In vitro micropropigation

[rootsnshoots]

Hey Farmers!

I am wanting to share my latest folly with you guys. I have taken a shabby stab at tissue culture once in the past. With new knowledge and much better/ newer equipment i am very excited to see this happen.

I've been doing a lot of reasearch on the subject for the last week or so, and am gaining confidecne and knowledge every day. My first attempt (over 5 yrs ago) was a major failure, but i gots all kinds of new shit!!!

If i see interest/ response in this thread i will definitely be more apt to go into further detail. Also if anyone has experience or information on micropropigation please chime in.

I will be ordering all my gelling agents and hormones hopefully this weekend. I am currently a little low on funds, but with all of my major equipment expenses made, I can see the light!

So far I have only found one experiment involving cannabis, and have not found much as far as forums. I havent looked that hard either. >>>> https://docs.google.com/viewer?a=v&...afe&zw&sig=AHIEtbSXhXs5Q83hM3jSDpG38_MEmGaYnA

.....Seems pretty good I will probably go with what phytohormones worked best for them, but TDZ is pretty pricy stuff so I might just supplement it with a different cytokinin until i have the money. I'm thinking of a 24 jar experiment. Murashige & skoog will be used for my gelling agent on all jars. 6 sets of 4 jars with different hormone ratios. Im hoping to have the ability, or atleast an idea on how to root, shoot and callus after this experiment. From what I've read you want to grow shoots, then transplant them to a rooting gel. So I figure if I can learn all three steps I will be able to start makin babies and storing genetics!!

Well I am more than excited at this point! Feel free to discuss and do some research for me ;)

 

[Thoth]

Hey Farmers!

I am wanting to share my latest folly with you guys. I have taken a shabby stab at tissue culture once in the past. With new knowledge and much better/ newer equipment i am very excited to see this happen.

I've been doing a lot of reasearch on the subject for the last week or so, and am gaining confidecne and knowledge every day. My first attempt (over 5 yrs ago) was a major failure, but i gots all kinds of new shit!!!

If i see interest/ response in this thread i will definitely be more apt to go into further detail. Also if anyone has experience or information on micropropigation please chime in.

I will be ordering all my gelling agents and hormones hopefully this weekend. I am currently a little low on funds, but with all of my major equipment expenses made, I can see the light!

So far I have only found one experiment involving cannabis, and have not found much as far as forums. I havent looked that hard either. >>>> https://docs.google.com/viewer?a=v&pid=gmail&attid=0.1&thid=137d7ca7bbdfcc72&mt=application/pdf&url=https://mail.google.com/mail/?ui=2&ik=47b4b01d34&view=att&th=137d7ca7bbdfcc72&attid=0.1&disp=safe&zw&sig=AHIEtbSXhXs5Q83hM3jSDpG38_MEmGaYnA

.....Seems pretty good I will probably go with what phytohormones worked best for them, but TDZ is pretty pricy stuff so I might just supplement it with a different cytokinin until i have the money. I'm thinking of a 24 jar experiment. Murashige & skoog will be used for my gelling agent on all jars. 6 sets of 4 jars with different hormone ratios. Im hoping to have the ability, or atleast an idea on how to root, shoot and callus after this experiment. From what I've read you want to grow shoots, then transplant them to a rooting gel. So I figure if I can learn all three steps I will be able to start makin babies and storing genetics!!

Well I am more than excited at this point! Feel free to discuss and do some research for me ;)

This is awesome bro! Micropropagation fascinates me. Please do post any and all results you have in this venture of yours!

Are you using a laminar flowhood or just a glovebox type arrangement?What were your major equipment expenses?

What is it you aim to achieve with micropropagation?

You should see if you can find a specific protocol for cannabis as that will help you be successful. Better not to have to guess on what is needed from the outset. Once you have a protocol that works, then you can tweak it if need be.

Look forward to hearing more of what you're doing bro!!

