Plant Tissue Culture

ok, thanks guys, ill try changing my medium to promote root growth.
the video that came with my kit said that the medium is supposed to promote stems, shoots, leaves and roots after 3-4 weeks growth. i guess this depends on the type plant grown.

if i remember correctly, the medium i mixed and used was all 3: BA, PPM, and NAA.
apparently it will take twice as long to get regular small plants with shoots and roots before planting them into hydro veg? i was really expecting to be able to have good shoots AND roots developed in a 30 day period. apparently i am supposed to grow shoots in 30 days, then transplant them into diff medium to promote roots?

i had about 66% success rate for my 1st time, not bad i think. only about half of the 66% are big enough to cut up for multiplication though, the others just didnt grow as much for some reason.

i will mix up more medium soon and try putting the better grown cultures into it, hoping to get good roots from the new medium.

ill let you guys know.

thanks!

scrxbandit: "when its time for another grow, cop a few rockwool slabs and root 200 of them, replace the jars and start over again."...

are you saying you simply take your new cultures out of the jar, with no roots on any of them yet, then put them into rockwool cubes and propagate them and root them that way?
what is your avg success rate of rooting the new cultures, and how long do they usually take to root?

The Benzylaminopurine (BAP) is a cytokinin. Cytokinins do three thing very well on their own, they are Adventitious shoot formation, Inhibition of root formation, and Promotes cell division.
The Plant Preservative Mixture (PPM) is a a broad-spectrum preservative/biocide, which effectively prevents or reduces microbial contamination in plant tissue culture.
The Naphthaleneacetic Acid (NAA) is an Auxin which is mainly used for Callus formation and growth.
You should be able to root the newly cultured plants in a clone machine with a little bit of rooting solution mixed in, or you can add the rooting solution to the new batch of media you are about to make.Add whatever the instruction on the rooting solution say to, if you have to calculate the right amount for 1 liter ( i'm assuming you are making up 1 liter of media), Or you can use the NAA hormone (not the best), or you can use Indole-3-Acetic Acid (IAA) which is for Adventitous root formation (high concen) i.e FAST ROOTING AND A LOT OF THEM, or Indole-3-Butyric Acid (IBA) which is for Adventitious shoot formation (low concen). I use IBA mainly because I can get it from HOME TISSUE CULTURE GROUP in 10 ml packages. I usually get the Plant Growth Regulator Pack - Basic 4+1, it comes with BAP, KINETIN, IBA,and NAA - 10 ml each - 1 mg/ml concentration); 10 ml PPM..
Fact is once you have a healthy stem with a few leaves you can collar it and root it just like you would your traditional clones, I personally like using the second culture just remove the BAP from the recipe for the second culture and if you have some hydro veg nutes throw in like 3 ml or a teaspoon in the media.
The thing i've found about tissue culture is that it takes a little longer than cloning to get a viable plant but you get like ten times a many when they are finally ready, and all of the new plants came from a fraction of on of the leaves.

iivan740:
You should be able to root the newly cultured plants in a clone machine with a little bit of rooting solution mixed in,

i havent been able to grow cultures that are tall / big enough to put in my ez-cloner aeroponic rooting machine, the cultures are simply way too small and short.
i have NEVER been able to propagate cuttings or cultures in regular rockwool cubes. for some reason. ive tried many times and nothing roots. maybe im located in the wrong environment? so putting my new, small rootless cultures in rockwool to root them is not happening for me here. the ez-cloner works great, but not for my short 1" to 1.5" tall cultures.

or you can add the rooting solution to the new batch of media you are about to make. Add whatever the instruction on the rooting solution say to, if you have to calculate the right amount for 1 liter ( i'm assuming you are making up 1 liter of media), Or you can use the NAA hormone (not the best), or you can use Indole-3-Acetic Acid (IAA) which is for Adventitous root formation (high concen) i.e FAST ROOTING AND A LOT OF THEM,

Where did you get the IAA??? i looked on the weblink / website you gave: "HOME TISSUE CULTURE GROUP", and i could not find any for sale. you say you use the Plant Growth Regulator Pack - Basic 4+1, but it doesnt come with any IAA... unless im supposed to use that pack, but leave out the NAA the 2nd round of medium i use to root them? but that still doesnt include the IAA you tell me is supposed to be great for growing roots!

or Indole-3-Butyric Acid (IBA) which is for Adventitious shoot formation (low concen). I use IBA mainly because I can get it from HOME TISSUE CULTURE GROUP in 10 ml packages. I usually get the Plant Growth Regulator Pack - Basic 4+1, it comes with BAP, KINETIN, IBA,and NAA - 10 ml each - 1 mg/ml concentration); 10 ml PPM..Fact is once you have a healthy stem with a few leaves you can collar it and root it just like you would your traditional clones, I personally like using the second culture just remove the BAP from the recipe for the second culture and if you have some hydro veg nutes throw in like 3 ml or a teaspoon in the media.

