Micropropagation through plant tissue culture.

[BobbyIronsights]

I like to read. and what I'm reading lately is on plant tissue culture, specifically

Plant Tissue Culture-Theory and Practice (Studies in Plant Science) By Dr.s Bhojwani and Razdan,

and

Plant Tissue Culture, Third Edition - Techniques and Experiments by Dr. Roberta H. Smith

Really exciting reading both, and the english of the two university of Dehli's professors is faultless, at least in the book.

I'm wheelchair bound, but while researching propagation techniques it occurred to me that I could dust off the old microscope and amuse myself with a part of the cannabis hobby that plays to my strengths (a pure sciences background) and not my weaknesses (physical limitations.).

I mean, I'm a chemist (bachelors) by training, but I still have a chemical supply house account and I know where to get autoclaves and other used scientific equipment.

I was particularly fascinated by "rescue" culture, where the embryo is freed from non-viable seed (because of extreme age or immaturity) and cultured into a mature plant via agar or broth based media.

I've been thinking that it may not be out of the realm of possibility to setup a small botany lab, and fiddle around a bit. For fun, and to stave off boredom.

Is anyone else here practicing cell culture? Are there other textbooks I shouldn't miss out on reading?

Thanks for your time.
Bob

 

[dmanexe]

Hey Bob,

I am looking into Tissue Culture as a means to propagate clones, I heard this is hands down the most effective way to produce massive numbers of clones. What have you learned since you posted this? I am very curious to see what else we can find out...I've found a few rookies on youTube but nothing pro...

 

[kasvi]

I am very interested in tissue culture of cannabis. I have read "Plants from test tubes".

I want to save some strains and cant keep any moms or grow for few months after plants are finished.
I will use technique described here -> https://ddd.uab.cat/pub/tfg/2014/119249/TFG_javierlidoylogrono.pdf

I am also interested trying embreyo rescue from imature seeds of Yumbolt x Maple leaf indica I got from my friend.

If you or any one else try embreyo rescue can you please share your technique.

 

[kasvi]

I forget to add that I don't have bioreactor so I just subculture callus to petri dishes.

 

[Phenome]

"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 regener- ants were better with thidiazuron (0.5 μM thidiazuron) than with benzyladenine or kinetin. Adding 7.0 μM of gibber- ellic 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 acclimatiza- tion, 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 transpira- tion 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 (Ci) and the ratio of intercellular CO2 concentration to ambient CO2 (Ci/Ca) 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://home.olemiss.edu/~suman/Directshootorganogenesis.pdf

 

[kasvi]

Thanks a ton Phenome for that paper. I will try that technique on some autoflowers.

 

[kasvi]

Do any one have this article as pdf? http://link.springer.com/article/10.1079/IVP2003454

 

[kasvi]

Do any one have this article as pdf? http://link.springer.com/article/10.1079/IVP2003454

I found it here https://www.icmag.com/ic/showthread.php?t=97494

I foud protocol for producing plantlets from leaf tissue.
http://www.ncbi.nlm.nih.gov/pubmed/20354950
I dont have full text but some info of it can be read on bage 35 onwards in this pdf ->https://zerista.s3.amazonaws.com/item_files/1348/attachments/31875/original/109.pdf
There is even protocol for producing synthetic seeds.(page 48)

Does anyone have any cool tissue culture projects going? Can you share pics pls?

 

[vermontman]

PLEASE HELP!

PLEASE HELP!

I like to read. and what I'm reading lately is on plant tissue culture, specifically

Plant Tissue Culture-Theory and Practice (Studies in Plant Science) By Dr.s Bhojwani and Razdan,

and

Plant Tissue Culture, Third Edition - Techniques and Experiments by Dr. Roberta H. Smith

Really exciting reading both, and the english of the two university of Dehli's professors is faultless, at least in the book.

I'm wheelchair bound, but while researching propagation techniques it occurred to me that I could dust off the old microscope and amuse myself with a part of the cannabis hobby that plays to my strengths (a pure sciences background) and not my weaknesses (physical limitations.).

I mean, I'm a chemist (bachelors) by training, but I still have a chemical supply house account and I know where to get autoclaves and other used scientific equipment.