 

[Thoth]

Here's an I just found. Perhaps someone with access to the journals might be able to download the full articles for free and post them here.

High Frequency Plant Regeneration from Leaf Derived Callus of High Δ9-Tetrahydrocannabinol Yielding Cannabis sativa L.

Hemant Lata1, Suman Chandra1, Ikhlas A. Khan1,2, Mahmoud A. ElSohly1,3
1 National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, MS, USA
2 Department of Pharmacognosy, School of Pharmacy, University of Mississippi, University, MS, USA
3 Department of Pharmaceutics, School of Pharmacy, University of Mississippi, University, MS, USA
Abstract

An efficient in vitro propagation protocol for rapidly producing Cannabis sativa plantlets from young leaf tissue was developed. Using gas chromatography-flame ionization detection (GC‐FID), high THC yielding elite female clone of a drug-type Cannabis variety (MX) was screened and its vegetatively propagated clones were used for micropropagation. Calli were induced from leaf explant on Murashige and Skoog medium supplemented with different concentrations (0.5, 1.0, 1.5, and 2.0 µM) of indole- 3-acetic acid (IAA), indole- 3- butyric acid (IBA), naphthalene acetic acid (NAA), and 2,4-dichlorophenoxy-acetic acid (2,4-D) in combination with 1.0 µM of thidiazuron (TDZ) for the production of callus. The optimum callus growth and maintenance was in 0.5 µM NAA plus 1.0 µM TDZ. The two-month-old calli were subcultured to MS media containing different concentrations of cytokinins (BAP, KN, TDZ). The rate of shoot induction and proliferation was highest in 0.5 µM TDZ. Of the various auxins (IAA, IBA, and NAA) tested, regenerated shoots rooted best on half strength MS medium (1/2 - MS) supplemented with 2.5 µM IBA. The rooted plantlets were successfully established in soil and grown to maturity with no gross variations in morphology and cannabinoids content at a survival rate of 95 % in the indoor growroom.

This one is a full article which give details for a couple of different protocols. I think this one is the best of the ones I've found so far.

Pak. J. Bot., 41(2): 603-608, 2009.

A MICROPROPAGATION SYSTEM FOR CLONING OF HEMP
(CANNABIS SATIVA L.) BY SHOOT TIP CULTURE
REN WANG1*, LI-SI HE1, BING XIA1, JIN-FENG TONG1, NING LI2 AND FENG PENG1
1Institute of Botany, Jiangsu Province & Chinese Academy of Sciences, (Mem. Sun Yat-Sen);
Jiangsu province key laboratory for plant Ex-situ conservation, Nanjing, 210014, P.R. China
2Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay,
Hong Kong SAR, P.R. China

Abstract
This study describes the standardization of an efficient in vitro propagation and hardening
procedure for obtaining plantlets from shoot tips of Hemp (Cannabis sativa L.). Hemp seedlings
were germinated on half-strength 1/2 MS medium supplemented with 10 g·L-1sucrose, 5.5 g·L-1agar
at a pH of 6.8 under light for 16 h per day. MS medium containing 0.2 mg·L-1TDZ, 0.1 mg·L-1NAA
supported the maximal auxiliary bud multiplication rate of 3.22 per shoot tip. The proliferated buds
were successfully rooted on MS medium supplemented with 0.1 mg·L-1IBA and 0.05 mg·L-1NAA
resulting in 85% of the plantlets rooting. The procedure requires a 54 days cycle for the In vitro
clonal propagation (14 days for shoot multiplication and 40 days for root induction) which includes
35-42 days for acclimatized plantlet production.

And check out this one! Has some pretty interesting stuff in it.

http://books.google.com.au/books?hl...v=onepage&q=micropropagation cannabis&f=false

 

[Thoth]

And some more:

In Vitro Cellular & Developmental Biology - Plant
Volume 45, Number 1 (2009), 12-19, DOI: 10.1007/s11627-008-9167-5
Published on behalf of Developmental Biology
Thidiazuron-induced high-frequency direct shoot organogenesis of Cannabis sativa L.

