT THERE ARE DEFIANTLY SOME THINGS U CANT GET AROUND,,,, PLANT PRESERVE MIXTURE IS A MUST.NADCC/POOL SANITIZER WILL WORK FOR A SHOT TIME,,BUT PPM HAS A WIDER SCALE FOR KILLING BACTERIA,,,,BUT NADCC MIGHT BE BETTER FOR UR RINSING AND CLEANING,,IN PLACE OF BLEACH BECAUSE OF ITS ABILITY TO BE A PPM SUBSTITUTE,,,70% ALCOHOL IS STILL USED,,NADCC DOES NOT HAVE TO BE RINSED OFF,,BUT I USED LIKE A 10% OF THE DILUTE TO TRANSFER TO MY AQUARIUM LUB-BOR-A-TORY,,
MIXES I USED:
--1/8 TEASPOON POOL/HOT TUBE SANITIZER ****(DONT BREATH THIS AT ALL,,UR POT WILL TASTE LIKE CHEMICALS FOR DAYS,,SERIOUSLY ITS DANGER DANGER SKULLXBONES
--CRUSH WELL,,POWDER "WELL" IT HELPS THE DILUTION
--FILL TO 750ML,,, SOMTHING WIDE YOULL NEED TO CRUSH THIS MORE,,TRY TO ALWAYS USE DISTILLED/COOKED WATER..
-THIS WILL BEE YOUR STUFF,,****DONT SMELL IT!!!!!!! ITS GOOD SHIT,, ****DONT WORRY!!*
*--I USED UP TO 1/2TSP- /750ML AND RINSED,,THEY ARE CLEAN,,CLEAN OH AND ALIVE.*
* BUT USE 30ML/L OF UR SOLUTION TO 1000ML OF THE WATER,THIS WILL BE YOUR MEDIUM WATER..*
*!!!!!!!!!!BUT THIS WILL NOT BE AS GOOD AS PPM!!!!!!!*

*-FOR ALL OF YOU THAT JUST WANT TO PUT UR COOKER UNDER COLD WATER TO "COOL FASTER" NO NO NO NO...THIS "WILL" HAVE AN EFFECT ON THE CONTAMINATION ENTERING WITH THE COOLING PROCESS..SO JUST LIKE THE MAN WITH THE PICS TO PROVE SAID,,, JUST LET IT COOL ON THE STOVE TOP...*
*I DID THIS..... AND MOST-LIKELY HAD AN AFFECT ON THE LENGTH OF TIME BEFORE THE NADCC COULD'NT HANDLE IT.*

*IF UR NOT IN IT TO WASTE TIME,,,,,,U KNOW,,,, "TILL THE BOSS GATHERS WORK FOR THE SHEEP" TIME...OR WANT RESULTS FASTER,,,,,,, THEN,,,,,, MAKING A SQUARE WHEEL ROLL,,,WELL THEN,, THERES MORE "ON" THIS THREAD THEN SOME FLUNKY MATH,,,,,,CONSIDER IF THERE WAS A PERSON WHO ALREADY DONE THIS FOR ALONG TIME, AND HAD A KIT THAT WAS FOR SALE SOME WHERE ON THE NET. .....* PROBABLY WOULDNT GET MY HOPES UP FOR ANY INGREDIENTS* TO THE MEDIUMS,,BUT,,WITH THE PICS TO PROVE/AND FOLLOWING DIRECTIONS, I WOULD TRUST THAT THIS DUDES KITS MAY WORK...I BET THAT GUYS CLOSE..SHIT HE MAY BE THE REASON MINE ARE STILL ALIVE AND GROWIN,,,, WITH PICS TO PROVE,,, HA HA!!!!!!* BUT I DIDNT BUY A KIT SO IF THIS IS WORKING,,,,LIKE I HEARD BEFORE,,,"JUST SAYIN!!"

IF ANY "PROS" KEEP HEARIN THE SAME QUESTIONS OVER AND OVER THEN SEND THEM TO THIS LINK...IM SURE SOME WHERE IN HERE THEY WILL FIND IT!!!!! AND THERE WILL BE MORE TO COME FROM THE:* 
CULTURE GRASSHOPPER*

LOVIN IT MAN,THANKS PHARMACOPING


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## pharmacoping (Feb 9, 2012)

wecome to the issue of Tissue my man !!


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## pharmacoping (Feb 9, 2012)

+ rep for the faucet and cool tile work on the counter....I'm gonna take a wild guess and suggest you did this yourself ?


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## pharmacoping (Feb 9, 2012)

heres some sultry culture snaps to drool over.... like I must have done on that moldy one. I 'm trying to capture the anomalies, rather than the normal ones. but the shooting/rooting pic is one I'll follow in this thread. shes strange as she got her shoot before her root. normally she would be culled from the herd, but for pix sake I'll keep her alive and fruiting. 

I'll get some shots of rootballs and the transitions to veg, then flower soon. I am really really busy gearing up for the spring nursery rush, and cant take a camera in there, so my private tissue stashes are what you get , and in some sort of a time line for understanding. 

the test tubes have agar in them mixed one month ago. none have molded, I'll keep them around for your entertainment. always used glass, but recently someone suggested these plastic alternatives. they suck, discarded after done, glass only man.


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## Bonkleesha (Feb 9, 2012)

heres a miniature rose... propagated on 1/25/12. i dont have pics from the other lab, but i have been doing photo documentation on this one


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## Bonkleesha (Feb 9, 2012)

can we talk about the totiopotency of cannabis for a second? what is it comparible to? does indica or sativa have more? THESE are beneficial questions... anyone know how to get the answer?


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## pharmacoping (Feb 9, 2012)

both are equally totipotent in my experience

the pic is so cool....care to share your rooting formula for roses? and what you cap your tube with ? is it a glass tube?


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## nitro20% (Feb 9, 2012)

Git a light with a bad ass bulb,,had a little under the counter plant/aquarium light...now w should see some more growth....
pharmacoping,,is that electrical tape???? so what about test tubes with good rubber corks and then tape...*grasshopper says*,,i trust baby jars with the metal lid (dont have the plastic) taped,,into sandwich zip locks,,then tape the zippy part...but that dont mean nothin got in during trasfer,,,do it????NOPE..

SOURCES for plant preserve,,and supplies like forceps,,and glass?

oh!


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## nitro20% (Feb 9, 2012)

nnnaaa,,,its came with the rental,,its a clean area to mix and cook...a glass pain is what i work off of..my work is outside and wondering if it should grow or not...this winter is WEIRD,,,everything has mildew,,and there is no snow..no ski park,,NO HOT ASS OUT OF TOWN SKI BUNNIES,,damn it...BETTER watch out for the mites in northern cali. this next summer....shit,,shouldnt trust a clone here if ur high depended on it..thats my experience..


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## Bonkleesha (Feb 9, 2012)

it was premade at some lab... the college orders em. im trying to get my hands on all the leftovers and even used ones with and without media they have had throughout the years.

i think its important to note that the cap is just thin plastic, but it goes over and around the tube, instead of in it like a cork. we made sure to sterilize the cap more than once, and we did it on the science lab under a high-suction HEPA hood. not the one with the enclosed gloves, but sterile enough. what i also want to get my hands on is the antioxidant that we soaked our explants in for 10 minutes directly prior to putting them into the media. do u want me to scan the lab instructions, dude?

oh and we just sealed the tube with some of that non adhesive plastic shit that stretches easy and sticks to itself.


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## pharmacoping (Feb 9, 2012)

. 10% bleach soak is standard in any culturing I've done in the nursery biz. thanks so much for the info...and stick aroundwtf?? of course I want the directions...what the heck was I thinking..sorrypost them or compress the or anything. you'll be sent love for any new info on the subject.


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## Bonkleesha (Feb 9, 2012)

how small would a cannabis explant have to be if you were going to microprop it to outgrow a disease?


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## pharmacoping (Feb 9, 2012)

I have no idea. I'm thinking that part is incidental for me, as I do this only to whip that plant count thing, without mothers even


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## Bonkleesha (Feb 9, 2012)

heres the info on definitions we got... im doing the rose lab now, and yes... the formula is one one of those pages (not these).
notice the antioxidant is plainly in view. i need to not go to class ripped :/


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## Bonkleesha (Feb 9, 2012)

heres the procedure and the formula for the media on the miniature rose. hope someone gets something out of it, if not, i just decided to scan all my handouts for easy reference in the future


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## nitro20% (Feb 10, 2012)

here we go on anther money rampage,,,maybe some thing will come to be....
got a REAL light,,this will help alot. Now off to carolina.com....
the pic is one i transferred,,,its doin well..way better to add about 3 drops D'n'P to the medium..


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## pharmacoping (Feb 10, 2012)

Bonleesha !!!you're efforts are appreciated. especially the last page ! many minds are about to open up to the inevitable future that tissue culture has in storethanks again !


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## pharmacoping (Feb 10, 2012)

nitro, that shoot is ready to be cut and transferred, and leave you with a healthy chunk of callus material, to shoot over and over, so you cna keep rooting the shoots, if plants are the intention of course. or you can do all kinds of crazy things with it too.


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## pharmacoping (Feb 10, 2012)

bonkleesha,
I dont need your lab contact, but could you tell me how much a rose culture costs from the lab. It may help people to put things into perspective in this endeavor.


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## Bonkleesha (Feb 10, 2012)

carolina.com is where the college gets its supplies for tissue culture. the class kit for microprop is about $150, BUT THEY WILL PREMAKE ANY MEDIA TO YOUR SPECIFICATIONS! just email them the formula and they will quote a price.

now... what in the formula needs to change to make it ideal for cannabis? if we can igure it out, i might actually ask for some custom culture tubes.


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## nitro20% (Feb 10, 2012)

thats the million dollar question that every body knows, but know body will say,,which in turn=who knows what? im sure you two could put it together,,...its defiantly not on the web..or not in the 1000 studies ive seen..not in a form that i can grasp anyway...didnt i hear carrot callus is pretty close to that med. for mj....for some reason mj and tomatoes come up as one in my brain,,,would a tomato medium have this.....quality? 

*I GREW A SHOOT, DID U HEAR, IT WORKS!!! I cultured that batch 1/30/12 ,,i can do this math,,,,,in 11 days i am able to split a shoot into 2 plantlets,,do the math 11DAYS,,and i doubled a culture,,,,OH and for all that skipped this whole thread,,, WITH NO KIT!! WITH PICS,, GO BACK AND SEE! 
*

*Pharmacoping*,,so thats CALLUS stage huh..i thought it may take longer "or" have to be more of a "plant" ,,,but seein how this works,,rooting a BROOMSTICK doesnt sound to hard..HAHA.. theres are about 10 like that, but i cant get pics..theres growth right out of the CUT ends,,little leaves...The P.P.M. is on the way,,hopefully the nadcc will hold out till it arrives...*fingers crossed*


i have been doing my homework and i have all the micro and macro nuts. plus i have iba/naa and seaweed extract for aux. and cytk. now i need to get the average ppms of micro & macro nuts. to the basic medium for mj or a plant like it..I knows there is a medium to buy that will solve all my guessing,,,but this will be way cooler when 1 X turns into 10+Xs in 30 days!!! IM almost there..*STAY TUNED FOR THE NEXT RESULTS,,SOON*


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## pharmacoping (Feb 10, 2012)

Bonkleesha....
carrot medium will get all your callus ready, and then its personal tweaking with cyo-aux to your liking. add both and watch what happens. it's fine, but if more branching is needed.....
just like the china report in this thread.. all of those results are repeatable, there is no one formula, it depends on what you want to do with your explant really. limeon and a bacteria could change things in a big way for you, or maybe you want small bonzai planur ts with 500 branches...all depends. but start with carrot, and basal salts no higher than 150ppm.

Nitro--

150ppm of pure basal salts, available here : http://www.ecogrow.com/16-oz-ecogrow-standard-10814-p-116.html use this exact choice. some will suggest using a bloom formula for all tissue culture, I have, and went bact to this. the nitrogen is sweet. for good.

check this out

and they said it couldnt be done just days ago !!!

To achieve genetic transformation in plants, we need the construction of a vector (genetic vehicle) which transports the genes of interest, flanked by the necessary controlling sequences i.e. promoter and terminator, and deliver the genes into the host plant. The two kinds of gene transfer methods in plants are:

*Vector-mediated or indirect gene transfer *

Among the various vectors used in plant transformation, the Ti plasmid of _*Agrobacterium tumefaciens*_ has been widely used. This bacteria is known as &#8220;natural genetic engineer&#8221; of plants because these bacteria have natural ability to transfer T-DNA of their plasmids into plant genome upon infection of cells at the wound site and cause an unorganized growth of a cell mass known as crown gall. Ti plasmids are used as gene vectors for delivering useful foreign genes into target plant cells and tissues. The foreign gene is cloned in the T-DNA region of Ti-plasmid in place of unwanted sequences.
To transform plants, leaf discs (in case of dicots) or embryogenic callus (in case of monocots) are collected and infected with _*Agrobacterium*_ carrying recombinant disarmed Ti-plasmid vector. The infected tissue is then cultured (co-cultivation) on shoot regeneration medium for 2-3 days during which time the transfer of T-DNA along with foreign genes takes place. After this, the transformed tissues (leaf discs/calli) are transferred onto selection cum plant regeneration medium supplemented with usually lethal concentration of an antibiotic to selectively eliminate non-transformed tissues. After 3-5 weeks, the regenerated shoots (from leaf discs) are transferred to root-inducing medium, and after another 3-4 weeks, complete plants are transferred to soil following the hardening (acclimatization) of regenerated plants. The molecular techniques like PCR and southern hybridization are used to detect the presence of foreign genes in the transgenic plants.
*Vectorless or direct gene transfer*
In the direct gene transfer methods, the foreign gene of interest is delivered into the host plant cell without the help of a vector. The methods used for direct gene transfer in plants are:

*Chemical mediated gene transfe*r e.g. chemicals like polyethylene glycol (PEG) and dextran sulphate induce DNA uptake into plant protoplasts.Calcium phosphate is also used to transfer DNA into cultured cells.

*Microinjection* where the DNA is directly injected into plant protoplasts or cells (specifically into the nucleus or cytoplasm) using fine tipped (0.5 - 1.0 micrometerdiameter) glass needle or micropipette. This method of gene transfer is used to introduce DNA into large cells such as oocytes, eggs, and the cells of early embryo.
*Electroporation* involves a pulse of high voltage applied to protoplasts/cells/ tissues to make transient (temporary) pores in the plasma membrane which facilitates the uptake of foreign DNA.
The cells are placed in a solution containing DNA and subjected to electrical shocks to cause holes in the membranes. The foreign DNA fragments enter through the holes into the cytoplasm and then to nucleus.
*Particle gun/Particle bombardment* - In this method, the foreign DNA containing the genes to be transferred is coated onto the surface of minute gold or tungsten particles (1-3 micrometers) and bombarded onto the target tissue or cells using a particle gun (also called as gene gun/shot gun/microprojectile gun).The microprojectile bombardment method was initially named as biolistics by its inventor Sanford (198. Two types of plant tissue are commonly used for particle bombardment- Primary explants and the proliferating embryonic tissues. 
*Transformation *- This method is used for introducing foreign DNA into bacterial cells e.g. E. Coli. The transformation frequency (the fraction of cell population that can be transferred) is very good in this method. E.g. the uptake of plasmid DNA by E. coli is carried out in ice cold CaCl2 (0-50C) followed by heat shock treatment at 37-450C for about 90 sec. The transformation efficiency refers to the number of transformants per microgram of added DNA. The CaCl2 breaks the cell wall at certain regions and binds the DNA to the cell surface.
*Conjuction* - It is a natural microbial recombination process and is used as a method for gene transfer. In conjuction, two live bacteria come together and the single stranded DNA is transferred via cytoplasmic bridges from the donor bacteria to the recipient bacteria. 
*Liposome mediated gene transfer or Lipofection *- Liposomes are circular lipid molecules with an aqueous interior that can carry nucleic acids. Liposomes encapsulate the DNA fragments and then adher to the cell membranes and fuse with them to transfer DNA fragments. Thus, the DNA enters the cell and then to the nucleus. Lipofection is a very efficient technique used to transfer genes in bacterial, animal and plant cells.

*Selection of transformed cells from untransformed cells*
The selection of transformed plant cells from untransformed cells is an important step in the plant genetic engineering. For this, a marker gene (e.g. for antibiotic resistance) is introduced into the plant along with the transgene followed by the selection of an appropriate selection medium (containing the antibiotic). The segregation and stability of the transgene integration and expression in the subsequent generations can be studied by genetic and molecular analyses (Northern, Southern, Western blot, PCR).



