Plant Tissue Culture

I was just wondering if anybody had an experience with cloning using plant tissue culture.

Briefly outlining, it's a method of propogation using a cells of a plant in a medium. From what i've seen the medium is like jello. Each unit of medium and each tissue culture is capable of producing like 50,000 specimens.

I saw a PTC kit online for 180- it includes all the stuff for sterilizing, the medium, dishes, and most important... Directions.

So anybody fux with this? I've read some how-to's and research published on the topic from OSU and Cornell, they seem to use this technique without problem.

It's not an easy method, expect at least a 50% if not a 75% failure rate. Also, you need specific hormones and levels of those hormones to initiate roots and shoots from the callus on the dish. Marijuana has it's own needed hormone levels, they are not easy to find but search online and you might get lucky. Also, a proper incubator is necessary, easily made with a heat-mat thermostat and a tupperware tub.
In all honestly it's not the best idea for those who do not know sterile lab procedure and takes a long time to form a fully functional plant. If you have a specific plant that you can't get clones from and you desperately want to save, buy the kit and make the cultures, but it should only be used by novices as a last resort...

Oh and if you know someone who is/was a biology major in college, they know sterile lab procedure and would be a big help.

Actually, the person who brought this to my attention, and the person who was initially going to get this is someone who has good knowledge of proper lab procedures.

I guess I could always try to get one of my Doctor siblings to do it... although something tells me they wouldn't . Squares.

But the sterile environment is actually one of the things I'm not too worried about, as I have also had laboratory experience as I am still in school.

Thanks though, 50%-75% failure rate was never specifically pronounced, but it makes sense as any microbial organism would thrive in the PTC environment.

To your knowledge has anyone actually endevoured to do this on any of the major forums?

I forgot to add... From the procedures I was looking at, they actually rigged a pressure cooker into a pretty functional pressure cooker. I think I pressure cooker would also protect against contaminants if initially properly sterilized.

Thoughts?

good god it's early... "They actually rigged a pressure cooker into a pretty functional INCUBATOR"

lol, if you think you can handle it, try it! We could use more information on this subject. What I know is from those I know who have tried (and classes) but most were laymen, it can't hurt to try! Worse comes to worse it fails, lessons learned.

I actually am a Biology major and Plant tissue culture is not worth the effort or time. Building or buying a sterile flow hood in my opinion is not worth it. You also have to buy the cytokinin or auxin to put the leaf tissue in. It's more of something you do for fun unless you plan on propagating plants on an extremely large scale. I have had decent success rates when using African violet plant tissue but I haven't tried it with cannabis.

yeh it seems like if your could perfect the art of PTC, then it would be worthwhile, especially since you can use pieces from the PTC to repropogate a new culture.

Just to say: The kit I was looking at came with all necessary products, including the medium. Although they do use a pressure cooker rather instrumentally.

I think I am going to throw down and pick up a kit, see if between my friends and I we can't work something out.

hahah... also african violet is the flower which is always mentioned on the college sites and research papers... makes you wonder if they haven't had any sizable success with anything else...

I'll fux with it tho, and post an update... i'm a little strapped for cash at the moment, and i still need a coupla things, so I would say two weeks or so? I['ll try to procure a camera too so I can show ya'll whats up.

:/ i wasnt sanitary at all making my mush cakes and they are all growing happily contaminant free

PTC isn't easy. Cloning via cutting is easy. We don't know if cannabis tissue cultures need a special rooting medium (different plants may need different mediums), and we can only guess that a midvein of a leaf will work.

Props to person above who had success with african violets because my first 2 attempts ended in fungus and mold (reverse flow hood and my aseptic technique must be off).

Just finished reading an article in The Growing Edge Magazine - Culture Club:It's not just for scientists anymore...

Very intresting read... =]

The writer recommends the following kit...

Kitchen Culture Kits - Catalog Ordering

Aside from the kit you will need:

Tools to handle material (its small, forceps and a scalpel etc)
Containers that can be sterilized (glass works best with plastic tops)
A way to sterilize tools and containers (pressure cooker)
A sterile still air cabinet (glass aquarium on side, plastic lined PVC frame)
Most importantly ASEPTIC technique (clean and sterile)

I think the beginner should expect a 50% or greater failure rate until that last item is perfected witch is the number one cause of failure... You end up growing fungi and bacteria witch destroy your delicate plant matter...

This really does sound intrigiging and exciting...

Yes cuttings are easier but where is the fun and sense of adventure in that...

I am sure i will try this at some point as being involved in my plants makes me happy but i just do not have the time, money or recourses for this right now...

FDD need a new project... =]

You know you want to be the one to do this... =]

There is a kit on eBay and on the hydroponic sites for Super Starts plant tissue culture and nearly every user is successfully tissue culturing plants, the rest have not been around to ask. Everything comes in the box including the jars, instruments, and lots of pictures and video.