I was particularly fascinated by "rescue" culture, where the embryo is freed from non-viable seed (because of extreme age or immaturity) and cultured into a mature plant via agar or broth based media.

I've been thinking that it may not be out of the realm of possibility to setup a small botany lab, and fiddle around a bit. For fun, and to stave off boredom.

Is anyone else here practicing cell culture? Are there other textbooks I shouldn't miss out on reading?

Thanks for your time.
Bob

Hello Bob
Maybe you can help? Just started tissue culture today. I have been very proficient at Orchid in vitro seed work but I am trying to make this fly. My query is I used M.S. Two batches formulations at full and half strength and I forgot to add Sugar, BUT do these need sugar if there is plenty of leaf to absorb light and produce there own? I also used IBA. rooting hormone. I would be perfectly happy if these would just set root for this batch, If anyone else has input I am all ears.
Pic in next post

 

[vermontman]

Stabbing atttempt

Stabbing atttempt

TISSUE CULTURE 002.jpg

 

[vermontman]

"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 regener- ants were better with thidiazuron (0.5 μM thidiazuron) than with benzyladenine or kinetin. Adding 7.0 μM of gibber- ellic 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 acclimatiza- tion, 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 transpira- tion 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 (Ci) and the ratio of intercellular CO2 concentration to ambient CO2 (Ci/Ca) 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://home.olemiss.edu/~suman/Directshootorganogenesis.pdf

Howdy
I suspect maybe you can help?

 

[Cannabologist]

Howdy
I suspect maybe you can help?

Vtman - this is what you want https://www.researchgate.net/public...ochemical_and_genetic_fidelity_of_micropropag

- DL using the DOI from Scihub. http://www.sciencedirect.com/science/article/pii/S221478611530019X
In vitro mass propagation of Cannabis sativa L.: A protocol refinement using novel aromatic cytokinin meta-topolin and the assessment of eco-physiological, biochemical and genetic fidelity of micropropagated plants

- Broken down SOP (standard operating procedure) for you pot heads like me

> Updated method using MS medium (3% w/v sucrose, 0.8% Type B Agar, 500 mgL-1 activated charcoal >available though sigma), and 2.0uM Metatopolin. ...
use 25mL's sterile medium mixture in glass culture vessels 4cm diameter by 9.5cm high with Magenta B caps..

Incubate @ 25degrees C on 16h photoperiod under fluros of 52 uMol-2s-1 ...

This is for nodal segments... The explants should be washed with surface disinfectant of 5% NaOCl (15% v/v bleach) and .1% Tween for 20 mins then washed in sterile distilled water 3 times for 5 min each prior to inoculation on medium...

Well rooted plants are removed from medium and washed with tap water to remove all traces of medium on roots...
(Harden off plants and) Place plants in growth medium for approx 10 days before transplant @ 700 uMol with 16 hr light, temp 25-30degrees C, 60% humidity.


I am sure I am missing some steps this is very general if anyone can do better updating this methodology for a general SOP for cannabis tissue culture please do so.
Use of Metatopolin comprises a one step procedure for tissue culturing of PLANTS

Most all the stuff is obtainable pretty cheap from SIGMA – ie MS medium Murashige and Skoog Basal Medium plant cell culture tested, with sucrose and agar | Sigma-Aldrich http://www.sigmaaldrich.com/catalog/product/sigma/m9274?lang=en&region=US Activated charcoal suitable for cell culture, suitable for plant cell culture | Sigma-Aldrich http://www.sigmaaldrich.com/catalog/product/sial/c9157?lang=en&region=US.................... As you can see... Something is nto woking riht ;-D ............... Ignore it and just have fun!

 

[Florere]

Nice

 

[detox²]

Are there other textbooks I shouldn't miss out on reading?

pbo.ftk.uin-alauddin.ac.id/e-learning/pbo_file/Edwin%20F.%20George,%20Michael%20A.%20Hall,%20&%20Geert%20Jan%20De%20Klerk%20-%20Plant%20Propagation%20by%20Tissue%20Culture%20Volume%201_The%20Background%20[2008].pdf

Sorry did not work as a link...