During the last decades, a tremendous progress has been made in the development of transgenic plants using the various techniques of genetic engineering. The plants, in which a functional foreign gene has been incorporated by any biotechnological methods that generally are not present in the plant, are called transgenic plants. As per estimates recorded in 2002, transgenic crops are cultivated world-wide on about 148 million acres (587 million hectares) land by about 5.5 million farmers. Transgenic plants have many beneficial traits like insect resistance, herbicide tolerance, delayed fruit ripening, improved oil quality, weed control etc. 
Some of the commercially grown transgenic plants in developed countries are: &#8220;Roundup Ready&#8221; soybean, &#8216;Freedom II squash&#8217;, &#8216;High- lauric&#8217; rapeseed (canola), &#8216;Flavr Savr&#8217; and &#8216;Endless Summer&#8217; tomatoes. During 1995, full registration was granted to genetically engineered Bt gene containing insect resistant &#8216;New Leaf&#8217; (potato), &#8216;Maximizer&#8217; (corn), &#8216;BollGard&#8217; (cotton) in USA. Some of the traits introduced in these transgenic plants are as follows:

*Stress tolerance*

Biotechnology strategies are being developed to overcome problems caused due to biotic stresses (viral, bacterial infections, pests and weeds) and abiotic stresses (physical actors such as temperature, humidity, salinity etc).

*Abiotic stress tolerance*
The plants show their abiotic stress response reactions by the production of stress related osmolytes like sugars (e.g. trehalose and fructans), sugar alcohols (e.g. mannitol), amino acids (e.g. proline, glycine, betaine) and certain proteins (e.g. antifreeze proteins). Transgenic plants have been produced which over express the genes for one or more of the above mentioned compounds. Such plants show increased tolerance to environmental stresses. Resistance to abiotic stresses includes stress induced by herbicides, temperature (heat, chilling, freezing), drought, salinity, ozone and intense light. These environmental stresses result in the destruction, deterioration of crop plants which leads to low crop productivity. Several strategies have been used and developed to build ressitance in the plants against these stresses.

*Herbicide tolerance*

Weeds are unwanted plants which decrease the crop yields and by competing with crop plants for light, water and nutrients. Several biotechnological strategies for weed control are being used e.g. the over-production of herbicide target enzyme (usually in the chloroplast) in the plant which makes the plant insensitive to the herbicide. This is done by the introduction of a modified gene that encodes for a resistant form of the enzyme targeted by the herbicide in weeds and crop plants. Roundup Ready crop plants tolerant to herbicide-Roundup, is already being used commercially.
The biological manipulations using genetic engineering to develop herbicide resistant plants are: (a) over-expression of the target protein by integrating multiple copies of the gene or by using a strong promoter., (b) enhancing the plant detoxification system which helps in reducing the effect of herbicide., (c) detoxifying the herbicide by using a foreign gene., and (d) modification of the target protein by mutation.

Some of the examples are:

Glyphosate resistance - Glyphosate is a glycine derivative and is a herbicide which is found to be effective against the 76 of the world&#8217;s worst 78 weeds. It kills the plant by being the competitive inhibitor of the enzyme 5-enoyl-pyruvylshikimate 3- phosphate synthase (EPSPS) in the shikimic acid pathway. Due to it&#8217;s structural similarity with the substrate phosphoenol pyruvate, glyphosate binds more tightly with EPSPS and thus blocks the shikimic acid pathway. 
Certain strategies were used to provide glyphosate resistance to plants.

(a) It was found that EPSPS gene was overexpressed in Petunia due to gene amplification. EPSPS gene was isolated from Petunia
and introduced in to the other plants. These plants could tolerate glyphosate at a dose of 2- 4 times higher than that required to kill wild type plants.
(a) By using mutant EPSPS genes- A single base substitution from C to T resulted in the change of an amino acid from proline to serine in EPSPS. The modified enzyme cannot bind to glyphosate and thus provides resistance.

(b) The detoxification of glyphosate by introducing the gene (isolated from soil organism- Ochrobactrum anthropi) encoding for glyphosate oxidase into crop plants. The enzyme glyphosate oxidase converts glyphosate to glyoxylate and aminomethylphosponic acid. The transgenic plants exhibited very good glyphosate ressitance in the field.
​Another example is of Phosphinothricin resistance 
Phosphinothricin is a broad spectrum herbicide and is effective against broad-leafed weeds. It acts as a competitive inhibitor
of the enzyme glutamine synthase which results in the inhibition of the enzyme glutamine synthase and accumulation of ammonia and finally the death of the plant. The disturbace in the glutamine synthesis also inhibits the photosynthetic activity. 
The enzyme phosphinothricin acetyl transferase ( which was first observed in Streptomyces sp in natural detoxifying mechanism against phosphinothricin) acetylates phosphinothricin, and thus inactivates the herbicide. The gene encoding for phosphinothricin acetyl transferase (bar gene) was introduced in transgenic maize and oil seed rape to provide resistance against phosphinothricin.
*Other abiotic stresses*
The abiotic stresses due to temperature, drought, and salinity are collectively also known as water deficit stresses. The plants produce osmolytes or osmoprotectants to overcome the osmotic stress. The attempts are on to use genetic engineering strategies to increase the production of osmoprotectants in the plants. The biosynthetic pathways for the production of many osmoprotectants have been established and genes coding the key enzymes have been isolated. E.g. Glycine betaine is a cellular osmolyte which is produced by the participation of a number of key enzymes like choline dehydrogenase, choline monooxygenase etc. The choline oxidase gene from Arthrobacter sp. was used to produce transgenic rice with high levels of glycine betaine giving tolerance against water deficit stress. 
Scientists also developed cold-tolerant genes (around 20) in Arabidopsis when this plant was gradually exposed to slowly declining temperature. By introducing the coordinating gene (it encodes a protein which acts as transcription factor for regulating the expression of cold tolerant genes), expression of cold tolerant genes was triggered giving protection to the plants against the cold temperatures. 

*Insect resistance*

A variety of insects, mites and nematodes significantly reduce the yield and quality of the crop plants. The conventional method is to use synthetic pesticides, which also have severe effects on human health and environment. The transgenic technology uses an innovative and eco-friendly method to improve pest control management.About 40 genes obtained from microorganisms of higher plants and animals have been used to provide insect resistance in crop plants
The first genes available for genetic engineering of crop plants for pest resistance were Cry genes (popularly known as Bt genes) from a _*bacterium Bacillus thuringiensis*_. These are specific to particular group of insect pests, and are not harmful to other useful insects like butter flies and silk worms. Transgenic crops with Bt genes (e.g. cotton, rice, maize, potato, tomato, brinjal, cauliflower, cabbage, etc.) have been developed. This has proved to be an effective way of controlling the insect pests and has reduced the pesticide use. The most notable example is Bt cotton (which contains CrylAc gene) that is resistant to a notorious insect pest Bollworm _*(Helicoperpa armigera)..*_ There are certain other insect resistant genes from other microorganisms which have been used for this purpose. Isopentenyl transferase gene from Agrobacterium tumefaciens has been introduced into tobacco and tomato. The transenic plants with this transgene were found to reduce the leaf consumption by tobacco hornworm and decrease the survival of peach potato aphid.
Certain genes from higher plants were also found to result in the synthesis of products possessing insecticidal activity. One of the examples is the Cowpea trypsin inhibitor gene (CpTi) which was introduced into tobacco, potato, and oilseed rape for develping transgenic plants. Earlier it was observed that the wild species of cowpea plants growing in Africa were resistant to attack by a wide range of insects. It was observed that the insecticidal protein was a trypsin inhibitor that was capable of destroying insects belonging to the orders Lepidoptera, Orthaptera etc. Cowpea trypsin inhibitor (CpTi) has no effect on mammalian trypsin, hence it is non-toxic to mammals. 

*Virus resistance*

There are several strategies for engineering plants for viral resistance, and these utilizes the genes from virus itself (e.g. the viral coat protein gene). The virus-derived resistance has given promising results in a number of crop plants such as tobacco, tomato, potato, alfalfa, and papaya. The induction of virus resistance is done by employing virus-encoded genes-virus coat proteins, movement proteins, transmission proteins, satellite RNa, antisense RNAs, and ribozymes. The virus coat protein-mediated approach is the most successful one to provide virus resistance to plants. It was in 1986, transgenic tobacco plants expressing tobacco mosaic virus (TMV) coat protein gene were first developed. These plants exhibited high levels of resistance to TMV. 
The transgenic plant providing coat protein-mediated resistance to virus are rice, potato, peanut, sugar beet, alfalfa etc. The viruses that have been used include alfalfa mosaic virus (AIMV), cucumber mosaic virus (CMV), potato virus X (PVX) , potato virus Y (PVY) etc.

*Resistance against Fungal and bacterial infections*
As a defense strategy against the invading pathogens (fungi and bacteria) the plants accumulate low molecular weight proteins which are collectively known as pathogenesis-related (PR) proteins.
Several transgenic crop plants with increased resistance to fungal pathogens are being raised with genes coding for the different compounds. One of the examples is the Glucanase enzyme that degrades the cell wall of many fungi. The most widely used glucanase is beta-1,4-glucanase. The gene encoding for beta-1,4 glucanase has been isolated from barley, introduced, and expressed in transgenic tobacco plants. This gene provided good protection against soil-borne fungal pathogen Rhizoctonia solani.
Lysozyme degrades chitin and peptidoglycan of cell wall, and in this way fungal infection can be reduced. Transgenic potato plants with lysozyme gene providing resistance to Eswinia carotovora have been developed. 

*Delayed fruit ripening*

The gas hormone, ethylene regulates the ripening of fruits, therefore, ripening can be slowed down by blocking or reducing ethylene production. This can be achieved by introducing ethylene forming gene(s) in a way that will suppress its own expression in the crop plant. Such fruits ripen very slowly (however, they can be ripen by ethylene application) and this helps in exporting the fruits to longer distances without spoilage due to longer-shelf life.
The most common example is the 'Flavr Savr' transgenic tomatoes, which were commercialized in U.S.A in 1994. The main strategy used was the antisense RNA approach. In the normal tomato plant, the PG gene (for the enzyme polygalacturonase) encodes a normal mRNA that produces the enzyme polygalacturonase which is involved in the fruit ripening. The complimentary DNA of PG encodes for antisense mRNA, which is complimentary to normal (sense) mRNA. The hybridization between the sense and antisnse mRNAs renders the sense mRNA ineffective. Consequently, polygalacturonase is not produced causing delay in the fruit ripening. Similarly strategies have been developed to block the ethylene biosynthesis thereby reducing the fruit ripening. E.g. transgenic plants with antisense gene of ACC oxidase (an enzyme involved in the biosynthetic process of ethylene) have been developed. In these plants, production of ethylene was reduced by about 97% with a significant delay in the fruit ripening. 
The bacterial gene encoding ACC deaminase (an enzyme that acts on ACC and removes amino group) has been transferred and expressed in tomato plants which showed 90% inhibition in the ethylene biosynthesis. 

*Male Sterility*

The plants may inherit male sterility either from the nucleus or cytoplasm. It is possible to introduce male sterility through genetic manipulations while the female plants maintain fertility. In tobacco plants, these are created by introducing a gene coding for an enzyme (barnase, which is a RNA hydrolyzing enzyme) that inhibits pollen formation. This gene is expressed specifically in the tapetal cells of anther using tapetal specific promoter TA29 to restrict its activity only to the cells involved in pollen production. The restoration of male fertility is done by introducing another gene barstar that suppresses the activity of barnase at the onset of the breeding season. By using this approach, transgenic plants of tobacco, cauliflower, cotton, tomato, corn, lettuce etc. with male sterility have been developed.


Clonal propagation refers to the process of asexual reproduction by multiplication of genetically identical copies of individual plants. The vegetative propagation of plants is labour-intensive, low in productivity and seasonal. The tissue culture methods of plant propagation, known as 'micropropagation' utilizes the culture of apical shoots, axillary buds and meristems on suitable nutrient medium.The regeneration of plantlets in cultured tissue was described by Murashige in 1974. Fossard (1987) gave a detailed account of stages of micropropagation. 
The micropropagation is rapid and has been adopted for commercialization of important plants such as banana, apple, pears, strawberry, cardamom, many ornamentals (e.g. Orchids) and other plants.The micropropagation techniques are preferred over the conventional asexual propagation methods because of the following reasons: (a) In the micropropagation method, only a small amount of tissue is required to regenerate millions of clonal plants in a year., (b) micropropagation is also used as a method to develop resistance in many species., (c) in vitro stock can be quickly proliferated as it is season independent,. (d) long term storage of valuable germplasm possible. 

The steps in micropropagation method are: a) Initiation of culture - from an explant like shoot tip on a suitable nutrient medium, b) multiple shoots formation from the cultured explant, c) rooting of _*in vitro*_ developed shoots and, d) transplantation - transplantation to the field following acclimatization.
The factors that affect micropropagation are: (a) genotype and the physiological status of the plant e.g. plants with vigorous germination are more suitable for micropropagation., (b) the culture medium and the culture environment like light, temperature etc. For example an illumination of 16 hours a day and 8 hours night is satisfactory for shoot proliferation and a temperature of 250C is optimal for the growth. 
The benefits of micropropagation this method are:
a) rapid multiplication of superior clones can be carried out through out the year, irrespective of seasonal variations.
b) multiplication of disease free plants e.g. virus free plants of sweet potato (Ipomea batatus), cassava (Manihot esculenta)
c) multiplication of sexually derived sterile hybrids
d) It is a cost effective process as it requires minimum growing space.

*Somaclonal variation*
The genetic variations found in the in vitro cultured cells are collectively referred to as somaclonal variation and the plants derived from such cells are called as &#8216;somaclones&#8217;. It has been observed that the long-term callus and cell suspension culture and plants regenerated from such cultures are often associated with chromosomal variations. It is this property of cultured cells that finds potential application in the crop improvement and in the production of mutants and variants (e.g. disease resistance in potato).
Larkin and Scowcroft (1981) working at the division of Plant Industry, C.S.I.R.O., Australia gave the term 'somaclones' for plant variants obtained from tissue cultures of somatic tissues. Similarly, if the tissue from which the variants have been obtained is having gametophytic origin such as pollen or egg cell, it is known as 'gametoclonal' variation.They explained that it may be due to: (a) reflection of heterogeneity between the cells and explant tissue, (b) a simple representation of spontaneous mutation rate, and (c) activation by culture environment of transposition of genetic materials. 
Shepard et al. (1980) also contributed by screening about 100 somaclones produced from leaf protoplasts of Russet Burbank. They found that there was a significant amount of stable variation in compactness of growth habit, maturity, date, tuber uniformity, tuber skin colour and photoperiodic requirements.
Somaclonal Variations has been used in plant breeding programmes where the genetic variations with desired or improved characters are introduced into the plants and new varieties are created that can exhibit disease resistance, improved quality and yield in plants like cereals, legumes, oil seeds tuber crops etc. Somaclonal variation is applicable for seed 
*Applications of Somaclonal Variations*
a) Methodology of introducing somaclonal variations is simpler and easier as compared to recombinant DNA technology.
b) Development and production of plants with disease resistance e.g. rice, wheat, apple, tomato etc.
c) Develop biochemical mutants with abiotic stress resistance e.g. aluminium tolerance in carrot, salt tolerance in tobacco and maize.
d) Development of somaclonal variants with herbicide resistance e.g. tobacco resistant to sulfonylurea
e) Development of seeds with improved quality e.g. a new variety of Lathyrus sativa seeds (Lathyrus Bio L 212) with low content of neurotoxin. 
f) Bio-13 &#8211; A somaclonal variant of Citronella java (with 37% more oil and 39% more citronellon), a medicinal plant has been released as Bio-13 for commercial cultivation by Central Institute for Medicinal and Aromatic Plants (CIMAP), Lucknow, India.
g) Supertomatoes- Heinz Co. and DNA plant Technology Laboratories (USA) developed Supertomatoes with high solid component by screening somaclones which helped in reducing the shipping and processing costs.

*Production of virus free plants*
The viral diseases in plants transfer easily and lower the quality and yield of the plants. It is very difficult to treat and cure the virus infected plants therefore te plant breeders are always interested in developing and growing virus free plants.
In some crops like ornamental plants, it has become possible to produce virus free plants through tissue culture at the commercial level. This is done by regenerating plants from cultured tissues derived from a) virus free plants, b) meristems which are generally free of infection - In the elimination of the virus, the size of the meristem used in cultures play a very critical role because most of the viruses exist by establishing a gradient in plant tissues. The regeneration of virus-free plants through cultures is inversely proportional to the size of the meristem used., c) meristems treated with heat shock (34-360C) to inactivate the virus, d) callus, which is usually virus free like meristems.e) chemical treatment of the media- attempts have been made to eradicate the viruses from infected plants by treating the culture medium with chemicals e.g. addition of cytokinins suppressed the multiplication of certain viruses. 
Among the culture techniques, meristem-tip culture is the most reliable method for virus and other pathogen elimination. 