Hemant Lata, Suman Chandra, Ikhlas Khan and Mahmoud A. ElSohly

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.

http://www.springerlink.com/content/3028210397611640/

Title
Evaluation of media for hemp (Cannabis sativa L.) in vitro propagation.
Foreign Title
Valutazione di terreni di coltura per la propagazione in vitro della canapa (Cannabis sativa L.).
Authors
Casano, S.; Grassi, G.
Journal article; Conference paper
Italus Hortus 2009 Vol. 16 No. 2 pp. 109-112
Conference Title
Comunicazioni e riassunti del Convegno Nazionale "La micropropagazione in Italia: stato attuale e prospettive future". Un incontro tra operatori del settore e della ricerca, Corte Benedettina, Legnaro (PD), Italia, 20-21 novembre 2008.
ISSN
1127-3496
URL
http://www.soihs.it
Record Number
20093155162
Abstract

The use of in vitro tissue culture techniques has revolutionized propagation, conservation and breeding strategies of several plant species. Hemp tissue culture has not yet been widely exploited by universities and breeding companies because of its difficult reintroduction as a legal industrial and pharmaceutical crop. Various media, different in their inorganic salt formulation, organic additives and plant growth regulators combination, were evaluated to improve the micropropagation rate of meristems excised from selected clones. The best results were obtained using a medium composed of Murashige and Skoog (MS) inorganic salts and Gamborg (B5) vitamins supplemented with 3% sucrose, 1 mg IBA/l, 0.25 mg GA3/l, 0.1% activated charcoal, 0.8% agar and pH=5.8 (MS-based medium). In the few published articles on hemp tissue culture, researchers have always used MS basal nutrient medium for the mineral composition of their media. Adaptation of the mineral composition of the medium to that of the plant necessities might result in better growth of meristems. In collaboration with CANNA Research we have developed a new adapted formula, called β. Formula β-based medium was compared to MS-based medium resulting in a significatively higher micropropagation rate of the meristems grown on adapted medium than on MS.

Title
In vitro culture and propagation of hemp (Cannabis sativa).
Authors
Yin PinXun; Yang Ming; Guo HongYan; Liu ZhengBo; Hu XueLi; Chen Yu
Journal
Southwest China Journal of Agricultural Sciences 2005 Vol. 18 No. 4 pp. 503-505
ISSN
1001-4829
Record Number
20063004147
Abstract

Cannabis sativa was cultured in vitro using stem segment with node as explant. The optimum medium for Yunma No. 1 was MS + 6-BA at 1.5-2.0 mg/litre + NAA at 0.4 mg/litre + GA3 at 0.1 mg/litre for producing buds, MS + ZT [zeatin] at 1.0 mg/litre + 6-BA at 1.0 mg/litre + NAA at 0.5-1.0 mg/litre + GA3 at 0.1 mg/litre for subculture, and 1/2MS + MS(full Fe2+) + IBA at 2.5-3.0 mg/litre for rooting.

At first I was a little surprised at how much info there is out there regarding cannabis micropropagation, but much of it is about hemp, so it makes sense considering the huge potential that hemp has as a cash crop.

I was unable to see the link you posted for some reason...

 

[sprizzle]

daaamn that is sum techinical shit! sound pretty kool tho.
would b sick to see yer work rootsnshoots.

 

[rootsnshoots]

Thoth, Nice work brotha!!

Are you using a laminar flowhood or just a glovebox type arrangement?What were your major equipment expenses?
Yezir! laminar flowhood is built and running. My Major expenses were the flowhood, pressure cookers, and clean room mostly.

What is it you aim to achieve with micropropagation?
My major goal is to be able to propagate pest and disease free ladies is good numbers. I have some good ties in the mmj industry and am looking to do cloning for them. Also I want to be able to store genetics for them as well.