The thing i've found about tissue culture is that it takes a little longer than cloning to get a viable plant but you get like ten times a many when they are finally ready, and all of the new plants came from a fraction of on of the leaves.

well, i bought my kit because it tells in the advertising video, that PTC kits come complete, with everything im supposed to need to grow cultures with shoots, leaves, stems AND roots, ready to plant in and grow! it doesnt say anything about me having to buy twice as much medium, jars, trays, lights, and use more space, in order to grow cultures the way its turning out to be.

how long do you grow your first round for (growth), and how long does it take you to root all your new cultures? should i expect my first grow round to be 30 days, then another full 30 days for the 2nd rooting round?

if i can just grow at least 1"+ of top stem & leaves, then grow at least 1"+ of really good roots...im fairly sure i can plant them in veg and they will do just fine.

i guess im just upset at the video advertisement telling me i will be able to use the PTC kit to grow shoots, leaves and roots, and take them out of the jar all ready to veg and grow...

I do not use IAA because the 4+1 from home tissue culutre group doesn't come with it, instead I use IBA. If you want IAA you can get it here http://www.phytotechlab.com/detail.aspx?ID=380 , I do not order solutions from them because the smallest shipping unit is 100ml. If I ordered 100 ml of anything besides PPM about 97ml would get thrown away. Most of the hormones in solution form only have a 30 day shelf life if refridgerated. That website also has phytocaps (plastic lids for babyfood jars), MS packets, and just about anything else that you will need.

How much do I let them grow? I normally let the shoots go to the top of a 6 oz baby food jar, then I transplant into a gall quart jar with new root media. The cultures/clones will continue to grow up and the roots start to develop, I put some hydro grow nutrients in the media so that the shoots will continue to develop and the stems will get stronger. By the time the roots form the shoots are developed and about 4 inches tall, the roots aren't that developed but the clone can be put into a cloner or if you do soil it can go into a rockwool or soil (I do aeroponic so if they die in rockwool or soil I do not know). After that a couple of weeks veg, and you know the rest.

The PCT kit does come with everything you need to get calluses, shoots and roots. However there are many different hormone combinations as puffone points in post #20.

Hope that I have been able to help.

You are difinitly right, all the adverts lead one to believe that it's a simple as simple as "buying the kit", putting "recipe" together, and waiting a week or so. THEN WALLA, presto chango; plant the new plants in the dirt and it off to the track........
Reality is that you kave to make a media to promote shoots. Then you wait 4 to 6 weeks (I usually wait a 7 or 8 weeks). Then you pull the clumps of shoots out of the shoot media, cut them into smaller pods (clumps) of 3-4 shoots and transplant them into the second rooting media. Then you wait ...... Well this one depends on the plant, the strain, the nutrients, the hormones, and on and on; but normally you will wait at least another 4 to 8 weeks for root development, leaf formation, and stem development. Now you should be ready to transplant into the garden, but wait if you do not give them special care they WILL DIE. they need high humidity for at least the first week outside of the culture jars, and lower than normal light. After the first week you can start to ween them off of the humidity and low light conditions because the roots should have adapted to their new enviroment and be ready to support the plant......
So you are definitly correct sir the adverts are a little misleading, but the simple fact remains that even at 3 to 4 months (culture to plant) you still cannot produce the same quantity in the same amount of time by traditional cloning. Once you have a rotation giong you will find that PCT will overgrow the space that you have for it and you will be throwing out cultures left and right....

iivan740 - how many cultures do you put in a jar each stage?
1- grow to multiply
2- cut up into smaller clumps, and put into shoot medium
3- transplant into root medium

so do you put 3+ into each jar for growing to multiply? then cut them up into smaller clumps and put 3-4 clumps into one jar with shoot medium, then transplant each of them into root medium, 3+ per jar until rooted?
or after you first cut the cultures up into smaller clumps, give each culture their own jar to grow shoots, then transplant into root medium, only one per jar?