Viruses have been eliminated from a number of economically important plant species which has resulted in a significant increase in the yield and production e.g. potato virus X from potato, mosaic virus from cassava etc. These virus free plants are not disease resistant so there is a need to maintain stock plants to multiply virus free plants whenever required.
*Production of synthetic seeds*

In synthetic seeds, the somatic embryos are encapsulated in a suitable matrix (e.g. sodium alginate), along with substances like mycorrhizae, insecticides, fungicides and herbicides. These artificial seeds can be utilized for the rapid and mass propagation of desired plant species as well as hybrid varieties. The major benefits of synthetic seeds are:
a) They can be stored up to a year with out loss of viability
b) Easy to handle and useful as units of delivery
c) Can be directly sown in the soil like natural seeds and do not need acclimatization in green house.
*Mutant selection*

An important use of cell cultures is in mutant selection in relation to crop improvement. The frequency of mutations can be increased several fold through mutagenic treatments and millions of cells can be screened. A large number of reports are available where mutants have been selected at cellular level. The cells are often selected directly by adding the toxic substance against which resistance is sought in the mutant cells. Using this method, cell lines resistant to amino acid analogues, antibiotics, herbicides, fungal toxins etc have actually been isolated. 

*Production of secondary metabolites*

The most important chemicals produced using cell culture are secondary metabolites, which are defined as&#8217; those cell constituents which are not essential for survival&#8217;. These secondary metabolites include alkaloids, glycosides (steroids and phenolics), terpenoids, latex, tannins etc. It has been observed that as the cells undergo morphological differentiation and maturation during plant growth, some of the cells specialize to produce secondary metabolites. The in vitro production of secondary metabolites is much higher from differentiated tissues when compared to non-differentiated tissues.
The cell cultures contribute in several ways to the production of natural products. These are: (a) a new route of synthesis to establish products e.g. codeine, quinine, pyrethroids, (b) a route of synthesis to a novel product from plants difficult to grow or establish e.g. thebain from Papaver bracteatum, (c) a source of novel chemicals in their own right e.g. rutacultin from culture of Ruta, (d) as biotransformation systems either on their own or as part of a larger chemical process e.g. digoxin synthesis.
*The advantages of **in vitro** production of secondary metabolites*
a) The cell cultures and cell growth are easily controlled in order to facilitate improved product formation.
b) The recovery of the product is easy.
c) As the cell culture systems are independent of environmental factors, seasonal variations, pest and microbial diseases, geographical location constraints, it is easy to increase the production of the required metabolite.
d) Mutant cell lines can be developed for the production of novel and commercially useful compounds.
e) Compounds are produced under controlled conditions as per the market demands.
f) The production time is less and cost effective due to minimal labour involved.

*Applications of secondary metabolites*
Many of these secondary products especially various alkaloids are of immense use in medicine. The yield of these chemicals in cell culture, is though generally lower than in whole plants, it is substantially increased by manipulating physiological and biochemical conditions. 
Shikonine is a dye produced by the cells Lithospermum erythrorhizon on a commercial scale. Besides this there are a number of secondary metabolite products that are being widely used for various purposes. Vincristine is used as anticancer agent, digoxin controls cardiovascular disorders, pyrithrins is an insecticide etc. The production of specialty chemicals by plants has become a multibillion industry.
Please refer to the table for some secondary metabolites and their uses.
*Table showing plant species and secondary metabolites obtained from them using tissue culture techniques*​
*Product**Plant source**Uses*Artemisin_Artemisia spp._AntimalarialAzadirachtin_Azadirachta indica_InsecticidalBerberine_Coptis japonica_Antibacterial, anti inflammatoryCapsaicin_Capsicum annum_Cures Rheumatic painCodeine_Papaver spp._AnalgesicCamptothecin_Campatotheca accuminata_AnticancerCephalotaxine_Cephalotaxus harringtonia_AntitumourDigoxin_Digitalis lanata_Cardiac tonicPyrethrin_Chrysanthemum cinerariaefolium_Insecticide (for grain storage)Morphine_Papaver somniferum_Analgesic, sedativeQuinine_Cinchona officinalis_AntimalarialTaxol _Taxus spp._AnticarcinogenicVincristine_Cathranthus roseus_AnticarcinogenicScopolamine_Datura stramonium_Antihypertensive

*Production of Somatic hybrids and cybrids*
The Somatic cell hybridization/ parasexual hybridization or Protoplast fusion offers an alternative method for obtaining distant hybrids with desirable traits significantly between species or genera, which can not be made to cross by conventional method of sexual hybridization. 

*Somatic hybridization*
Somatic hybridization broadly involves in vitro fusion of isolated protoplasts to form a hybrid cell and its subsequent development to form a hybrid plant. The process involves: a) fusion of protoplasts, (b) Selection of hybrid cells, (c) identification of hybrid plants. 
During the last two decades, a variety of treatments have been used to bring about the fusion of plant protoplasts. Protoplast fusion can be achieved by spontaneous, mechanical, or induced fusion methods.. These treatments include the use of fusogens like NaNO3, high pH with high Ca2++ ion concentration, use of polyethylene glycol (PEG), and electrofusion. These inducing agents used in protoplast fusion are called &#8216;fusogen&#8217;.
PEG treatment is the most widely used method for protoplast fusion as it has certain advantages over others. These are : (a) it results in a reproducible high-frequency of heterokaryon formation., (b) The PEG fusion is non specific and therefore can be used for a wide range of plants., (c) It has low toxicity to the cell and (d) The formation of binucleate heterokaryons is low.
*
Mechanism of fusion*
The fusion of protoplasts takes place in three phases- agglutination, plasma membrane fusion and formation of heterokaryons. When the two protoplasts come in close contact with each other, they adhere to each other. This agglutination can be induced by PEG, high pH and high Ca2+. The protoplast membranes get fused at localized sites at the point of adhesion. This leads to the formation of cytoplasmic bridges between protoplasts. High pH and high Ca2+ ions neutralize the surface charges on the protoplasts which allows closer contact and membrane fusion between agglutinated protoplasts. The fused protoplasts become round as a result of cytoplasmic bridges which leads to the formation of spherical homokaryon or heterokaryon.
*Selection of hybrid cells* 
The methods used for the selection of hybrid cells are biochemical, visual and cytometric methods using fluorescent dyes. The biochemical methods for selection of hybrid cells are based on the use of biochemical compounds in the medium. The drug sensitivity method is useful for the selection hybrids of two plants species, if one of them is sensitive to a drug. Another method, auxotrophic mutant selection method involves the auxotrophs which are mutants that cannot grow on a minimal medium. Therefore specific compounds are added in the medium. The selection of auxotropic mutants is possible only if the hybrid cells can grow on a minimal medium. The visual method involves the identification of heterokaryons under the light microscope. In some of the somatic hybridizations, the chloroplast deficient protoplast of one plant species is fused with the green protoplast of another plant species. The heterokaryons obtained are bigger and green in colour while the parental protoplasts are either small or colourless. The cytometric method uses flow cytometry and flourescent-activated cell sorting techniques for the analysis of plant protoplasts.

*Applications of Somatic hybridization* 

a) *Creation of hybrids with disease resistance *- Many disease resistance genes (e.g. tobacco mosaic virus, potato virus X, club rot disease) could be successfully transferred from one species to another. E.g resistance has been introduced in tomato against diseases such as TMV, spotted wilt virus and insect pests.
b) *Environmental tolerance* - using somatic hybridization the genes conferring tolerance for cold, frost and salt were introduced in e.g. in tomato.
c) *Cytoplasmic male sterility *- using cybridization method, it was possible to transfer cytoplasmic male sterility.
d) *Quality characters *- somatic hybrids with selective characteristics have been developed e.g. the production of high nicotine content. 

*Chromosome number in somatic hybrids*
The chromosome number in the somatic hybrids is generally more than the total number of both of the parental protoplasts. If the chromosome number in the hybrid is the sum of the chromosomes of the two parental protoplasts, the hybrid is said to be symmetric hybrid. Asymmetric hybrids have abnormal or wide variations in the chromosome number than the exact total of two species.

In 1972, Carlson and his associates produced the first inter-specific somatic hybrid between Nicotiana glauca and N. langsdorffii. In 1978, Melchers and his co-workers developed the first inter-genetic somatic hybrids between Solanum tuberosum (potato) and Lycopersicon esculentum (tomato). The hybrids are known as &#8216;Pomatoes or Topatoes&#8217;.
*Limitations of Somatic Hybridization*
a) Somatic hybridization does not always produce plants that give fertile and visible seeds.
b) There is genetic instability associated with protoplast culture.
c) There are limitations in the selection methods of hybrids, as many of them are not efficient.
d) Somatic hybridization between two diploids results in the formation of an amphidiploid which is not favourable therefore haploid protoplasts are recommended in somatic hybridization.
e) It is not certain that a specific character will get expressed in somatic hybridization.
f) Regenerated plants obtained from somatic hybridization are often variable due to somaclonal variations, chromosomal elimination, organelle segregation etc.
g) Protoplast fusion between different species/genus is easy, but the production of viable somatic hybrids is not always possible.

*Cybrids*
The cytoplasmic hybrids where the nucleus is derived from only one parent and the cytoplasm is derived from both the parents are referred to as cybrids. The process of formation of cybrids is called cybridization. During the process of cybridization and heterokaryon formation, the nuclei are stimulated to segregate so that one protoplast contributes to the cytoplasm while the other contributes nucleus alone. The irradiation with gamma rays and X-rays and use of metabolic inhibitors makes the protoplasts inactive and non-dividing. Some of the genetic traits in certain plants are cytoplasmically controlled. This includes certain types of male sterility, resistance to certain antibiotics and herbicides. Therefore cybrids are important for the transfer of cytoplasmic male sterility (CMS), antibiotic and herbicide resistance in agriculturally useful plants. Cybrids of Brassica raphanus that contain nucleus of B. napus, chloroplasts of atrazinc resistant B. capestris and male sterility from Raphanus sativas have been developed. 


*In vitro** plant germplasm conservation*
Germplasm refers to the sum total of all the genes present in a crop and its related species.
The conservation of germplasm involves the preservation of the genetic diversity of a particular plant or genetic stock for it&#8217;s use at any time in future. It is important to conserve the endangered plants or else some of the valuable genetic traits present in the existing and primitive plants will be lost. A global organization- International Board of Plant Genetic Resources (IBPGR) has been established for germplasm conservation and provides necessary support for collection, conservation and utilization of plant geneic resources through out the world. The germplasm is preserved by the following two ways:
(a) *In-situ** conservation*- The germplasm is conserved in natural environment by establishing biosphere reserves such as national parks, sanctuaries. This is used in the preservation of land plants in a near natural habitat along with several wild types.
(b) *Ex-situ** conservation*- This method is used for the preservation of germplasm obtained from cultivated and wild plant materials. The genetic material in the form of seeds or in vitro cultures are preserved and stored as gene banks for long term use._*
In vivo*_ gene banks have been made to preserve the genetic resources by conventional methods e.g. seeds, vegetative propagules, etc. _*In vitro*_ gene banks have been made to preserve the genetic resources by non - conventional methods such as cell and tissue culture methods. This will ensure the availability of valuable germplasm to breeder to develop new and improved varieties.

The methods involved in the in vitro conservation of germplasm are: 

(a) Cryopreservation- In cryopreservation (Greek-krayos-frost), the cells are preserved in the frozen state. The germplasm is stored at a very low temperature using solid carbon dioxide (at -790C), using low temperature deep freezers (at -800C), using vapour nitrogen (at- 1500C) and liquid nitrogen (at-1960C). The cells stay in completely inactive state and thus can be conserved for long periods. Any tissue from a plant can be used for cryopreservation e.g. meristems, embryos, endosperms, ovules, seeds, cultured plant cells, protoplasts, calluses. Certain compounds like- DMSO (dimethyl sulfoxide), glycerol, ethylene, propylene, sucrose, mannose, glucose, praline, acetamide etc are added during the cryopreservation. These are called cryoprotectants and prevent the damage caused to cells (by freezing or thawing) by reducing the freezing point and super cooling point of water.

(b) Cold Storage- Cold storage is a slow growth germplasm conservation method and conserves the germplasm at a low and non-freezing temperature (1-90C). The growth of the plant material is slowed down in cold storage in contrast to complete stoppage in cryopreservation and thus prevents cryogenic injuries. Long term cold storage is simple, cost effective and yields germplasm with good survival rate. Virus free strawberry plants could be preserved at 100C for about 6 years. Several grape plants have been stored for over 15 years by using a cold storage at temperature around 90C and transferring them in the fresh medium every year.

(c) Low pressure and low oxygen storage- In low- pressure storage, the atmospheric pressure surrounding the plant material is reduced and in the low oxygen storage, the oxygen concentration is reduced. The lowered partial pressure reduces the in vitro growth of plants. In the low-oxygen storage, the oxygen concentration is reduced and the partial pressure of oxygen below 50 mmHg reduces plant tissue growth. Due to the reduced availability of O2, and reduced production of CO2, the photosynthetic activity is reduced which inhibits the plant tissue growth and dimension. This method has also helped in increasing the shelf life of many fruits, vegetables and flowers.

The germplasm conservation through the conventional methods has several limitations such as short-lived seeds, seed dormancy, seed-borne diseases, and high inputs of cost and labour. The techniques of cryo-preservation (freezing cells and tissues at -1960c) and using cold storages help us to overcome these problems.

I think its going to be a good year for some !


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## Bonkleesha (Feb 10, 2012)

+++++++++++++rep!!!!!!!!!!!!!!!!!!!!!!!!!!!


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## nitro20% (Feb 10, 2012)

holy shit dude!!!not another one...This one is for the pros...(u guys) i like the china mans dumb dummy study.....BUT tell me???? Does this mean that taking a x-plant down to its nubbin does not take it back to "a seed" like quality??? or will this JUST ensure the quitting of any local things like bugs and mold..ha ha (pharamacopying ssshhhh.) so maybe one could p.m. me ...... or just put this into GRASSHOPPER terms,,,,obviously,,its to much for one to handle at first, but, all the info helps, the start,of what im tryin to learn about...this is the reason i am interested, to purify a plant (not just mj)


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## nitro20% (Feb 10, 2012)

*

QUOTE;;; and they said it couldnt be done just days ago 


WERE IS GUILE ?? ​
​
*


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## pharmacoping (Feb 10, 2012)

heres my take. during and after these cells are washed and sterilized, with success and failures noticed within day to the eye, and then cut up, under sterility, divided, amongst a hundred more vessels, all sterile, over and over, your tru genetics, or at least the ones already affecting the very first plant you excised, are now able to express themselves for the babes they are. Under no genetic stress, caused by the mutagens continuing an attack on your hunny throughout the grow, unnoticed, altering its dna code every time(note the bumps in your stalk) and on trees, this is where the "Genetic Throw Down" occurs, leaving the remainder of its life cycle affected in different ways....these ladies are able to fully report their entire dna code back to you, given their environment is top notch, (described in detail in above post link). Every generation produced is a supergirl compared to the last generation. every leaf is thicker, stalks are shiny and strong from birth and till harvest. the leaves are super prounounced in detail and terpine profiles scream their potential. the very point of this is to accomplish repeatable results, which is a must imo in the medical mj field. every grower knows that there are subtle if not pronounced differences in every clone taken from one mom, seed to seed, generation to generation. A clone fucks up bad eventually, compared to when it was your "best in the world weed". the best way to keep her is in this suspended animation, or a hundred of her in case something goes wrong. not a mom ever changing with clones of varying acceptance(lab results) clones dont get better after that first cut, and even that cut changes many many clones for many growers, as it introduces viruses instantly. In contrast you may infect a plant that grows three fingers and a nose, and may like that trait caused by a viral hijacking somewhere in an ancestor(and could even report more anomalies later!) so you can keep it, purify it, and stop the viral dna hijacking for good, rather than continuing changing traits with every cut, and moment in growth. The finish date of 100% of my flowers is precise, everytime, for years,their finish weight is within 1/4oz evertime, and each one looks exactly like the last of its kind. all are pest resistant, no mites here ever in the room(3 yrs)never a death, or strange plant reaction, or ph issue, no nutrient lock ups, no additives except nutes,silica,zone. the tissue culture has been my link to success. the missing link. its not for many in the field, but the ones growing for their maximum patients, full time, and providing professional results to professional people, know about this, and are now taking a serious look. I've been screaming tissue culture for 20 yrs, to everybody. with very similar results, as here. 
so, I would like to thank you guys for chiming in, getting your feet wet, and seeing some possibilities. there are more than we can imagine. 


for instance,

If someone(with a card) in MI will pay me for a bio luminescence gene inserted into the dna of a young plant, maybe of your choice. bid now. no pics
until sold, if permissable then.

what do you think nitro ? how high do you think it'll go?