You can grow as many plants as you want. The ingredients make 30 jars or 15 deli tubs at a time, and that makes about 240 plants from only a litle bit of work. Tissue culture plants are sterile so there are no molds or pests in the containers except for afew and that is they are being handled sloppy or just bad luck. You will definetley have as many plants as you need. I have seen jars given away because it only takes a few jars to produce all rooted clones needed.

You dont have to have any more sterile handling area that an plastic storage tub turned on its side. I have seen all the parts carried into the house and the plants being tissue cultured right on the counter. You have not seen cool clones until you have rooted tc clones. They have chunky roots and branching nodes, and only a few inches tall-but they get bigger.

You onlly need a pressure cooker like from mushroom culture or canning and ingredients like bleach and alcohol. These are mother plants, cloning and rooting in the space of a microwave and uses only a small fluorescent. Go to www.planttc.com, www.hydroasis.com, or txhydroponics.com.

So.... Anyone try this out with cannabis?... with any success? If so, please share your experience.

Cheers

There has to be someone here who tried this, I have two books on the subject and believe that it is definately possible and probably not to difficult, (although probably not financially feasible, but fun) the two books are plants from test tubes,and experiments in tissue culture these two books are very different from each other and both have alot of good information. Most of todays commmercial horticulture operations especiall in the household plants and flower industry use tissue culture, currently there are thousand of plant laboratories throughout hte country.

there is also another tissue culture method I would like to try called meristem culture, with this technique I think that you could pull 30 plus clones every 3-4 days from one mother instead of raising a callus and stimulating it to become a plant you cut off the very tip of the meristem (the tip meaning the very top couple of cells with a stereo microscope) and in a tissue culture you clone a plant. Imagine the possibilities, instead of ordering seeds from overseas you could order a premo clone in a test tube or petri dish, with very accurate discription from the grower since the plants they are selling are the same ones they grew.

I think the bottom line is, it definately can be done and I am sure someone somewhere is doing it if not for commercial purposes for entertainment.

in the very near future I plan on trying this (with the assistance of my sister with a masters in plant science) If there is someone else getting ready to try this send me an email and maybe we could experiment at the same time and share notes as we progress.

Thanks all

uwhcmw76 said:

There has to be someone here who tried this, I have two books on the subject and believe that it is definately possible and probably not to difficult, (although probably not financially feasible, but fun) the two books are plants from test tubes,and experiments in tissue culture these two books are very different from each other and both have alot of good information. Most of todays commmercial horticulture operations especiall in the household plants and flower industry use tissue culture, currently there are thousand of plant laboratories throughout hte country.

there is also another tissue culture method I would like to try called meristem culture, with this technique I think that you could pull 30 plus clones every 3-4 days from one mother instead of raising a callus and stimulating it to become a plant you cut off the very tip of the meristem (the tip meaning the very top couple of cells with a stereo microscope) and in a tissue culture you clone a plant. Imagine the possibilities, instead of ordering seeds from overseas you could order a premo clone in a test tube or petri dish, with very accurate discription from the grower since the plants they are selling are the same ones they grew.

I think the bottom line is, it definately can be done and I am sure someone somewhere is doing it if not for commercial purposes for entertainment.

in the very near future I plan on trying this (with the assistance of my sister with a masters in plant science) If there is someone else getting ready to try this send me an email and maybe we could experiment at the same time and share notes as we progress.

Thanks all

Click to expand...

I know bill gram, the inventor of superstarts, subdivision of hydrosolutionz. We were doing our tissue cultures last night, and we will have 100% success rate. we've got 3 genetics 7 weeks into flower, and performed leaf cultures on all of them. I've got another 4 genetics in my mother room that need to be put to rest. with the culture kits, ima hang onto my pk, ak, hs, ss, JR, plo and special breed for as long as i need to. they wont be sucking off 54w t5s. i dont have to worry about infestations and i keep them all under a cfl. when we start reworking our plants, each male gets its own jar, the females get their own jars, you can even germinate seeds in culture and get multiplication. when its time for another grow, cop a few rockwool slabs and root 200 of them, replace the jars and start over again.

Wow that is a great idea to use a bin as a laminar hood I think I will try that.

i just started my 1st round of cultures, and i didnt know what i was doing, first time, didnt use the best materials, couldve been a little more sterile, etc.
so far i am getting 10 out of 15 jars doing fairly well! (66% success rate so far). the only thing im concerned about is, they are all producing leaves and growing on top, and i have yet to see one single root growing. maybe i can grow massive amounts of them by splitting them all up into more and more jars, but if i cant get some roots growing out the bottom of them, its all a waste of time as far as im concerned.
my cultures are at day 18 today, and like i said they are looking pretty good so far, but still no roots!

scrxbandit - how do you get yours to root?

scrxbandit said:

I know bill gram, the inventor of superstarts, subdivision of hydrosolutionz. We were doing our tissue cultures last night, and we will have 100% success rate. we've got 3 genetics 7 weeks into flower, and performed leaf cultures on all of them. I've got another 4 genetics in my mother room that need to be put to rest. with the culture kits, ima hang onto my pk, ak, hs, ss, JR, plo and special breed for as long as i need to. they wont be sucking off 54w t5s. i dont have to worry about infestations and i keep them all under a cfl. when we start reworking our plants, each male gets its own jar, the females get their own jars, you can even germinate seeds in culture and get multiplication. when its time for another grow, cop a few rockwool slabs and root 200 of them, replace the jars and start over again.