You should see if you can find a specific protocol for cannabis as that will help you be successful. Better not to have to guess on what is needed from the outset. Once you have a protocol that works, then you can tweak it if need be.
That is what I've been hunting for. I would love to not have to run too many experiments with no results. Being the phytohormone ratios are so plant specific it could take a long time to get good results. I have a pretty good idea of what works to get shoots, roots, and callus so my first exp. will involve doing all three. Also the web site i am ordering from has been very helpful. I can't say which site because these guys are straight up Phds and I dont want to step on any toes here. Or cost anyone their job.

This link, The one you said you think was the best you found so far >> was the one I tried to post on my first post. I probably have shitty software on my laptop here and am pretty fuckin stoooopid when it comes to computers haha. I think what i posted was something i have downloaded on my comp.

Any ways i havn't read through the other links you hooked me up with yet, but will get on it later this evening. Thanks for the help I appreciate the hell out of it. uda man!

 

[YarraSparra]

This is really cool Rootnshoots.
I've read into plant tissue culture a bit, even applied for a job doing it once but didn't get it. The place pumped out hundreds of thousands of TC plants every year. That blew my mind!
Other than producing pest and disease free ladies, will you try doing anything else? I met an orchid grower you made entirely new types of orchids through plant TC. Some of them were incredibly beautiful.
Perhaps you could make a fruit salad strain! :D

YS

 

[rootsnshoots]

This is really cool Rootnshoots.
I've read into plant tissue culture a bit, even applied for a job doing it once but didn't get it. The place pumped out hundreds of thousands of TC plants every year. That blew my mind!
Other than producing pest and disease free ladies, will you try doing anything else? I met an orchid grower you made entirely new types of orchids through plant TC. Some of them were incredibly beautiful.
Perhaps you could make a fruit salad strain! :D

YS

Thanks for the love yarra,

Out of curiousity what was the company you applied for? I have heard a lady on a video cinfidently say " you can turn one plant into a million in under ten months" which by no means I have that type of facility/dollars/reason to do numbers like that, but I believe it is possible no doubt.

I have heard of in vitro breeding, but currently am not trying to accomplish or even think about (though as soon as i heard it i was like WAAAA!!!!) that untill I have the basics down to a science. I will definitley be taking things slow in this operation, experiment by experiment. I have a major issue with trying to jump forward too fast and try new things before I even know the basics. ( even my first grow many many years ago was a very complicated aeroponics system, which even now as a pretty experienced grower would have a hard time keeping up with) so i will be keeping the horse in front of the cart on this one. Meaning this thread should be around for a long time to come.

If there is any information you know of please share it with me, I am far from a scientist, but maybe one at heart.

much love,
rns

 

[Ythor]

My major goal is to be able to propagate pest and disease free ladies is good numbers. I have some good ties in the mmj industry and am looking to do cloning for them. Also I want to be able to store genetics for them as well.

Then you will want to make certain that your stock material has been indexed, i.e.: tested for viruses, prior to placing it into axenic tissue culture. Pretty much all the big producers do this with susceptible material; for example, strawberries are all indexed for virus prior to initiation.

Good luck with your work.

 

[rootsnshoots]

Then you will want to make certain that your stock material has been indexed, i.e.: tested for viruses, prior to placing it into axenic tissue culture. Pretty much all the big producers do this with susceptible material; for example, strawberries are all indexed for virus prior to initiation.

Good luck with your work.

Stock material meaning ex-plants or say my auxin cytokinin stocks? If ex plants any ideas on who would be willing to test ganja!