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## pharmacoping (Feb 10, 2012)

I know, a tall order, but I have seen one, and now can make one ! It's very difficult, with many failures, and a ton of time, for sure an expensive endeavor. But imagine having a mother plant that floresces, as well as all of its cuttings, seedlings, etc. I would venture to say priceless, especially by the way the security of the plant I saw was handled.

I know there are non believers, and I'm a tease...so here is proof that this is being done, and has been done , in a home lab. this is wicked, with my boys(Scott, in Georgia) videos, and some far out stuff. take notes, no secrets divulged, but you'll see a walnut tree, make light. I'm still in school learning these types of manipulations, and their potential applications. I bring marijuana to the lab table, years ago even, and am here now. 

Nitro, this wil get you off BIG time !! and is exactly how I will floresce an amazing purified strain hybrid called Ace Of Spades. It may already be the best mj on the planet, it is for me and every person who has used it thusfar. 

enjoy the b'jeezus out of this one, and follow the links, all of them...you might even get a glimpse of something special http://www.esf.edu/chestnut/tissue culture.htm#

the one ingredient you're not seeing is PEG. its the link between all of this home lab magic. it will allow layering , like grafting of several different plant species on top of eachother in vitro. this may prove to be a very quick way of growing a multi strain plant/100plants even , with a few different kinds of weed on it for your dialed in pleasure, customized. carrot roots on pot plants

would sure be easier to remove from these damn gro rocks when done !


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## pharmacoping (Feb 10, 2012)

I can rot fresh shrimp for a bit, and culture the bacteria from its skin. much of this bacteria is bioluminescent in nature,stuck on the skin, and will group together in the soup, until grown, and if taken care of in a jar or petri dish(needs lots of 02), will turn into a nice little herd of glow in the dark stuff.. if anyone has taken this any further, please let me know your experience. rabbits, mice, fish, and other animals are already born and some for sale with these type of bio markers present and visible.

nitro...the medium i used for the shrimp stuff was a snip of beef boullion ! I knew you'd love that !!


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## nitro20% (Feb 10, 2012)

Justus von Liebig's *Law of the Minimum* states that yield is proportional to the amount of the most limiting nutrient, whichever nutrient it may be. From this, it may be inferred that if the deficient nutrient is supplied, yields may be improved to the point that some other nutrient is needed in greater quantity than the soil can provide, and the Law of the Minimum would apply in turn to that nutrient









 SHIT,,, this guy thinks anything is possible!!!
*

OK,,,,,,,,,,, DO IT UR WAY "DUDES"!?!?!?! THIS IS WHAT I THINK I KNOW. HA HA *,,* JUST LIKE CLONES,, YOU NEED TO GET THEM GOIN,,,BUT WHEN CLONES GET GOIN U GET TO ADD SOME N/P/K/......WHEN YOU "MAKE A HOME-MADE MEDIUM,,,, U NEED TO DO ALOT OF RESEARCH,,,,,,THEN THE IDEA IS TO GET ALL OFF THE NUTRIENTS THAT U DISCOVERED AND PUT AS MUCH AS YOU CAN BUT NOT TO MUCH,,,,YUH YUH,,,I KNOW "JUST LIKE ANYTHING",,,,,,NO NO NO!!!,,,THIS TIME YOU WILL NOT BE ABLE TO "ADD" ANYTHING TO IT UNLESS U RE-JAR UR PLANT LET (I THINK)...A CLONE HAS LEAVES AND MORE STEM THAT WILL HELP THE PRODUCTION OF IT TO A POTABLE PLANT,,,A BASIC CULTURE HAS NONE OF THIS,, AND WILL NEED "ALL" THE HELP IT CAN GET THROUGHOUT THE PROCESS..... AND I GATHER THAT THE PLANTLET WILL LEARN HOW TO,,, AND WHEN TO USE THE FOOD SOURCE....SO YOU NEED TO COME UP WITH THAT PERFECT SOLUTION THAT U THINK WILL "DO IT FOR YOU" **OR DONT BE A "GRASSHOPPER" AND BUY A MEDIUM THAT U CAN EXPERIENCE THE "GOLD" WITH!!
* 

PHARMACOPING,,,MAN thats cool that u are into all kinds of TC..almost sounds like u ARE a pro...(scientist)..ok i want a 6 tri tip'ed cow!!!GRASS FED PLEASE! one thing at a time,,plants,,then animals...


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## nitro20% (Feb 10, 2012)

*I MIGHT BE THE LAST STONER TO FIND THIS BUT I THOUGHT IT WAS FUN.. *http://www.firstrays.com/fertcalc.htm *OH AND HELPFUL,,TOOK ALOT OF GUESSING RIGHT OUT OF MY PLANS!!!*


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## Bonkleesha (Feb 11, 2012)

lebigs law doesnt apply anymore. school isnt telling me this, my brain is. we can give these plants 100% of EVERYTHING just about, and the colas arent infinitely big. im sure science people can see the problem with it in the present. plant tissue culture media is a PERFECT example. look at the formula for the mini rose media, and talk to lebig about minimums. no offense to the dude. most scientists were right.... IN THEIR TIME.


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## pharmacoping (Feb 11, 2012)

This is a standard law in the grow room, however outdated, and provides a set of parameters that need to be addressed. Most continue trying to add things to the mix when they're results are not whats desired, when in fact its most likely one of the major ones needing help- air-water-light-temp-hum-food.

Bonkleesha is right, sorta, about old news. however, food is not the only parameter that is limited in the lab, I'm guessing. A perfect grow atmosphere is described in a bigbuds mag link earlier. ONce all these are alll met, your cola will grow as tall as genetics will allow. But in a shitty room, the best food in the world provided cant make up for the lack of c02,o2 in the root zone,ph imbalances,weak lighting not photosynthesizing enough to completely uptake and burn carbs, etc. I quote the law of minimum often, to anyone at the store trying to "fix" an issue

look at the guitar colas in my pics, they were given the perfectly balanced conditions, and produced perfectly, as expected, each time the same.. 
not he is not working in a test tube that does not require photosynthesis to flourish. adding nute ppm 's higher than 200ppm(which willl yield slower results from burn) will end the life of the culture. example. One drop of a quality nute, mixed in one liter of water will support callus material for a month in 30 jars!.

worry about your nutrient demands once the plantlet has been released into the veg room. simply stop mixing your own formulas, and buy a professionaly formulated off the shelf one thats already made the wheel turn, Dutch master, advanced nutes, and several others. make sure you follow the instructions from marijuana tested nutes only. My tomatoes do great with the wasted run off nutrients alone, in dirt, outside, or inside. 

lesson=more nutrients are never better than less. quality and completeness of your nute system is key. eyes closed, the easiest grow room issue to solve is the nutrient one.


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## pharmacoping (Feb 11, 2012)

nice calculator for nutrient formulating professionals nitro !!!!

after done fuggin around with spoons and weights, dial this up, leave the rest to the pros, and use that time to experiment with in vitro body parts?

http://www.dutchmaster.com.au/nutrient_calculator.php?language=english


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## Bonkleesha (Feb 11, 2012)

if more people bought the media, i bet they would make it more readily available and it would be cheaper.


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## pharmacoping (Feb 11, 2012)

I've thought the same thing Bonkleesha, so I started to sell it specifically for mj, about year or two ago. it really is cheap, even retail costs. but the mixing/formulating is a little tricky, and the reason I'm here. Many ways to skin this cat, and I am biased, so these fresh perspectives, unbiased by experience or teachers, is a refreshing way to see new possibilities. working in the lab does not allow these one off types of activities, which is where this all came from in the beginning ! so I take this opportunity to learn form the inexperienced hopefully, working without the constraints of protocol and technique.

it has been great for me, and my lab, with new trial every day. 

agar is sold at the health food stores cheap, in bulk. the rest is linked.


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## pharmacoping (Feb 11, 2012)

In recent years, knowledge of anthocyanin pigments has undergone unprecedented expansion. Indeed, the molecular genetic control of anthocyanin biosynthesis is now one of the best understood of all secondary metabolic pathways. Advances in analytical technology have led to the discovery of many novel anthocyanin compounds, dramatically enriching the palette used by plant breeders to introduce vibrant new colors into horticultural crops. I found these bio flavanoids and anthocyanin pigments used in tissue culture to permanently alter the colors of the flower of plants for sale. salysylic acid(aspirin, willow branch) is used in vitro to induce these, as well as UV light, or heat, for the home lab. 

What we are doing is called Bio Hacking


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## nitro20% (Feb 12, 2012)

yeah i see where you are at...I thought he meant that IF we put the RIGHT amount,, of the RIGHT things,,, then,,, that plant would FLOURISH,,thought about allllllll the different recipes in TC for every plant....there not the same. WOW ALL the things that i know now......

U ARE RIGHT about NEW nutrients for hydro/dirt,,, IF used correctly,,all the TECH. that we know from TC is changing the way to grow plants (ANY)... changes the way I SEE it!!!

*PHARAMACOPING * DUDE is there a label on the medium u suggested with a basic macro-nutrient count,,ive looked for days!!! seriously,,thought i saw one that said 10-10-10 n-p-k..and you said to get the carrot medium mix and mix to a no more then 150 ppm,,,right....WELL!!! (I DIDNT GIVE UP)its coming and cant seem to find out how to do that with out a ppm meter.......???
OR just put the whole pack in a liter??done....STILL WANT TO KNOW A LOW AND A HIGH n-p-k ppm for my average medium..IF anything just a ppm on the nitrogen......have a recipe @ 90 ppm total macro-nuts. IS THIS TO MUCH? IT has 33 ppm nitrogen???? backed out of my math and it was 11.2% more then a seedling.....OR WHATS STRONGER? seedling or a just cultured "culture"
*
THIS IS WHAT I THINK I SEE!!!! GRASSHOPPERS*......... *THE CULTURES ARE DOING WELL AND IT SEEMS LIKE THEY ARE PERKY AND WANT TO GROW THINGS,,THE PROBLEM IS,,, THE NUTRIENTS,,,I ADDED ONLY 2 DROPS OF LIQUID KARMA WITCH ONLY IS ABOUT 6-9 PPM OF A SEAWEED EXTRACT THAT IS .1-.1-.5 N.P.K. BUT IS FULL OF AUXINS AND CYTOKININS THAT HELP PLANT CELL DIVISION,,,BUT IF THE PLANTLET HAS NO OTHER MEANS OF SUPPORT FOR GROWTH...WWWWWWEEELLLLL...THIS IS WERE THE LA-BUR-I-TORYS DO THAT WORK FOR YOU,,,THEY KNOW WHAT LIKES WHAT,,,THIS HAS BEEN GOING ON SINCE THE 50s..........
**
**
AND THATS WHAT THIS GRASSHOPPER THOUGHT IT SAW WHEN IT JUMPED*


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## nitro20% (Feb 12, 2012)

IF YOU NEVER HAD BUGS OR MOLD.... THEN YOU SEEM LIKE A NEWBIE TO ASK HOW TO KILL IT!!!! WE ALL KNOW THAT THERE IS NO CURE ALL FOR BUGS OR WHATEVER...NUTRIENTS,,,SHIT FIND YOUR "THANG" FOR UR "GANG" AND SICK TO THE SAME "THING"


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## nitro20% (Feb 12, 2012)

There is so much to learn AND remember before taking on a TC formula...like read ur dudes messages before asking a question!!! ALRIGHT then..now im some where..NO where can i find a dumb dummy ppm for these callus mediums..there secret..thought the secret was in the other things they added... oh well,,, so 150 ppm...one drop quality nute..is about 3-20 ppm..right..RIGHT!!! 3 days 3DAYS!!! I looked!!!!


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## nitro20% (Feb 12, 2012)

OK FUKRS!!! I SEE WERE U ARE BOTH AT WITH THIS PHILOSOPHER DUDE,,AND I CAN SEE WERE WE THINK THAT THIS IS THE "END" BUT LOOK AT IT THIS WAY,,,WE HAVE PUSHED ALL PLANTS TO THE LIMIT,,IN A WAY,,RIGHT, TO THE POINT WHERE "OUR" TECH SEES NO MORE OPTIONS FOR FASTER GROWTH OR NOT SO BIG OF A DIFFERENCE TO CALL HOME OVER..RIGHT? WELL THERE WILL BE THAT ONE MORE THING THAT WE WILL DISCOVER,,,THAT WILL HELP A PLANT USE MORE OF A SUBSTANCE THAT "WAS" ALL "WE" COULD GIVE IT FOR THE MINIMAL LAW...WE SEE THAT "THIS IS THE END" 20 YEARS FROM NOW,,SOME ONE WILL SAY THE SAME THING..."THAT PLANT CANT GROW ANY FASTER" I DONT KNOW BUT EVEN THE "THEORY OF RELATIVITY" IS IN QUESTION!!! SAME WITH HUMANS,,,WE'RE ONLY AS STRONG AS OUR WEAKEST PART,,, IQ, BROKEN LEG, NO DRIVERS LICENSE, A WIFE,E.C.T. ALL THESE THINGS CAN CHANGE IF WE ADD THE RIGHT "OTHER" THING!!!!!*I LOVE TO THINK*


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## pharmacoping (Feb 12, 2012)

[h=3]16 oz. Ecogrow Standard (10-8-14)[/h]Ecogrow Standard (10-8-14 N-P-K) was developed for the Pacific N.W. Climate (temperate zone). It is ideal for our most popular hydroponic plants -..


ms carrot culture will arrive at around 150-200 ppm, if its a one litre kit. more will kill. hormones just make more things happen, at varying speeds, not the nutrients, assuming they are well balanced and complete. I'm guessing that liquid karma alone would not grow a healthy plant through its cycle(but I dont know). if thats tru, then it wont hellp in tc either. use eco nutes (period) or buy pre mixes like murishage and skooter for carrots.

if you use basal salts, you must have a ppm meter. hanna, 29$ amazon. batteries last for years, my hanna is 15 years old and cost me 39$. ph paper is amust also...6.5 is good. adjust before you hormone though. if food is right, your growth speed is caused/limited by the hormones used, hosts original health, and contaminants in vitro.


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## wyteboi (Feb 12, 2012)

guile an pharm , thank you very much for the info ..... dont worry you didnt scare me away .....yet , i just been a lil busy. im readin every word and am gonna start testing in a few months , once i get enough knowledge to at least try.




soil


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## pharmacoping (Feb 12, 2012)

wyteboiiiiii !,

thanks for stopping by man, cant wait to see your creations ! I threw eggs(fresh, from chickens, even, not sure which came first though, dont ask) in a jacket pocket this morning, with my digi camera, and one broke ! otherwise I'd end this note with some cultured cuties. I think the cam will be ok. 

peace


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## nitro20% (Feb 12, 2012)

got fresh eggs too...man oh man,,,i eat 2 dozen a week,,of GOOD chicken eggs...4.95 a dozen...it gets expense...


i read back through this a little,,,some where on the rule list is: dont over think it!!! 

and maybe that simply hydroponics you tube vid. may have a working mix...IM sure glad nobody reads this shit!!! if u go on the net.. and start lookin for a tc recipe/dip n grow you will end up seeing these threads all over...


no... liquid karma can hurt u with to much/with no n/p/k..it is just a general additive... starting to see another way of looking at plant growth...


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## pharmacoping (Feb 12, 2012)

the nutrient mix is a no brainer. quit thinking about it at all, buy the basal salts, or a muri skoog mix and do it right without a thought. You could mix ANY complete hydro nute, very weak, under 200ppm, and it will work well !!. if its got iron, a multivitamin works great even. the hormone mix will need your duty. 

we keep free range chickens forever. we dont eat the chix, but we do breed and eat/gift the organic eggs. if people knew what an egg went through to get to their plate, they would do the same I think. rabbits to, got a buck and two does, they very intimate, when visiting eachother, but we do eat their young, right after they get off the nipple !, for free, without feeding them. they'll never taste any better, and we have hundreds in a couple months if we want. 

for the cost of three dozen eggs, you could have all the hormones you need for a year you know......