Click to expand...

do you mean to say that you are taking clones (tissue) from the plants during flowering? wow. this is an amazing technique.

splitmanj what is your media mix for shoot formation? Have you tried making a new mix without BAP? BAP can inhibit root formation use naa or iba for root formation, or maybe this mix will work...
""Home Style Medium"
In a quart jar filled with water, mix:
1 teaspoon hydroponic fertilizer
(Peter’s NPK 20-20-20)
2 tablespoons sugar
a multivitamin pill
1 ml PPM

In Vitro Cellular & Developmental Biology - Plant

ISSN1054-5476 (Print) 1475-2689 (Online)

Abstract:
Induction of high-frequency shoot regeneration using nodal segments containing axillary buds from a 1-yr-old mother plants of Cannabis sativa was achieved on Murashige and Skoog (MS) medium containing 0.05–5.0 μM thidiazuron. The quality and quantity of regenerants were better with thidiazuron (0.5 μM thidiazuron) than with benzyladenine or kinetin. Adding 7.0 μM of gibberellic acid into a medium containing 0.5 μM thidiazuron slightly increased shoot growth. Elongated shoots when transferred to half-strength MS medium supplemented with 500 mg l−1 activated charcoal and 2.5 μM indole-3-butyric acid resulted in 95% rooting. The rooted plants were successfully acclimatized in soil. Following acclimatization, growth performance of 4-mo-old in vitro propagated plants was compared with ex vitro vegetatively grown plants of the same age. The photosynthesis and transpiration characteristics were studied under different light levels (0, 500, 1,000, 1,500, or 2,000 μmol m−2 s−1). An increase in photosynthesis was observed with increase in the light intensity up to 1,500 μmol m−2 s−1 and then decreased subsequently at higher light levels in both types of plants. However, the increase was more pronounced at lower light intensities below 500 μmol m−2 s−1. Stomatal conductance and transpiration increased with light intensity up to highest level (2000 μmol m−2 s−1) tested. Intercellular CO2 concentration (C i) and the ratio of intercellular CO2 concentration to ambient CO2 (C i/C a) decreased with the increase in light intensity in both in vitro as well as ex vitro raised plants. The results show that in vitro propagated and hardened plants were functionally comparable to ex vitro plants of same age in terms of gas and water vapor exchange characteristics, within the limits of this study.

------------------------------------------------------------------------------------------------------------

Tissue culture and Agrobacterium-mediated transformation of hemp (Cannabis sativa L.)
FEENEY M. (1) ; PUNJA Z. K. (1) ;

ABSTRACT:
Hemp (Cannabis sativa L.) is cultivated in many parts of the world for its fiber, oil, and seed. The development of new hemp cultivars with improved traits could be facilitated through the application of biotechnological strategies. The purpose of this study was to investigate the propagation of hemp in tissue culture and to establish a protocol for Agrobacterium-mediated transformation for foreign gene introduction. Stem and leaf segments from seedlings of four hemp varieties were placed on Murashige and Skoog medium with Gamborg B5 vitamins (MB) supplemented with 5 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 µM kinetin, 3% sucrose, and 8 g l-1 agar. Large masses of callus were produced within 4 wk for all cultivars. Suspension cultures were established in MB medium containing 2.5 µM 2,4-D.

Publisher: CAB International


Yes you have to transplant into different media for roots...
Replace the kinetin in the second abstracts formula with the thidiazuron and there u go for the veg stage. To promote root growth transplant to the indole-3-butyric acid formula in the first abstract.
and no the first abstract doesn't include the other essential ingredients like: the vitamins, sugars etc. that the second does but it does show the rooting chemistry variation you will need when you transplant your XPLANTS. On a completely different note, well maybe not as I am really stoned, I use a fish tank I picked up at a garage sale for 3 dollars.

If you find it too difficult for what ever reason here is an old recipe that works good too.
For veg: MS medium
.5 mg./Liter 11z(zeatin)
.1 mg./L IAA
1.5 teaspoon/ L sugar
.2mg./L thiamine

for roots: 1/2 strength MS
.1 mg./L IBA
.01 mg./L NAA

That was my old way and worked ok.


Other links:
This guy did a really good youtube series on the subject.
http://www.youtube.com/watch?v=KYvJByYrSPg&feature=related

also:http://www.scribd.com/doc/3544465/Plant-Tissue-Culture for general info and a basic understanding.

http://www.hos.ufl.edu/mooreweb/TissueCulture/tcclass.htm basically a full course in TC

also see:
http://www.ib.uj.edu.pl/abc/pdf/47_2/145-151.pdf
The results of this study were mediocre compared to the first but is a really good source of what combos don't work so well. If you plan on experimenting it is worth the read.

GC Rocks

Happy trails;
puff