i was hoping to have 3 per jar, through the entire growing process, with 3 per jar, then cut them up, put 3 per jar for shoots, then transplant 3 per jar in rooting medium...are the cultures going to be too big to end up having 3 per jar after they root?

also, (so many questions, i know!), 1- what light do you use over your lights, and 2- how close to the tray do you hang it?
im using a regular cheap 80w cfl shop light over mine now, only one tray (starting small), and hanging it about 1 ft above the top of the jars.
im also planning to use one 80w shop light over 3-4 stacks of trays (5 trays per stack x 3-4 stacks side by side)
will this be ok concerning stacks and amount of light, or too much light? light too close to jars?

splitmanj
how many cultures do you put in a jar each stage?
I start with one or two leaf cuttings that are about the size of a hole punch. I place these leaf cuttings into a medium that contains BAP or other shoot promoting hormone.
1- grow to multiply
Once I get shoots from the original cultures and leaves start to form on the shoots I can start culturing from the original culture. This is where PCT wins over cloning. I can multiply my plants exponentially. Just take a few shoots with leaves and put them into the original shoot media.
2- cut up into smaller clumps, and put into shoot medium
When you PCT from leaves you get "clumps" of shoots. Normally you can start rooting the shoots as soon as you see the first leaves, however if you do this you WILL stress the new shoots and you WILL kill a few. If you wait about 6 days after the first sign of leaves you will have a better success rate.
I PCT raspberry and blackberry VINES. That being said when I cut up my clumps of shoots, I usually cut them down to 2 to 4 shoots and let them root together. Seeing as how they are vines I can do it this way, I do not know how this would work for you. If I were doing bushes or vegetables I would cut the clumps down to individuals. I one could do the 2 to 4 shoots per clump and then only keep the best shoot when transplanting. This way you would get the advantage of multiply shoot rooting with only one stem to support.
3- transplant into root medium
When transplant the shoots into Rooting media I usually transplant 2 or 3 sets of shoots. Instead of using baby food jars I will use larger wide mouth mason jars, and put the cultures as far apart as I can. This helps keep the roots from becoming entangled once they start to grow. Also instead of the usual 30-45 ml of media, I put 75-90 ml in the larger jars.
so do you put 3+ into each jar for growing to multiply?
I will put multiply shoot cultures in the same container. I will sometimes put multiply cuttings in a container. You must have good aseptic technique and a steril working enviroment for this to work. If only one part of one cutting is contaiminated it will normally destroy the entire culture container. This can happen with single specimen cultures as well, but you only loose one specimen per container.
then cut them up into smaller clumps and put 3-4 clumps into one jar with shoot medium, then transplant each of them into root medium, 3+ per jar until rooted?
Yes. Different plants will be treated differently at this stage. Vines and ferns are ok with multiply shoots per rooting culture. vegetables such as cabbage or celery would be done individually at the shoot stage. The differance is in the end result. Thing about what you want to acheive with your cultures and thin the shoots accordingly.
or after you first cut the cultures up into smaller clumps, give each culture their own jar to grow shoots, then transplant into root medium, only one per jar?
This is the easiest way to make sure that contaminated cultures do not effect other cultures. You can only kill what is inside the contaminated cultures container. If each culture is in its own container and one culture becomes contaminated you can only loose that one.
i was hoping to have 3 per jar, through the entire growing process, with 3 per jar, then cut them up, put 3 per jar for shoots, then transplant 3 per jar in rooting medium...are the cultures going to be too big to end up having 3 per jar after they root?
I do this after I have my first round of shoot cultureing completed. There is a much higher risk of contamination from "parent plant" cuttings than there is from cultured cuttings. Once I have cultured shoots I take my next batch of cuttings from the cultured shoots, and culture multiply cuttings within the same container.
also, (so many questions, i know!), 1- what light do you use over your lights, and 2- how close to the tray do you hang it?
I use 4 foot 4 bulb t-5 ho fixtures that are adjustable from 12 inches high to right on top of the culture container. I have never mover my lights lower than 12 inches.
im using a regular cheap 80w cfl shop light over mine now, only one tray (starting small), and hanging it about 1 ft above the top of the jars.
I think that those will work... cultures do not need a lot of light.
im also planning to use one 80w shop light over 3-4 stacks of trays (5 trays per stack x 3-4 stacks side by side)
will this be ok concerning stacks and amount of light, or too much light? light too close to jars?
I do not stack my cultures, however I do not see why you couldn't. I use vented caps and can not stack cultures my cultures. As long as there is a little light for the container I would think that you could stack them.

Hope that this helps.