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## Bonkleesha (Feb 12, 2012)

i was reading somewhere that someone was crushing up a B-vitamin and throwing in a drop of superthrive with good results. i wonder if he was using an antibiotic.


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## pharmacoping (Feb 12, 2012)

I used to use a multi, before basal salts were available in the 70'ss-80's, did have results of live tissue, but would never calll them good results. no results using just those for nutes have ever produced anything that left the culture vessel, that I know of, or ever seen. the culutre may live for sure, but will not yield any of the token results we're looking for, except a chunk of callus materiL, OR A SPROUT, FOR OBSERVATION.


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## dontexist21 (Feb 13, 2012)

How long can the root balls be stored before you are forced o put them in veg?


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## nitro20% (Feb 13, 2012)

*deleting myself,,, 420 peace*


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## pharmacoping (Feb 13, 2012)

plant preservative is a given, a standard, and should not be given a second thought. just get some, use it, and never look back. I dont know what it does, but I cant operate my way without it. most bio hackers use it in vitro for better results.

explants will never look like mj, until hardened and planted in medium outside of the vessel. they are bonzai's , I think due to lack of photosynthesis. hope that helps

*Big Tom Callahan*: Of course, I can get a hell of a good look at a T-Bone steak by sticking my head up a bull's ass, but I'd rather take the butcher's word for it.


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## pharmacoping (Feb 13, 2012)

dontexist21 said:


> How long can the root balls be stored before you are forced o put them in veg?


I'm never forced to put any in veg. my rootballs thrive indefinately(can be divided over and over too) after a month or two sometimes negative changes can be seen, suggesting it be transferred into a freshly mixed vessel. The root ball continues to grow, so theoritically it could outgrow the vessel too. 
My rootballs are stored until they need to be sprouted, then hardened off then to veg.


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## Bonkleesha (Feb 13, 2012)

off topic, but have u propagated cannabis from a root cutting ever?


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## pharmacoping (Feb 13, 2012)

yes I have(hundreds)in vitro, to complete harvest and use. The biggest difference I see, which is huge to me, is a uniformity thats lost eventually when traditional clones are taken, and a very pure, more pronounced growth,pattern,terpine profiles, without any kind of issues, including pests, and no knots on the stems, so I think they are super girls, caused by a purified dna, free of any replication infection/dna changing virus. also, the vegging is much more efficient, due to roots already being present at sprouting, and tighter foliage, ending in a sort of self pruning action, as all but the fan leaves are gone inside the tight flowers. lab results show consistancy on the tc plants not seen with clones/seeds of the same variety. 

but I think you're talking about cloning from a root snip sor so? I have tried this too(hundreds) with (hundreds) many failures, and some success. but again, success is what? I saw sprouts of plants that grew slow, were obviously infected, and yielded very low. not sure, but I think the reason is because those roots, without the protection of photosynthesis, or a sterile environment, are susceptible to disease. And, sugar must be provided, as there is no photosynthesis without leaves, and sugar in your roots/medium come with all sorts of problems,pests/disease unless sterile/in vitro. 

when harvested, if you leave one tiny branch at the bottom, and a bit of the thick stalk cut correctly, and vegged again, will result in another full size plant soon, complete with multiplying problems that may or may not have been evident in the first fruiting. they often look like a clone taken from a "topping" of a plant, which is not ideal, at least for me. this area has the most infectious contaminated material, right down to the dna, of the whole plant. thats why they appear differently. 

peace


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## Bonkleesha (Feb 13, 2012)

i was just reading that root cuttings of varigated plants will never produce a varigated clone. also, was reading its easier for a root to grow chutes than it is for a chute to grow roots. just got my mind spinning.


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## nitro20% (Feb 13, 2012)

*deleting myself,,, 420 peace*


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## nitro20% (Feb 13, 2012)

*deleting myself,,, 420 peace*


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## nitro20% (Feb 13, 2012)

*deleting myself,,, 420 peace*


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## pharmacoping (Feb 13, 2012)

Bonkleesha said:


> i was just reading that root cuttings of varigated plants will never produce a varigated clone. also, was reading its easier for a root to grow chutes than it is for a chute to grow roots. just got my mind spinning.


never tc'd a variegated houseplant, but my understanding is that the ones that are chimeras, actually containing tissues of genetic make up growing adjacent to eachother, that one would have difficulty exact cloning. however I may give this a whirl on a chimera in my home. the aberrant characteristic is limited to epidermal tissue, which is already commercially cultured this way.

You are correct though, a root snip from a chimera(some thornless berries,seedless oranges,grapes) will not contain both genes(maybe if lucky ??). Marijuana is not a variegated chimera(YET !!) so no worries.But if one did, for instance, make a bioluminescence marijuana plant into a variegated chimera, they could all but guarantee a "clone only " strain, increasing its value immensely I imagine, just sayin. 

great idea in the name of corpocracy !


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## pharmacoping (Feb 13, 2012)

nitro20% said:


> thats a question i had on that other tc thread,,,how come all the pics of MJ look like they just got jared,,clean and look just like a potted MJ plant....not the ones of real results though,,,,they look like kinda what u describing! And u never hear from those dudes having pic of a perfect looking, fresh jared culture...i aint scared to show a screw up..or even lame results,,,i-aint-a-lyin-2-myslf.....
> 
> I have tryed to find definitions for plant preserve,,but the same thing is found,,,hydrates,,and has a wide soectrum for killing contamination in TC..a must for tissue cultures...I knew this was somthing that was not replaceable,,,and i hope that the 30ml bottle will be here this week!!



here ya go. I threw in some mold, for good, honest measure. but the shooting/rooting one has been cut and is hardening/or in veg already. maybe i get some pics of hardening if you want? or vegging?
that callus produces clumps of roots and shoots with roots, continually, for me to cut off of. I scalpel the mini plant right off the callus and carefully ,on its side, cut through the roots, to preserve roots with the shoot, into another jar to grow more roots if I have time or straight to hareden/veg. but first, a dip into dutch master Replicator gel and into rockwool in a dome tray.

that ppm is a mystery, but it works. i dont want to know. i dont care. i'll support their biz and allow their function of profitability, because its the american way, and they did all the work. 

peace


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## WolfZen (Feb 13, 2012)

That problem is easily solvable through _in vivo _(not _in vitro_) chromosomal differentiation. Innate parthenogenisis-oriented plasticity methodology is a requirement though.


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## pharmacoping (Feb 13, 2012)

wow, you smart..thanks.. what does this mean though?

peace


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## nitro20% (Feb 13, 2012)

*deleting myself,,, 420 peace*


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## nitro20% (Feb 13, 2012)

*deleting myself,,, 420 peace*


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## pharmacoping (Feb 13, 2012)

nitro20% said:


> thats what i mean, those dont look any thing like plants to me,,,,were is the stem u cut to split...were is the roots? why is it green all the way through ur gel? so many questions..those are real progress pics..not just jared cuttings/bleached and clean...this is actually getting to the point were i might be SCARED....a culture with roots,,the one u might be getting ready to pot..pics.


cut on the line, transfer the callus into another shooting rooting compound for more divisions. this explant has small roots I cant seem to focus on, beneath the gel. she has lots of time to develop roots in another stronger rooting jar, or dipped in rooting compound and put in rockwool. all my cultures are green, for the love of proper nutrition, including sugar. when roots fully develop in vitro mine need to be lite tight for some reason, so no photos of those, but I shoot some hardening/vegging soon. maybe get a real digital microscope too!


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## pharmacoping (Feb 13, 2012)

ok, now I see what you've done. the sticks you're starting with need to be scraped, and trimmed to a nub the size of a marijuana seed. look at one of those new shoots on your stick..thats the part you want to start with from the host plant, a new meristem growth shoot, about mid way down the stalk, but grown away from it, like the tip of a branch used to clone. just the very tip of new growth, not the whole stick. near impossible to support this stick, or sterilize it. Currently you are making simple clones in a jar, expensive time consuming ones, but they are clones, and can be dipped into gel and rooted as such. host moms should be clean, dust and mold free before explants are taken, mornings only, just like clones.growth shoot TIPS only, will do what you're looking for. scrape the cambium layer because.....mitosis takes place in the shoot apex (meristem), root tip meristems, and the meristematic cambium layer of the stalk. we using that knowledge to hasten this procedure. kit worked awesome, but I was bought out. sorry.


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## pharmacoping (Feb 13, 2012)

your pics prove to you that your mix is supporting growth, let them grow and see what kind of growth they show, keep you r notes, yo umay like what you find. I always take alot of cuts from callus, hoping I get that freak someday.also, I found the gene that bioluminecses in plants and animals. its available to me, pretty expensive and so far the bids for my offer are too low.


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## WolfZen (Feb 13, 2012)

Where did you find it and where do you keep it currently?


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## pharmacoping (Feb 13, 2012)

supplier contact, recumbant genes-r-us type of outlet. I'll store the flock in a heated area, um, but I never said I had them yet dude.


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## pharmacoping (Feb 13, 2012)

as said earlier, anyone can culture bioluminesce genes, some are on raw fish, just waiting to be cultured. not sure if those can be used for anything other than study though, however, the one from the jellyfish has already been used on plants and several animals, fish, cats, rabbits, mice, etc.


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## nitro20% (Feb 13, 2012)

*deleting myself,,, 420 peace*


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## nitro20% (Feb 13, 2012)

*deleting myself,,, 420 peace*


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## WolfZen (Feb 13, 2012)

pharmacoping said:


> as said earlier, anyone can culture bioluminesce genes, some are on raw fish, just waiting to be cultured. not sure if those can be used for anything other than study though, however, the one from the jellyfish has already been used on plants and several animals, fish, cats, rabbits, mice, etc.


What are you going to do once you have cultured some bacteria that can luminesce? That doesn't really get you anywhere except for having some bacteria on an agar plate.


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## nitro20% (Feb 13, 2012)

*deleting myself,,, 420 peace*


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## pharmacoping (Feb 13, 2012)

nitro20% said:


> holly shit man thats a lot of words,,,,,i dont think it will go for long..plus i need to get something together now...so scrap all the outer layer off and start with a part out of the nod,,, not the nod itself?mmmmmm just a 1/4 inch of that sprout of the mother nod,,brand new little things..how do you push those WIMPY things into the gel? that ppm tech. store hasnt sent or processed my order yet..they have the order but nothing happened,,,i called but,,their closed...now,,,,I have a desperate feeling,,,that nadcc will not last much longer,,, i can see it wearing off now,,,i cant re-jar them again with out that ppm,,it makes no sense,,,till i think about it!!!! *ALL YOU WANNA BEESSSS LIKE ME DONT START WITHOUT [email protected]#$#@*


Yes, part of the node, the newest part of it. I dont push them in, I ease them down, and settle them, or make a little piece of paper like a bridge, two sides in, loop up and out, and then set the culture on top of that. I'm jsut starting to play with this suspended technique. evidently they sell jars made to empty and refill without disturbing the suspended culture. might be cool.


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## pharmacoping (Feb 13, 2012)

WolfZen said:


> What are you going to do once you have cultured some bacteria that can luminesce? That doesn't really get you anywhere except for having some bacteria on an agar plate.



yep, especially if you weren't paying attention in class, tissue culture 101. Then, we eat the bio luminescent material for the glow in the dark poop silly, what else ? nitro might have some ideas, but I'm too busy to play.


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## pharmacoping (Feb 13, 2012)

nitro20% said:


> HELL YEAH. AND THAT SOUNDS FUN TO..GROWING THING FROM CELLS IS COOL......BETTER THEN LOOKING OUT THE WINDOW ALL DAY.....
> 
> Ordered a set of 10in. stainless forceps friday at midnight from this aquamarine place 4.95..and they have the bend at the end,,,,,they are already here...WERES MY PPM.....
> 
> but,,,, thats cool though huh???that i got one of the hardest parts to start spouting,,,and im doing it ALL WRONG!!!TAKE THAT!!! one for the *GRASSHOPER!!!*


those forceps will make things easier for sure, and a good price too ! you may as well keep those cultured clones going, they are possibly viable, and definately study worthy.


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## nitro20% (Feb 13, 2012)

*deleting myself,,, 420 peace*


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## WolfZen (Feb 13, 2012)

pharmacoping said:


> yep, especially if you weren't paying attention in class, tissue culture 101. Then, we eat the bio luminescent material for the glow in the dark poop silly, what else ? nitro might have some ideas, but I'm too busy to play.


That's the most constructive thing you could do with it. There is nothing else you could do without a lot more knowledge and equipment than you have access to - so why are you asking me 'what else' like it's obvious? You're presenting yourself as someone doing something with genetics, so why not discuss something specific about how you plan on making use of lux genes taken from the bacteria. How are you going to isolate those genes? How are you going to copy them? PCR? What kind of PCR equipment do you have? How are you going to vector them into a plant if successful? How would you get the theoretical plant to then express LuxR?


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## nitro20% (Feb 13, 2012)

*deleting myself,,, 420 peace*


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## Bonkleesha (Feb 13, 2012)

update on mini rose. headed for chute splitting and rooting media friday.


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## pharmacoping (Feb 14, 2012)

WolfZen said:


> That's the most constructive thing you could do with it. There is nothing else you could do without a lot more knowledge and equipment than you have access to - so why are you asking me 'what else' like it's obvious? You're presenting yourself as someone doing something with genetics, so why not discuss something specific about how you plan on making use of lux genes taken from the bacteria. How are you going to isolate those genes? How are you going to copy them? PCR? What kind of PCR equipment do you have? How are you going to vector them into a plant if successful? How would you get the theoretical plant to then express LuxR?



Get back to your "computer monitor clone warmer" or your xbox,or whatever you kids do while dreaming of producing marijuana for the masses. 
are you culturing? please share some experiences, like wacky flowering times, or homemade additives, or something. nobody has time to cater to your negativity in this forum, until they get out of school, maybe at 3pm. everyone has read your posts thusfar, and must say, not so impressed by your information. . If you're so sure about these foundations of yours, why are you here taunting peolple with real life experiences. 
If you are genuinely interested in my bio lumi mj plant, PLACE A BID NOW, public or private. 
I will not give you these specific answers, as I might be here just to sell something, remember? 

fyi, everyday, when each of us clones,or cultures, we are genetically modifying plant material, albeit near impossible on an Xbox warming plate, or a desk light for illumination. 

PICS or GTFO wolfzen, really. we dont read words well, so if you feel the need to type more, just dont! and for the record, I Do think your mom is hot !


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## pharmacoping (Feb 14, 2012)

nitro20% said:


> Dust and mold free??? isnt that the reasoning for all the bleaching and alcoholing before jar???


right, but heres some food for thought. a dusty, dirty, moldy room/plant is much more likely to carry ???? who knows what to the jar. may not even report as mold? might just die, or mutate or? so its just a good measure.
I'm way less technical than many posters here. maybe an outside of the box look is why I'm able to actually perform this with success, and the smarty pants are afraid to even read the one book that puts an end to their fears, or even mix some agar ! 

if it was read, me, you, and bonkleesha would be the only ones here, until they assembled their lab !


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## pharmacoping (Feb 14, 2012)

Plants From Test Tubes-name of the most comprehensive do it yourself culture book ever written

One person here, thinking outside the box, without any admitted biology experience, fearless, and determined, in less than a month, has success in tissue cultured marijuana, with off the shelf supplies. Need I say more ?

Congratulations Nitro20%


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## WolfZen (Feb 14, 2012)

pharmacoping said:


> Get back to your "computer monitor clone warmer" or your xbox,or whatever you kids do while dreaming of producing marijuana for the masses.
> are you culturing? please share some experiences, like wacky flowering times, or homemade additives, or something. nobody has time to cater to your negativity in this forum, until they get out of school, maybe at 3pm. everyone has read your posts thusfar, and must say, not so impressed by your information. . If you're so sure about these foundations of yours, why are you here taunting peolple with real life experiences.
> If you are genuinely interested in my bio lumi mj plant, PLACE A BID NOW, public or private.
> I will not give you these specific answers, as I might be here just to sell something, remember?
> ...


Why are you so upset by any specific questions about what you are trying to do?


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## Bonkleesha (Feb 14, 2012)

WolfZen said:


> That's the most constructive thing you could do with it. There is nothing else you could do without a lot more knowledge and equipment than you have access to - so why are you asking me 'what else' like it's obvious? You're presenting yourself as someone doing something with genetics, so why not discuss something specific about how you plan on making use of lux genes taken from the bacteria. How are you going to isolate those genes? How are you going to copy them? PCR? What kind of PCR equipment do you have? How are you going to vector them into a plant if successful? How would you get the theoretical plant to then express LuxR?


not genetics. plant tissue culture.