 

[rootsnshoots]

Ok im slackin and I have not been able to purchase the hormones or MS yet. I dont want to let this thread go stagnant so just a quick update here. Im broke.... But I did get ahold of the book Plants from test tubes. I have skimmed over the whole book and gotten in depth on the importance of it. Its pretty basic but got a few tid bits on some of the protocal to going about this. I have said this before but i hope it is true... I am going to order the goods this weekend and get a start on this. I have changed my first exp. to just growing auxillary shoots in all 24 jars. Which means 24 ex plants will be used and then i will probably take half of those shoots and root them on the next exp. I plan to take the other 12 jars and re shoot them bitches to keep the goods going.

 

[neverbreak]

very cool bro! totally amped about seeing this happen! reckon it's worth the investment to play around with this idea if you already have most of the equipment.

i had that same book at one stage but never did anything with it so passed it on. looked pretty decent.

keep up the good work bro.

neverbreak

 

[Ythor]

Stock material meaning ex-plants or say my auxin cytokinin stocks? If ex plants any ideas on who would be willing to test ganja!

The material (plants) from which you collect your explants should be indexed. Testing your mother plant for viruses will help reduce the incidence of viruses in plants grown today.

This is used for plants of much lower value (strawberries) than cannabis, for the simple reason that if you're going to crank out hundreds of thousands of berry plants, they'd better be "clean." The technology hasn't trickled down to cannabis TC for the simple reason that nobody has that kind of demand. TC of cannabis is futile in the sense that you can quickly get into the territory of >99 plants. If the demand were there for 10,000 cannabis plants in TC, the major labs would quickly squash hobbyists from doing it, but it's still illegal at the federal level.

In fact, most commercial labs wouldn't touch anything less than 10,000 or 100,000 starts- simply not enough to merit the outlay. Cannabis is too easy to grow in tissue culture, so the margins would be very thin.

 

[Thoth]

Any progress Rootsnshoots?

 

[homebrew420]

This has always been intriguing, however I feel it sort of impractical for the regular grower. Rootsnshoots sounds as though it would be for profit which may be a very lucrative where cuts are always dirty and pest ridden. I wish you the best of luck and hope to see some progress. Great luck in all you do

Peace

 

[pork]

i hear the TC freshens genetics to their original form...sort of power washes the history of the plant and starts fresh with fresh DNA...so, for instance, doing a TC on a 91 Chemdog would bring it back to it's 1991 form...is that just hearsay or legit?

 

[rootsnshoots]

Thoth,
not really, ive had my hands tied with work and work and a major achohol abuse problem haha. i am waiting for a check to clear and am buying the stuff asap. my partner is losing his patience as well so its time to get it off the ground here... plus im stoked !

pork,
"freshins genectics" can probably be translated into many different things, but no your genetics will be the same as what ever your ex plant tissue contains. But!...... if some one had made callus from 91 chem in 91!!.... then yes ablosutley you could have it stored with those original genetics.

 

[Ythor]

i hear the TC freshens genetics to their original form...sort of power washes the history of the plant and starts fresh with fresh DNA...so, for instance, doing a TC on a 91 Chemdog would bring it back to it's 1991 form...is that just hearsay or legit?

That's just broscience. There's no "resetting" DNA through tissue culture.

 

[rootsnshoots]

Shits in the mail! hopefully I will have a chance to prep and pour plates this weekend!

 

[rootsnshoots]

Sorry for the long wait here fellers! I scored a new job and have been busy as hell. Any who I have almost everything i need to get this party started except for a dissolving agent for the cytokinin TDZ. Which i need to do the first step lol. It is in the mail though, hopefully here by the weekend. If so i will have time next sunday to at least get the stock solutions made up.

THE GOODS:
Plant growth regulators; cytokinin= Thidiazuron (TDZ). Auxin= Indole-3-butyric acid (IBA). Gibberellic acid.

sucrose, plant TC grade agar, Activated charcoal, MS basal salt mixture with and with out Gamborg vitimans. and i think thats it? oh Koh to dissolve the IBA, turns out i just checked and the TDZ can be dissolved in it as well? so maybe i can get started before the DMSO which i thought i needed for the TDZ.

ill keep you guys posted with pics and step by step action on everything.