Hey iivan740, how much of each hormone do you use per stage of development?

Also, I do not fully understand Callus. Does all PTC start with Callus which then turns into shoots? If not, what is the benefit of inducing Callus?

OLD`ish THREAD STILL NEW TO MOST !

I have heard alot about this and am looking into this now.
Great thread and loads of good links and info !
I think with MM laws, this would be the best way to "store" genetics and potentially send via post.

Couple of questions ;
How long can the cultures store for ?
How about sending a culture in the mail ?
Have any of you got pictures of MJ cultures rooted ? Or close ups ??

Thanx in advanced !

its entirely possible with cannabis but i am no pro in this department but know a little... i know it can be done and done with quite good success in a good sterile environment etc. hell greg green mentions it in his book though he doesn't go into it much either. the problem being is that deep tissue culture is just too much work or too expensive to do propperly for most people to even think about..

if you can in fact get a kit rather cheap and feel like looking into it then thats great. the plus side is if you only need one leaf and some time on your hands and you can make many, many clones once you have it down pat.

ive tried to talk about this on this forum before and people think your just full f shit when its really there just uneducated and not willing to learn

Jester88 said:

its entirely possible with cannabis but i am no pro in this department but know a little... i know it can be done and done with quite good success in a good sterile environment etc. hell greg green mentions it in his book though he doesn't go into it much either. the problem being is that deep tissue culture is just too much work or too expensive to do propperly for most people to even think about..

if you can in fact get a kit rather cheap and feel like looking into it then thats great. the plus side is if you only need one leaf and some time on your hands and you can make many, many clones once you have it down pat.

ive tried to talk about this on this forum before and people think your just full f shit when its really there just uneducated and not willing to learn

Click to expand...

IMFAO - Its seem the ONLY way for the future (subject to restrictions in the MM possesion laws), yes it takes longer. But if you can do 1000`s of clones from one leaf that's a no brainier. YES it would take effort and time to setup a cycle. But I think once your setup the worlds the limit.
Taking a culture and storing the EXSACT genetics with out having to keep a mother is IMO the SHIZZ. And dream of a fridge full of cultures with 100`s of different top notch proven genetics, ready to be broken out of there petri dish !

the idea for the mainstream is once the processs is over you have your mother plant.
you then resume taking cttings as you would if you had the mother there from the beginning or start breeding.

well thats what i always thought.

Tell me more.

Protoplast (cells without cell walls) fusion is novel technique used by advanced tissue culturists. The cell wall of a plant is broken down until only the protoplasts remain. These are then cultured until they form a clump and can be mixed with other clumps of protoplasts from the same species, or foreign species. The protoplasts are encouraged to recombine with each other via chemicals or electric shock and form new cell walls around the fused center by weaving together both sets of plant instructions to create what is called a somatic hybrid. This can produce results such as true female-female combinations of prized clone only plants (ex. Cypress O.G. Kush X pre-98 Bubba) without the use of drawn out breeding programs. Combinations of cannabis sativa and other plant species like grapes, mint, succulents, etc. are all also possible, but the results are highly unpredictable.

IMHO, this would be the BY FAR THE BEST way to share genetics with friends across the country... just my opinion, so much safer than driving them, no selection needed for a seed packet, the best of all worlds. This is a paitient man's game, so why worry about your time frame to have the best? Just a tought... I have seen stuff that has to be re-vegged in the past because it was looked over in the clone room, or a problem occured during cloning... just a thought.

PLUS, you can keep that special male for ever without needing space...

Dunno why this thread died, as there's lots of amazing information here.

here's my two pesos to add to the subject.

PDF on the Plant Tissue Culture of seed bank Cannabis strains.

http://www.pakbs.org/pjbot/PDFs/41%282%29/PJB41%282%29603.pdf

It was this tread that encouraged me to buy a kit and give it a try myself. I am three days into this and have a health callus growing from stem tissue I was throwing away. (this was to be my fallback!) Can't wait for the real cultures to take off.

This will end my days of empty flowering space because I don't have the clones because I don't have the space for mothers!!


Question for everyone...

Is there any risk associated by ordering plant hormones from biological supply companies? I need to order TDZ.

Looked at the recipes in the pdf and the second one using BA is just as good. That is the exact recipe in the tissue culture kit sold at www.planttc.com.

They have TDZ if absolutely needed but dont go to the trouble.

All of the claimed advantages are true. That is why all commercially cloned nursery plants are cloned from tissue culture. The kits work for users at home, without a lab needed.