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## WolfZen (Feb 14, 2012)

Bonkleesha said:


> not genetics. plant tissue culture.


What about plant tissue culture? What does that have to do with bioluminescing bacteria for a start?


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## pharmacoping (Feb 14, 2012)

life's short man, google for this information, it's like pedaling backwards with you.


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## WolfZen (Feb 14, 2012)

Google what information? You won't even state what it is you are trying to do. First it's something about discovering genes and bioluminescing bacteria, then it's nothing to do with genetics. Then what are you talking about? Connect the dots and make some sense by following through with your thoughts.

And again, is it really so hard for you to answer any questions without getting upset and being insulting please?


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## Bonkleesha (Feb 14, 2012)

he trolls it long time. so sad.


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## pharmacoping (Feb 14, 2012)

check these out;

http://www.makeitbreak.com/bioconst-demo/
http://www.sciencedirect.com/science/article/pii/S0168945202000444

Creeping bentgrass (_Agrostis palustris_ Huds.) is a cool season grass widely used on putting greens in golf courses. Transformation of creeping bentgrass has been conducted using microprojectile bombardment and protoplast electroporation. The objective of our study is to develop an alternative and more efficient approach in transforming the grass using _Agrobacterium_ (strain EHA 101). This technique was effective in transforming 40-day old calli derived from mature seeds cultured on MS medium supplemented with 2,4-D, kinetin, and sucrose. Dozens of transgenic plants have been produced from two independent transformed calli. Presence of functional green fluorescence protein (GFP) was detected in leaves, stems, and roots of transgenic seedlings. Four putative transgenic plants and two control plants were randomly chosen and analyzed by Southern blot analysis. Bands corresponding to the GFP gene were clearly shown in transgenic plants. These results indicated that _Agrobacterium_ transformation can successfully be applied to creeping bentgrass.

http://aob.oxfordjournals.org/content/85/6/831.full.pdf
http://www.biomedcentral.com/1471-2229/10/165


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## pharmacoping (Feb 14, 2012)

more than one callus= calli...nice


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## pharmacoping (Feb 14, 2012)

no, I wont connect the dots for you. you're not a tissue culturist, you're an antagonist. you were very insulting in the beginning of this thread. I dont want to answer your questions because I do not like your tone. 

peace


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## WolfZen (Feb 14, 2012)

How are you going to conduct microprojectile bombardment and/or protoplast electroporation?


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## pharmacoping (Feb 14, 2012)

imagine the impossibilities. like plants that lite up when they need something, maybe different colors for diff needs? christmas trees/no lights, lumi trichomes, wow, I'm so bummed it's only sci fi, in your closet


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## nitro20% (Feb 14, 2012)

*deleting myself,,, 420 peace*


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## nitro20% (Feb 14, 2012)

*deleting myself,,, 420 peace*


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## WolfZen (Feb 14, 2012)

Read it and still have questions. Foremost being how are you planning on going from culturing bioluminescing bacteria to having glowing plants (even if it's just for fun)?

There's no need to get upset at questions being asked.


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## nitro20% (Feb 14, 2012)

=*deleting myself,,, 420 peace*


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## WolfZen (Feb 14, 2012)

pharmacoping said:


> I have never tissue cultured anything, or grown marijuana, or infringed upon any patents. Everything I say is a lie and every photo I post has been photo shopped a million times even. just sayin'


Be that as it may, could you answer any question directly without getting upset, changing the subject, or otherwise avoiding answering?

How are you planning on going from culturing bioluminescing bacteria to having glowing plants (even if it's just for fun)?


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## nitro20% (Feb 14, 2012)

=*deleting myself,,, 420 peace*


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## WolfZen (Feb 14, 2012)

Ok, electricity usage is top secret if you say so. What protoplast material are you referring to? From the flourescing bacteria you've cultured? How did you extract, isolate, and copy it for use?


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## pharmacoping (Feb 14, 2012)

...millions of dollars are spent to create GMO Fusarium fungi that kill marijuana. A former employee of an "anti-drug" agency says biotech scientists are trying to genetically modify marijuana to create zero-THC cannabis that genetically terminates high-THC varieties. Growers in Hawaii and elsewhere tell spooky stories about helicopters dumping GMO insects on marijuana cropsseemingly indestructible voracious insects that ate the marijuana to the ground virtually overnight.
No, those people who reported that weren't hallucinating off some Puna Gold from the Big Island of Hawaii. Those GMO insects, and a lot of other scary stuff are real parts of the GMO brave new world. It's like science fiction come true... 
http://bigbudsmag.com/grow/article/discover-how-you-stop-gmos-harming-your-medical-marijuana-february-2012


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## WolfZen (Feb 14, 2012)

Yeah that's really interesting and all. But let's get back to talking about tissue culturing. You're right, it IS fun and I'm glad you have this thread to discuss it. So can you answer some of my questions?


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## WolfZen (Feb 14, 2012)

pharmacoping said:


> dude, I apologise for being elusive. This is delicate for me, and I simpy cannot give you the answer I know you're looking for. but it is in the book you said you already read, maybe read it again, or google. when you find it, you can post it, but I cannot.


No worries, can you show some of the results of your super secret processes with micrografting if you don't want to talk about the process itself?


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## Bonkleesha (Feb 14, 2012)

first i smoke 2 joints. then i take a nice plant... about 4 feet and take off all of the lateral maristems and leaves so that there is only one apical maristem shooting straight up. then i cut it out of the pot and whoop the shit out of this WolfZen character with it. 

rinse and repeat.


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## WolfZen (Feb 14, 2012)

Bonkleesha said:


> first i smoke 2 joints. then i take a nice plant... about 4 feet and take off all of the lateral maristems and leaves so that there is only one apical maristem shooting straight up. then i cut it out of the pot and whoop the shit out of this WolfZen character with it.
> 
> rinse and repeat.


No need to get upset and try to be rude. Why do you keep switching to different accounts?


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## Bonkleesha (Feb 14, 2012)

???????????


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## alphawolf.hack (Feb 14, 2012)

man forget about these guys. im in the sit. i want to learn to micro-culture but these guys aren't sharing shit. best to read as much about as you can and learn about it another way..... so far im still in the process of building hood with gloves and filters and and positive pressure and all that. first you got to learn the science part then build a small lab that can preform more functions than micro-culture than start experimenting getting the chemicals the you can experimentally micro-culture

this is the process im going about to get there i cant see any other way to do because you cant just jump right into it. even if you know all the ingredients to add to a culture it wont survive if the conditions are not correct. so i guess my idea is if you can build the environments and make the chemicals then you can culture.


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## alphawolf.hack (Feb 14, 2012)

that bug shit is some scarey shit and of course it absolutely true. on the other hand there are better organisms to design bugs to eat.


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## Bonkleesha (Feb 14, 2012)

alphawolf.hack said:


> man forget about these guys. im in the sit. i want to learn to micro-culture but these guys aren't sharing shit. best to read as much about as you can and learn about it another way..... so far im still in the process of building hood with gloves and filters and and positive pressure and all that. first you got to learn the science part then build a small lab that can preform more functions than micro-culture than start experimenting getting the chemicals the you can experimentally micro-culture
> 
> this is the process im going about to get there i cant see any other way to do because you cant just jump right into it. even if you know all the ingredients to add to a culture it wont survive if the conditions are not correct. so i guess my idea is if you can build the environments and make the chemicals then you can culture.


although the gloves arent a bad idea, we have been doing our microprop. in the science building under HEPA hoods.... no enclosed gloves or whatnot. to each their own!

as far as sharing microprop info, its all in this thread. go pick it out. i even uploaded lab manuals from school, dude. have at it.


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## nitro20% (Feb 14, 2012)

*deleting myself,,, 420 peace*


----------



## nitro20% (Feb 14, 2012)

*deleting myself,,, 420 peace*


----------



## nitro20% (Feb 14, 2012)

*deleting myself,,, 420 peace*


----------



## alphawolf.hack (Feb 14, 2012)

im re-reading there is some useful info for sure... but i still have alot of ?'s i will just go at this by myself for now .i have alot equipment to purchase b4 i make any real statements around here. i wasnt trying to be a jerk and say to u guys wont help. but you guys are on another level with this shit its pointless for him to come in here without any prior knowledge and expect you guys to give some real answers. because those answers probably run a page or so long and are somewhere else already written for him. im the same way with electronics i know so much shit it would make your head spin. its like asking how do i use a capacitor? there is a long answer to that along with alot of variables and possibilities.


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## alphawolf.hack (Feb 14, 2012)

i agree once i figure this out i probably wont be sharing


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## nitro20% (Feb 14, 2012)

DUDE,go buy a kit,,, im tellin ,,,, that if you dont,,,,, you will buy a half of one before you could even split something...I WOKE UP 3 WEEKS AGO AND DECIDED TO TC,, DONT ASK WHY I DONT KNOW....DIDNT KNOW SHIT BEFORE THIS!!!

GO BY A KIT AND FOLLOW DIRECTIONS,,YOU ALREADY KNOW TO BE CLEAN,, YOU ARE HALF WAY TO YOUR FIRST AGAR FAILURE... SERIOUSLY.....I THINK I GOT LUCKY...BUT KNOW IM INTO IT,,,BUT IM INTO IT FOR 400+ AND I HAD 500$ WORTH OFF STUFF TO START WITH.. SO TAKE THE INFO THATS HERE AND DO WHAT I AM NOT,, SHUT UP AND LISTEN,,,I/WE WILL DO THE REST....UNLESS YOU HAVE A PAGE LIKE MY CHINA MEN PAGE.....I SAY THIS WITH A HUGE CLOUD,,AND A BIG BONG SMILE!!!


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## nitro20% (Feb 14, 2012)

*deleting myself,,, 420 peace*


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## alphawolf.hack (Feb 15, 2012)

you are right. im about to buy a kit i have done the math the costs to build your own are about the same as the kit kinda like aeroponics. but man its so off-putting knowing i cant get some of the chemicals/genes more easily.(yeah i stay up doing math and shit all night i spend most of my time designing systems i will never use or destroying old electronics for components)


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## PetFlora (Feb 15, 2012)

pharmacoping said:


> Get back to your "computer monitor clone warmer" or your xbox,or whatever you kids do while dreaming of producing marijuana for the masses.
> are you culturing? please share some experiences, like wacky flowering times, or homemade additives, or something. nobody has time to cater to your negativity in this forum, until they get out of school, maybe at 3pm. everyone has read your posts thusfar, and must say, not so impressed by your information. . If you're so sure about these foundations of yours, why are you here taunting peolple with real life experiences.
> If you are genuinely interested in my bio lumi mj plant, PLACE A BID NOW, public or private.
> I will not give you these specific answers, as I might be here just to sell something, remember?
> ...


_*OMG LMFAO!*_ But it points out why I do journals, where I can delete idiots, but then I would not have seen your awesome reply


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## pharmacoping (Feb 15, 2012)

PetFlora said:


> _*OMG LMFAO!*_ But it points out why I do journals, where I can delete idiots, but then I would not have seen your awesome reply



wish I would have started a journal instead, but am a forum newby. wonder if it can be moved to a journal?? 
thanks for the yell

peace


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## Bonkleesha (Feb 15, 2012)

but dude. give me a roadmap to making media....

im sure its in here... in the pages i missed.


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## alphawolf.hack (Feb 15, 2012)

okay im ordering the classroom kit with dvd for African violets (im gonna start there stupid to jump in with out an a good example) then im gonna fabricate my own media from what i learn. so in this process of making you plants glow it a cultured virus correct? could it where off as a plant grows? or has the virus literally changed the genetics of the plant? and in the second scenario how would one go about designing a virus to do this, or is there a virus that does this already?


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## WolfZen (Feb 15, 2012)

Making a plant glow requires _genetic_ engineering methods, knowledge and equipment. _Tissue culturing_ is not genetic engineering. Why do you keep implying otherwise?


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## pharmacoping (Feb 15, 2012)

WolfZen said:


> Making a plant glow requires _genetic_ engineering methods, knowledge and equipment. _Tissue culturing_ is not genetic engineering. Why do you keep implying otherwise?


ok, to settle this for you;
humans have been tampering with genetics for centuries. selective harvesting,hand pollinating or cross pollinating, or inducing mutants, with mutated, genes, that may have been engineered by a plethora of bacteria/virus at many different stages in growth, engineered by combining its own dna, with the hosts' dna, creating recombinant dna. this is done with great success in my lab now, and many other home labs. yes, more knowledge than you are willing to accept, or posess, is necessary to perform these small miracles. 

So, you see, just because we may not have created(engineered) genetic material, does not mean that we are not intentionally culturing it, harnessing it, selecting it, breeding it, and combining it with other dna, becoming the final engineer. Tissue culture is a tool for genetic engineering. genetic engineering takes place in tissue culture. genes may be altered during your sloppy clone cutting/warming practices. 

I know this is rhetoric for you, since you've already read the book I've suggested, perhaps a reread is in order. This information is near the beginning, before the words get bigger.


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## alphawolf.hack (Feb 15, 2012)

okay got ya. about the meadia im not sure i understand. the meadia is agar with nutes and hormones added right? so i under stand how to get NAA and auxins but i have yet to see a conventional cytokin hormone (i have heard of coconuts milk but thats all i have found so far dont plan on using it mind you.) ii i understand what your saying is i will use agar as the media and my mixture of hormones and nutes will be what im fabricating.


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## nitro20% (Feb 15, 2012)

*deleting myself,,, 420 peace*


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## alphawolf.hack (Feb 15, 2012)

all right well most of my preliminary ?'s have been answered im on-board and will get back with you guys when i get some stuff up and running. super helpful too. my bad again for kinda raggin on you. im just gonna sit back a pay attention to the info from here on out or at least till im in the middle of my process and go oh fuck.


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## nitro20% (Feb 15, 2012)

*deleting myself,,, 420 peace*


----------



## nitro20% (Feb 15, 2012)

*deleting myself,,, 420 peace*


----------



## nitro20% (Feb 15, 2012)

*deleting myself,,, 420 peace*


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## alphawolf.hack (Feb 15, 2012)

that is one hilarious statement. if true i will be stopping buy a local store to try and find my diamond.


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## TaoWolf (Feb 15, 2012)

pharmacoping said:


> ok, to settle this for you;
> humans have been tampering with genetics for centuries. selective harvesting,hand pollinating or cross pollinating, or inducing mutants, with mutated, genes, that may have been engineered by a plethora of bacteria/virus at many different stages in growth, engineered by combining its own dna, with the hosts' dna, creating recombinant dna. this is done with great success in my lab now, and many other home labs. yes, more knowledge than you are willing to accept, or posess, is necessary to perform these small miracles.
> 
> So, you see, just because we may not have created(engineered) genetic material, does not mean that we are not intentionally culturing it, harnessing it, selecting it, breeding it, and combining it with other dna, becoming the final engineer. Tissue culture is a tool for genetic engineering. genetic engineering takes place in tissue culture. genes may be altered during your sloppy clone cutting/warming practices.
> ...


You haven't had any success that you've shown nor do you have enough knowledge to discuss your claims without getting upset by direct questions. Again, tissue culturing doesn't alter DNA or manipulate DNA no matter how obtuse you want to get about it. Tissue culturing is simply culturing tissue in a media. 

Trying to compare tissue culturing to genetic manipulation/engineering is like equating knowing how to fill a car's gas tank to being an automotive engineer. What's the point in misleading people like that unless you are selling gasoline?


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## nitro20% (Feb 15, 2012)

*deleting myself,,, 420 peace*


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## nitro20% (Feb 15, 2012)

*deleting myself,,, 420 peace*


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## nitro20% (Feb 15, 2012)

*deleting myself,,, 420 peace*


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## alphawolf.hack (Feb 15, 2012)

why didnt you consult a dictionary b4 you came to this thread



Definition of GENETIC ENGINEERING merrium-webster

: the group of applied techniques of genetics and biotechnology used to cut up and join together genetic material and especially DNA from one or more species of organism and to introduce the result into an organism in order to change one or more of its characteristics
See genetic engineering defined for English-language learners »
See genetic engineering defined for kids »

so according to this definition grafting and tissue culture are genetic engineering.


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## nitro20% (Feb 15, 2012)

*deleting myself,,, 420 peace*


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## alphawolf.hack (Feb 15, 2012)

im actually changing my account. its devil now


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## nitro20% (Feb 15, 2012)

*deleting myself,,, 420 peace*


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## nitro20% (Feb 15, 2012)

*deleting myself,,, 420 peace*


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## alphawolf.hack (Feb 15, 2012)

you are taking specific DNA and culturing it thus genetic engineering. there is no way this cutting would survive with your genetic intervention of adding hormones to alter DNA of cell to allow them propagate and create roots and shoots.


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## WolfZen (Feb 15, 2012)

This is a forum, wild claims will probably be questioned, whether for fun or not. That's the way forums are.

You could always sell your TC kits legitimately and pay for advertising if you don't want interaction with forum members?


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## nitro20% (Feb 15, 2012)

*deleting myself,,, 420 peace*


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## nitro20% (Feb 15, 2012)

*deleting myself,,, 420 peace*


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## WolfZen (Feb 15, 2012)

alphawolf.hack said:


> you are taking specific DNA and culturing it thus genetic engineering. there is no way this cutting would survive with your genetic intervention of adding hormones to alter DNA of cell to allow them propagate and create roots and shoots.


Would have been a good point except that you aren't altering or manipulating basic cellular DNA by using hormones. Even by layman's terms using hormones is not 'genetic engineering'.


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## nitro20% (Feb 15, 2012)

*deleting myself,,, 420 peace*


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## nitro20% (Feb 15, 2012)

*deleting myself,,, 420 peace*


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## WolfZen (Feb 15, 2012)

nitro20% said:


> That is how much u know,,think this is a kit thing....uuuuuuuuuuubbbbbbbbmmmmmmmbtbtbtbtbtbtbtb..
> Just growin plants son!!!!





pharmacoping said:


> *I do this very well, and assemble kits for sale too /prepared solutions/pre filled sterile containers/proper nutrients, and even genetic specimens, for observation purposes. Some samples include Thyme, Rosemary, Tahoe OG Kush,PlushBerry,Kandy Kush, and hundreds of other non plant dna samples. The study of these samples will can prepare you for the art of tissue cultivation/cloning. From a single spec thousands of master programmed clones can be taken monthly ! Imagine no more mother plants or clones using your plant spaces? How important is that to you? These are stored/divided as masses of tissue or unusable roots. Only in the last few days do they become a "plant". You can easily have roses with thc, or mj without leaves, or even glow in the dark, really, I've seen it ! It's not the future, it's now ! Patents are already being granted for different mj types grown this way. suicide genes,inability to clone/seed..are some of the programing in these super corpopharma plants. Instructions are available online, but the right mixes are almost impossible to find and invaluable to growing your own rare herbs successfully, and storing their dna forever. With these supplies you'll learn how to master this fast because the work is done !! Plan on spending a couple hundred bucks or less, depending on the equipment you have at home...jars/mixer/pressure cooker,etc.
> 
> I'll answer some q's here, but private message me for instructions for above.*




Want kind of rice car do you have by the way?


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## nitro20% (Feb 15, 2012)

*deleting myself* 420peace


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## nitro20% (Feb 15, 2012)

wolfzen said:


> [/font][/b]
> 
> want kind of rice car do you have by the way?



30.5cc hpi usa made fully moded from dave's motors,,,3 months old.. 2700.00...90mph 2 speed all the goodies..fuk off what u got bitch!


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## pharmacoping (Feb 16, 2012)

nitro20% said:


> *deleting myself* 420peace


Nitro ?? I know you're in there....come out and play. dont get mad, get even !


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## PetFlora (Feb 16, 2012)

FYI: A few decades back I worked a company named AccuDerm. They were 'famous' within the Dermatology for their version of agar colloid. I believe they are located in Fort Lauderdale, but not sure you can buy without being a dr.


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## pharmacoping (Feb 16, 2012)

TaoWolf said:


> You haven't had any success that you've shown nor do you have enough knowledge to discuss your claims without getting upset by direct questions. Again, tissue culturing doesn't alter DNA or manipulate DNA no matter how obtuse you want to get about it. Tissue culturing is simply culturing tissue in a media.
> 
> Trying to compare tissue culturing to genetic manipulation/engineering is like equating knowing how to fill a car's gas tank to being an automotive engineer. What's the point in misleading people like that unless you are selling gasoline?



ASSHOLES LISTEN UP !

John Kleyn-chapter one, tissue culture overview; phd cornell university, plants from test tubes, again.. this is why q's are not answered. first you must understand the fundamentals of culture, and it's practices, then a grasp of it's reality may share a glimpse

"Tissue culture is a clean and rapid way to grow material for identifying and -manipulating- genes, or to -tranfer individual characteristics from one plant to another"

you are wrong sir.


radiation causes genetic manipulation, uv light causes it to, electricity causes it also, so does a magnetic field....all of which may or may not be happening within our home labs. 
if a plant is infected, it's genetics are changed, even if slightly, but if you purposely infect a plant, changing is dna, or mixing other foreign dna, you have become the genetic engineer of that creation, and furthermore, you would be hard pressed to locate a botanical genetic engineer that does not perform most of his lab duties in tissue culture.

dammit all, get over it !


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## Bonkleesha (Feb 16, 2012)

HEY, DUDE! i got my test tube from the microprop lab (the chuting one.. we transferred our new chutes into the rooting media). anyways theres like 2 inches of media in it. if i am careful and sterile, and i take about half an inch out where my miniature rose was and use it for cannabis?


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## alphawolf.hack (Feb 17, 2012)

whats up pharm. hey man okay so i just finished book 3 on this subject got a couple a ?'s for you... do you sterilize the media nute hormone ect.? or just the container? isn't all that stuff supposed to com prepacked and sterilized or is this just a safety measure? also once you reach the stage where plantlet is cultured in hormone free media how long is shelf life? as far as degrading genetics does this only apply to callus cultured cells? damn i learned alot.

bankleesha you werent around he said togo4it.


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## pharmacoping (Feb 17, 2012)

PetFlora said:


> FYI: A few decades back I worked a company named AccuDerm. They were 'famous' within the Dermatology for their version of agar colloid. I believe they are located in Fort Lauderdale, but not sure you can buy without being a dr.


They have this bad ass seed encapsulation system, for our artificial seeds. when we make these seeds, they cannot be stored except in agar, and AccuDerm solved that with a wrapper ! I use colloidal agar also, and am damn tempted to culture human cells, but I'm scared. I've done the glo on the animals found on on raw shrimp and its pretty wild bacteria. played with the glo in fireflies for a long time in vitro. I used colloidal agar but got huge results with a beef brine. bioluminescence was occupied my thoughts since childhood. it was the very first reason I cultured over 30 yrs ago, never stopped.

thanks for sharing this, it shed some light on my materials..haha, literally. heres a shot for you, recognize the agar ??


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## pharmacoping (Feb 17, 2012)

alphawolf.hack said:


> whats up pharm. hey man okay so i just finished book 3 on this subject got a couple a ?'s for you... do you sterilize the media nute hormone ect.? or just the container? isn't all that stuff supposed to com prepacked and sterilized or is this just a safety measure? also once you reach the stage where plantlet is cultured in hormone free media how long is shelf life? as far as degrading genetics does this only apply to callus cultured cells? damn i learned alot.
> 
> bankleesha you werent around he said togo4it.


sterilize the whole mix. dont tighten the lids on the containers in the pressure cooker, until it cools naturally. I dont culture in hormone free media until in the vegging room. callus production division/rooting chuting/hardening off, then to veg.


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## pharmacoping (Feb 17, 2012)

Bonkleesha said:


> HEY, DUDE! i got my test tube from the microprop lab (the chuting one.. we transferred our new chutes into the rooting media). anyways theres like 2 inches of media in it. if i am careful and sterile, and i take about half an inch out where my miniature rose was and use it for cannabis?



Do It ! Do it now !!!! for real, should be cool.


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## Bonkleesha (Feb 18, 2012)

pharmacoping said:


> Do It ! Do it now !!!! for real, should be cool.


tomorrow. i got everything ready to go. so i can just use canning stuff (fruit fresh) as the antioxidant? what ppm?

better to use a new or old mother???


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## pharmacoping (Feb 19, 2012)

old mother/new mother, either one....never heard of fruit fresh in tissue culture??? nutrition=ppm=between 150-200ppm. plus some sugar.


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## pharmacoping (Feb 19, 2012)

El Ray,

Your a couple years late on the pineberry, it's kind of in the past. 
I dont own a microwave, no comments on using it.

If shortcuts are on your mind at this stage, you're in for a ton of moldy jars, and un usable plant material. Follow the directions exactly, to hope for similar results described in the kit, ohterwise its all experimentation, which is cool too !!


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## Bonkleesha (Feb 19, 2012)

looks like this is put off while i find antioxidant.


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## Bonkleesha (Feb 19, 2012)

no, i decided to try without. as of right now, my explants are sterilixing


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## Bonkleesha (Feb 19, 2012)

big buddha cheese is loaded up. now we wait.


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## el rey (Feb 19, 2012)

is the pineberry strawberry plant available here in US ? I could not find it anywhere.

No shortcuts are in mind, just have a microwave but don't have a pressure cooker. But I will go buy one no prob. Can't wait to get started. Is there a special media mix for cannabis? Like more of one thing than another?



pharmacoping said:


> El Ray,
> 
> Your a couple years late on the pineberry, it's kind of in the past.
> I dont own a microwave, no comments on using it.
> ...


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## pharmacoping (Mar 16, 2012)

>>>>>poof<<<<<<


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## pharmacoping (Mar 16, 2012)

http://www.ox.ac.uk/media/news_stories/2011/110729.html


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## el rey (Mar 16, 2012)

pharm.... what is this thing doing ? Just seems to be bubbling


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## pharmacoping (Mar 17, 2012)

thats pretty strange. gassing, dont ever sniff cultures, no matter how tempting. these molds are really nasty. my first guess would be some sort of strange mold/fungus/pathogen/bacteria taking hold. I'd bleach it and dump it. I saw live animal/human tissue do this before, maybe an eye lash,skin flake,not sure? toxic though..


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## el rey (Mar 17, 2012)

ok will do, it was just a pretty small piece of a stem.


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## pharmacoping (Mar 18, 2012)

any visible cloud/mold


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## el rey (Mar 26, 2012)

pharm i did it! Bammmmm....i have roots starting on a leaf! They were in my green formula i'll post a pic when i can focus on the roots iphopne cant shoot past jar.


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## pharmacoping (Mar 26, 2012)

thats tits !! the most difficult part of a plant for me to culture is the leaf. I definately want to watch that one !!
then try that same formula on all the other parts too, and compare ......man, life is so short


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## el rey (Mar 26, 2012)

Kind of hard to see but the white things are roots poking out and from the looks of the other stuff it looks like a callus is forming? Or is it mold lol. The white things look like spikes coming out (roots) and they even have tiny hairs on some of them. Whats everyones thoughts on this?


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## pharmacoping (Mar 27, 2012)

this looks like rooting callus ,aterial, with some mold trying hard to win. If it is mold, it can be washed again and transferred. older samples have abetter chance of surviving the mold


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## el rey (Mar 27, 2012)

awesome! so do just pull it out of the media and rinse in 10% bleach like i did before? Or go weaker? Will it kill my callus? Could i just take a small piece of the callus and transfer it to new medium to start one mold free and have more success to get growing? What would you do now? I have another strain that has a callus as well.


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## pharmacoping (Mar 27, 2012)

take the cleanest piece without mold, quickly rinse in 2% bleach, then sterile water, then transfer. take several excisions just in case. then wash the original when you're sure some of the others will survive.


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## el rey (Mar 27, 2012)

which part is the mold lol? can i just take a small piece of callus and transfer it?


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## pharmacoping (Mar 27, 2012)

the moldy part is the one with the callus attached silly. The callus is the one with the mold attached

only you can tell


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## el rey (Mar 27, 2012)

ok well one part looks like a callus like a clump of goo growing...then at the tips is looks like white roots are growing from it. so just work with the callus i take it?


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## el rey (Mar 27, 2012)

also how would you take a clump of callus just with some tweezers and dip in bleach ?


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## pharmacoping (Mar 27, 2012)

dip scalpel in alcohol, flame off, then cut that callus, maybe use tweezers(flamed) to help and you wont have to transfer the original piece out of the jar. hold your breath, do it fast.

try to get a root included when cutting, but watch that mold goo stuff.


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## el rey (Mar 27, 2012)

ok i'll see what i can do. I'll post a pic once i have it out of the tube to get a good pic for you and others to see hopefully help someone down the line.


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## el rey (Mar 27, 2012)

ok so i put three seperate parts of the leaf into new media. lets see how that goes now. I dipped in 2% bleach then sterile water then transferred next to a candle while holding breath....best i could do for now


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## pharmacoping (Mar 28, 2012)

definately some roots there !! 
when you can, ditch the candle, and get a alcohol burner(cheap @ amazon)


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## el rey (Apr 3, 2012)

Pharm, So now i have cells growing and in my blue media that i made for shoots i now have shoots. What do I do next? In my red agar i made for roots it seems its making a callus then making roots. How would i go about taking a small cutting that has been making shoots and getting some roots on it?


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## pharmacoping (Apr 4, 2012)

well, first you have to decide what your goals are. If it's just new fresh cuttings, then scalpel a chunk off of any material that is growing roots/sprouts. Put the rooting calli into the sprouting mix, and the sprouts in the rooting mix if their slow. Then for a cutting, take a piece of callus with roots(at least) and root them like you would a clone, either in rockwool, or into another tube without hormones, but with sugar.


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## el rey (Apr 4, 2012)

thanks...i'm going to post some pics to give you a better idea of where i am....thanks for your time pharm.


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## pharmacoping (Apr 4, 2012)

can even use a commercial rooting compound at this stage .
no thanks necessary, my jollies are gotten watching your success ....your first time out !!


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## el rey (Apr 4, 2012)

ok here is 1 ...chem dog 91...i have a callus forming at the bottom like around the trunk. I'd like to get to the point where when i need 100 cuttings i can make them that is my goal. So do you recommend just making a lot of callus's? this one is in the shooting formula. But i'm kind of confused...my thinking when i did this was the roots would grow in the agar just like how you would take a cutting and it would form roots. But seems even my rooting formula it makes a callus then roots off of that..is that correct? if so then i would take that rooting callus and place it on top of the agar but in my shoot formula to make a shoot? Once that is done i then take the whole rooting/shooting callus and plant like normal? after letting it aclimate to the climate?


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## pharmacoping (Apr 5, 2012)

you are exactly correct. your root mix is a little hot, evedenced by the curling leaves, but thats ok. the rest of your plan is right on.
At this stage callus material would be your goal, however, experimenting with a little less hormones would be helpful. Your mix will work for sure, 
but may have some mixhaps. sometimes I expect roots and get shoots instead, its ok, both can happen in a rockwool cube too.

great pic !!


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## el rey (Apr 6, 2012)

what should i back off of to make it not so hot? ms or the hormones?


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## pharmacoping (Apr 8, 2012)

el rey said:


> what should i back off of to make it not so hot? ms or the hormones?


reducing the hormone levels by half never hurt me, when I saw symptoms. If your salts are at or under 150ppm they should be fine, if not, adjusting them a little will expedite your growth, and lessen the chance for a freaky failure


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## readysetgro (May 13, 2012)

Intresting thread, I have been following it from the beginning all morning. Are there any more picture updates to those that have been posting? I will be purchasing my materials to build a lab this wknd, & hopefully be proceeding to tc next week or so thanks to al the info ive found here! Never has it been made as plain for me. Anywho I plan to tc some big buddha blue cheese & some th seeds s.a.g.e. I also have some stalks from some local strawberry cough that a friend saved for me. Im most excited about these bc the stems are a year or so old & no longer green, so i cant wait to experiment on bringing them back to life  Great thread, great info i will have plenty of ?s & lots of pics coming up! Thanks!!


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## jujubee (May 14, 2012)

I think you will need live plant matter to tc.

I haven't read the whole thread yet. I've had some tc gear for a little while, but haven't got around to trying it out. I'm still not sure on the hormones. I have NNA, IBA, BAP and Kinetin. I also have agar, ms and ppm. Is there something else I should have?


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## readysetgro (May 16, 2012)

The strawberry cough stems are rehydrating now. Week 2. Theyre in an experimental setup Ive got going, pretty much a dwc bucket. One of them is visibly starting to show green, most of the others just "feel" like theyre going from sticklike to live plant stem like but arent showing any visual signs. I read in a Skunk a yr or so ago how to tc & they mentioned taking specimens from dead plants. Either way i never planned to just try to tc a peice that i hadnt rehydrated but i think i will now for control purposes. Its all experimental though


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## readysetgro (May 16, 2012)

Read the whole thing its pretty informative, nutrients, recipes, etc, these guys rock.


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## readysetgro (May 16, 2012)

also im experimenting with agar and gelatine as well. Before i got my agar i found classroom recipes for gelatine & started a few plates & have them upside down in the fridge. Well see how it goes!!


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## dvs1038 (May 19, 2012)

Cloning MJ tissue samples, well we all saw the rat with the human ear growing on his back, that would be really awesome if next u see a rat running around with a MJ plan growing off its back!!! Lol ok well it sounded funny when I said it to myself.


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## ADR3NALINE3x (May 21, 2012)

should these TC be burped for fresh air?


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## pharmacoping (May 24, 2012)

ADR3NALINE3x said:


> should these TC be burped for fresh air?


Nope, but the lids should be the type that breathe a bit, or I use micron filter patches over a hole drilled in a babayfood jar lid. the only time you open the jar is to transfer your material, I even syringe inject through the micro proe filter down into the agar beneath the calli, to avoid a couple transfers. 

peace


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## pharmacoping (May 24, 2012)

dvs1038 said:


> Cloning MJ tissue samples, well we all saw the rat with the human ear growing on his back, that would be really awesome if next u see a rat running around with a MJ plan growing off its back!!! Lol ok well it sounded funny when I said it to myself.



still funny man !!

I've seen human replacement tissue growing on plants in the lab, and even plants producing artificial poisons. morgellons disease should be of interest to anyone playing with growth hormones.

I no longer use hormones in my process. Its much slower, but works for me. Cutting specific areas of a plant, at different maturity stages, or culturing anthers is much safer, and challenging. I no longer own hormones, except my trusty Replicator cloning gel.

peace


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## polyarcturus (May 24, 2012)

hey pharm been a while just wanted to let you know my progress. i have pretty much given up on a DIY setting for my tissue culture setup and decided to save the cash to do a real set up in the future have a lot of materials collected(hepa filter/fan, jars pressure cooker alchhol burner, forceps, stainless steel table small t5 light set up tyvek filters, and more) but still have a way to go(still have to order my hormones and such). do you know any places to get glass petri dishs cheap? or do you use canning jars only?

long time no see too.


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## pharmacoping (May 24, 2012)

readysetgro said:


> Read the whole thing its pretty informative, nutrients, recipes, etc, these guys rock.



Dude, thanks for the so kind words. I took a new direction after years of this. funny I started in the seventies as a kid with nothing but gelatin, crushed vitamins, sugar and did have some success. when the internet came I was awesome with hormones.....now...back to the cave man, no artificial anything here !!! no more hormones either


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## pharmacoping (May 24, 2012)

polyarcturus said:


> hey pharm been a while just wanted to let you know my progress. i have pretty much given up on a DIY setting for my tissue culture setup and decided to save the cash to do a real set up in the future have a lot of materials collected(hepa filter/fan, jars pressure cooker alchhol burner, forceps, stainless steel table small t5 light set up tyvek filters, and more) but still have a way to go(still have to order my hormones and such). do you know any places to get glass petri dishs cheap? or do you use canning jars only?
> 
> long time no see too.


good to see you !!

I use babay food jars(bbj) and their lids. screw on loose and tape with a breathable tape, or drill the lid and stick a micropore patch on it. I get mine from coffee bags and prolly have 1000 in a can now ! soak in alcohol for a second and use them with hot glue. I dont use hormones anymore, but it sounds like you have everything you need to play.

peace


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## pharmacoping (May 24, 2012)

readysetgro said:


> Intresting thread, I have been following it from the beginning all morning. Are there any more picture updates to those that have been posting? I will be purchasing my materials to build a lab this wknd, & hopefully be proceeding to tc next week or so thanks to al the info ive found here! Never has it been made as plain for me. Anywho I plan to tc some big buddha blue cheese & some th seeds s.a.g.e. I also have some stalks from some local strawberry cough that a friend saved for me. Im most excited about these bc the stems are a year or so old & no longer green, so i cant wait to experiment on bringing them back to life  Great thread, great info i will have plenty of ?s & lots of pics coming up! Thanks!!


I've been fascinated with this concept before Jurassic Park. I so wanted to bring life to the dead !. I can do it, sort of, but not from several year old material, although that is certainly able to be done, but I need a half million dollars more to do it. 

I've done it with a week old mj with success, and a little magic. Remember that when you cut a sample to culture, you soak it in alcohol, and bleach, and it lives on.......everyday after the plant is cut, the chances of my successful culture is diminished


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## readysetgro (May 24, 2012)

Im as femme as my clones----- (girl) Newho so i soaked in a bleach solution then peroxide cuz i had no alcohol & im just all for the experiment, & i took cuttings & put them in a homemade bubbler. Some of the stems changed by color & feeling, a few seemed hollow inside & didnt do anything. Ive taken 6 cultures from 3 stems that are under a 75 watt?? Im not sure the wattage but its a long tube fluor with some kinda grow bulb (from home depot). I see the calus & a little mound of "stuff" forming under the cutting, I see growth in size, no new leaves, & it looks like a gas may be in the jars?...Is this normal? Also I have them in these little 4oz ball canning jars, but it looks like theyll grow out of them in a few weeks, do i transplant them to new jars or how do i know when theyre ready to be hardened & put into a medium? Hope I didnt lose u lol


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## pharmacoping (May 24, 2012)

readysetgro said:


> Im as femme as my clones----- (girl) Newho so i soaked in a bleach solution then peroxide cuz i had no alcohol & im just all for the experiment, & i took cuttings & put them in a homemade bubbler. Some of the stems changed by color & feeling, a few seemed hollow inside & didnt do anything. Ive taken 6 cultures from 3 stems that are under a 75 watt?? Im not sure the wattage but its a long tube fluor with some kinda grow bulb (from home depot). I see the calus & a little mound of "stuff" forming under the cutting, I see growth in size, no new leaves, & it looks like a gas may be in the jars?...Is this normal? Also I have them in these little 4oz ball canning jars, but it looks like theyll grow out of them in a few weeks, do i transplant them to new jars or how do i know when theyre ready to be hardened & put into a medium? Hope I didnt lose u lol


If you're just storing/growing clones no need to harden. Not sure bout the gas, guessing its the beginning of a new life form though. My explants are smaller than a pencil eraser and widdled down to a thread. These will grow into undifferentiated callus very quickly, then I cut pieces off of this and root/chute them, then to the veg room, keeping the original culture(s) as mother plants to cut from. 
Your flouro will work great. I transplant when changes stop rapidly occuring inside the vessel. i store rooting callus for months in ajar, and even cut samples from these to immediately sprout. rooted mini bonzai sprouts are in a jar for a week, then planted in wet ph'd rockwool in a humidity dome for 2 days, then to the veg room.

I love newho's, welcome, and lots of pics are expected form you...


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## pharmacoping (May 24, 2012)

the culture sprouted, without roots on callus. I cut this off, continue the ulture of the callus, and place the chute into rockwoo with store bought gel. In a week its bonzai structure is evident, this one took a bit longer because it had no roots to start in rockwool yet, so after two weeks shes ready for the veg room and a real live mj bonzai is born.


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## Saldaw (May 24, 2012)

just a blazed question but would it be possible to clone a bud and grow it in a tissue culture?


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## pharmacoping (May 24, 2012)

Saldaw said:


> just a blazed question but would it be possible to clone a bud and grow it in a tissue culture?


thats exactly what we're doing. any part of the plant, leafs, stalk, bud, anthers, stamen, pistil, root can be cloned, because the plant is totipotent, it has all the genetic make up necessary to produce. Of course you you could have a stick with a bud on it, but why? may as well grow out an auto flowering strain...which also can be cloned in vitro and kept like a mother plant.


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## Saldaw (May 24, 2012)

pharmacoping said:


> thats exactly what we're doing. any part of the plant, leafs, stalk, bud, anthers, stamen, pistil, root can be cloned, because the plant is totipotent, it has all the genetic make up necessary to produce. Of course you you could have a stick with a bud on it, but why? may as well grow out an auto flowering strain...which also can be cloned in vitro and kept like a mother plant.


so you can clone autos in vitro? how does that work? + rep btw


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## polyarcturus (May 24, 2012)

umm where did you get your baby food jars?

and your saying i should just forgat about hormones and learn the old fashioned way still because MJ is totipotent?


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## pharmacoping (May 25, 2012)

polyarcturus said:


> umm where did you get your baby food jars?
> 
> and your saying i should just forgat about hormones and learn the old fashioned way still because MJ is totipotent?


I get my (gerber only,lids) from the grocery store.

without hormones a normal culture experience is common, but with hormones/chems, anything is possible. I despise monsanto.......so its all natural for me now.


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## pharmacoping (May 25, 2012)

Saldaw said:


> so you can clone autos in vitro? how does that work? + rep btw


yep, besides making a plants bioluminesce successfully, cloning/storing autoflowers has been my funnest experience. In vitro they'll slow vegetation down, to a near stop, divide, sprout, root normally, and I constantly use my santa maria auto mom in a test tube !


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## pharmacoping (May 25, 2012)

autoflower culture is no different than any other culture here, except when removed form the jar, these bits take hold fast, and produce twice as much weight as the seeded ones. I breed autoflowers invitro for fun, because the results can be seen so quickly, F1's and IBL can be produced in months rather than years. The punnets square is necessary for proper trait isolation and successful breeding.


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## readysetgro (May 25, 2012)

I have to figure out how to post pics lol, im on my phone so maybe it doesnt offer it in mobile version, but i have taken plenty so when i figure out how to post ill be sure to post a bunch. Also, so i CAN tc autoflowers, now THAT is cool. I have 60 day wonder & world of seeds lowryder kush going right now, gonna take plenty of samples!!! Also harvested my bbbc its been about 4 days in a huge paper grocery sack & the stems arent "snapping" yet, (48g) newho; is it safe for me to put it in the jar yet? Normally after 2 or 3 days its ready to be jarred, I harvested sun/mon. Its friday...sn...if i dont get it in a jar its good as vaporized cuz were smoking off it daily wet still lol...


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## pharmacoping (May 25, 2012)

safe....properly dried(before any jars, or none at all) marijuana wil lose about 75% of it's(water) weight while drying. Although it can be used earlier, othing gets bagged or jarred until stems are loud snappers, /or you culd weigh your fresh cut(or even the largest cola drying, and when it weighs 75% less, its ready to cure or use. Most of my product is demanded before it ever sees a jar, and some mj gets nasty after sealed, like hay, useless for smoking, extracts only. Jars and curing made many garden products smell like pocket piss weed, acrid, which is very simply mold, very common ! Good weed will be good weed after about a week in a proper drying room. The hanging nets work awesome and mine is in a controlled room, not the garden, inside a grow tent. Your finished buds, spongy, crisp, dense, will be able to be stored in a vac sealed bag indefinately, but never in the freezer or refridgerator. I've sealed up samples for more than 2 years and when opened they smelled like a fresh harvest.
The drying process is also a chemical one, carbs, starches, cbd, cbn are all being changed into more psychoactive compounds. 

If it doesnt smell good, or taste good, or get you high after a week of drying properly, it never will. 

If it does smell good, taste good, and get you high, bring some over, and we'll share a  together.
.


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## readysetgro (May 26, 2012)

Stems snapped! & Jarred. And just since my first 12 hour "burp" it smells way more "sharp". I think it had something to do with most the buds not being on the stems (im revegging) as to why they didnt dry fast as usual. Just a theory..were getting high smoking it wet!! is that not normal? I know bagseed reefer isnt any good till its cured (to me) smells good tastes good...no high (I have a homegirl that insists that what she finds in the bag was once some "purp or kush" & was ruined from being in the "mexiwild" so she grows them & trys to identify their strain & just calls them what she thinks they are lol. So she has diesely fuelly smelling sativas, minty smelling indicas but they dont get me high!!! Theyll roll 5 or 6 blunts back to back to have a "little" buzz. Do u think its bc its wet & the cure incites the potency or genetics?


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## pharmacoping (May 27, 2012)

If it doesnt smell good, or taste good, or get you high after a week of drying properly, it never will.


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## pharmacoping (Jun 4, 2012)

While classic experiments have demonstrated that plants are able to adjust the timing of their flowering in response to environmental conditions, such as light, temperature and the availability of nutrients, very little has been known about what exactly triggers plants to make flowers instead of leaves, under various environmental conditions.
Now, a team of researchers from the National University of Singapore (NUS) has discovered how this happens. The team, led by Associate Professor Yu Hao from the Department of Biological Sciences at the NUS Faculty of Science, has identified a protein that is essential for flowering under normal light conditions. The team's findings are published April 17 in the online, open-access journal _PLoS Biology_.
To identify the element that triggers the process of flowering in plants, Prof Yu and his colleagues undertook a study that spanned around five years, in which they scanned for proteins in plants using a process called yeast two-hybrid screening. After scanning around 3 million samples, the researchers identified a molecule they dubbed FT-INTERACTING PROTEIN 1 (FTIP1).
The researchers found that plants with mutant, non-functional versions of the FTIP1 gene flowered much later under normal light conditions (around 16 hours of light per day). When such mutants were given a working version of this gene, their flowering time was restored largely back to normal.
These findings suggest that FTIP1 is key to how flowering is controlled by light and imply that FTIP1, and genes similar to it, could be used as molecular markers for both classical plant breeding and for targeted genetic modification for desirable flowering traits, with the aim of increasing crop yields in changing environments.


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## pharmacoping (Jun 10, 2012)

readysetgro said:


> Stems snapped! & Jarred. And just since my first 12 hour "burp" it smells way more "sharp". I think it had something to do with most the buds not being on the stems (im revegging) as to why they didnt dry fast as usual. Just a theory..were getting high smoking it wet!! is that not normal? I know bagseed reefer isnt any good till its cured (to me) smells good tastes good...no high (I have a homegirl that insists that what she finds in the bag was once some "purp or kush" & was ruined from being in the "mexiwild" so she grows them & trys to identify their strain & just calls them what she thinks they are lol. So she has diesely fuelly smelling sativas, minty smelling indicas but they dont get me high!!! Theyll roll 5 or 6 blunts back to back to have a "little" buzz. Do u think its bc its wet & the cure incites the potency or genetics?



when the trichs are milky, the thc is ripe, when its amber, it is degrading. ripe is more energetic, and amber is more sleepy, generally. Weed does not need to be cured, and the "sharp" smell you detect is called acrid, and it is the beginning of spore formation/acid ph value


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## Western5 (Oct 1, 2013)

Marijuana from Test Tubes - A Guide to Micropropagating 
Cannabis For The Rest of Us


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## pharmacoping (Oct 2, 2013)

Western5 said:


> Marijuana from Test Tubes - A Guide to Micropropagating
> Cannabis For The Rest of Us


 after reading, and wondering what to do with your clone in a gel cup, turn to "Plants from Test Tubes" also available from Amazon. The first is an introduction mere 58 pages of gel cups and cuttings. Culturing the genus to full expression will be a whole new program. She is a good writer though, but it seems MJ was a shill in for her new book. good find


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## billy4479 (Oct 2, 2013)

haven't seen this thread in awhile


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## Vincent VonBlown (Oct 6, 2013)

Flavor, is a component of "Dank" and it is a hard thing to capture for the most part... Most genetics don't have the ability to be very dank. They will range from Ditchy woody tastes, to sickly sweet. It is very rare to have classic flavors that stand up to the taste of actual food, like say fruit for example.


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## Vincent VonBlown (Oct 6, 2013)

Real dank, "dank", like the Wonder Thunderstruck clone, (which is where T99 is produced from). You can cut a bud that isn't even done, microwave it. And it still will have a decent flavor. You can't do that with very many strains I'll guarantee you that


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## Trousers (Oct 23, 2013)

Cannabis is the best fucking plant ever. 
I need to go back and read this entire thread


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## hank123 (Oct 24, 2015)

dang where did this dude go? He did it with auto flowers and they produced better?!


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## hank123 (Dec 7, 2015)

come on bump on this. this is really good stuff!


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## Marijuannoisseur (Dec 23, 2015)

looks intense... I prefer the classic version personally


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## hank123 (Dec 28, 2015)

Marijuannoisseur said:


> looks intense... I prefer the classic version personally



That is a CUTTING not a true clone. Difference.


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## jackblaster (Feb 18, 2016)

pharmacoping said:


> yep, besides making a plants bioluminesce successfully, cloning/storing autoflowers has been my funnest experience. In vitro they'll slow vegetation down, to a near stop, divide, sprout, root normally, and I constantly use my santa maria auto mom in a test tube !


Can I see the biolumeplants you created? do you have photos?


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## hank123 (May 3, 2016)

Someone please update. Please share something


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