This a very well know plant tissue cuture book by George, he is a little bit of the godfather of tissue culture book as far as I know.
I don't know why this book is free. Is it?

I have done a lot of tissue culture. It is really cool.
But I do not believe that one should use it for commercial propagation if not needed. Simply cos it will be more expensive considering the total amount of energy required and working time. It is true that in an early stage of propagation process less space is required. Propagation by adventitious shoots requires two steps. Producing shoots and let them root, let's say 8 weeks or at least 6 weeks. You will need two types of media. I think with the same amount of energy (and money) you will get more biomass on motherplants and rooted cuttings faster. Just my two cents, this is something that easily can be investigated. Maybe you can benefit from tissue culture in cannabis propagation.

Anyway it is cool like i said. Bringing plants into in vitro is easy.

1. You need sterile nodes. Put them in MS medium with 20g/l of sucrose. Add 3mg/l cytokinin to your media.

2. Shoot growth, same media without cytokinin and 10g/l sucrose.

3. Rooting works good with IBA with 5mg/l. You can try muss less salts and sucrose. Depending on that root morphology changes. Especially if phosphate is low.

Have fun, any questions, feel free...

Cheers

 

[Darpa]

Plant tissue culture of nodal explant is only relevant for mass production of plantlet. Within its field of application this technique offers important advantages compared to traditional propagation technique for well-established Authorized Licensed Producers, but the method is not suited for the hobbyists and small-scale grower (cost \ time \effort) unless you are a science enthusiast. Plant tissue culture of nodal explant is really close to traditional cloning except that everything need to be sterile. The real advantages of plant tissue culture require the use of callus induction. When you master this technique, there is no limit to your imagination. (Creating true-breeding (homozygous) lines instantly from doubled-haploid technique \ making sterile seeds \ creating new strains through Protoplasm fusion of two distinct strains or plant species \ plant cell transformation… let say overexpressing trichome gene expression…)

Here’s a good publication on plant regeneration from leaf derived callus:

High frequency plant regeneration from leaf derived callus of high Δ9-tetrahydrocannabinol yielding Cannabis sativa L.
Lata H1, Chandra S, Khan IA, Elsohly MA

Also, for those who don’t know where to start, PhytoTechnologies Laboratory are selling Hemp/cannabis micropropagation kit which is quite complete.

 

[P. Dondrub]

pbo.ftk.uin-alauddin.ac.id/e-learning/pbo_file/Edwin%20F.%20George,%20Michael%20A.%20Hall,%20&%20Geert%20Jan%20De%20Klerk%20-%20Plant%20Propagation%20by%20Tissue%20Culture%20Volume%201_The%20Background%20[2008].pdf

Sorry did not work as a link...

This a very well know plant tissue cuture book by George, he is a little bit of the godfather of tissue culture book as far as I know.
I don't know why this book is free. Is it?

I have done a lot of tissue culture. It is really cool.
But I do not believe that one should use it for commercial propagation if not needed. Simply cos it will be more expensive considering the total amount of energy required and working time. It is true that in an early stage of propagation process less space is required. Propagation by adventitious shoots requires two steps. Producing shoots and let them root, let's say 8 weeks or at least 6 weeks. You will need two types of media. I think with the same amount of energy (and money) you will get more biomass on motherplants and rooted cuttings faster. Just my two cents, this is something that easily can be investigated. Maybe you can benefit from tissue culture in cannabis propagation.

Anyway it is cool like i said. Bringing plants into in vitro is easy.

1. You need sterile nodes. Put them in MS medium with 20g/l of sucrose. Add 3mg/l cytokinin to your media.

2. Shoot growth, same media without cytokinin and 10g/l sucrose.

3. Rooting works good with IBA with 5mg/l. You can try muss less salts and sucrose. Depending on that root morphology changes. Especially if phosphate is low.

Have fun, any questions, feel free...

Cheers

Having also performed tissue culture, I have to agree it is both expensive and time consuming, unless specific results are desired.
It can be an excellent way of getting rid of pathogens without the use of systemic agrochemicals, for example.

Everyone should be clear though, as was said earlier in this thread, the cytokinin of choice that works better for cannabis is meta-topolin, don't use other cytokinins and you can likely avoid IBA that way too. Meta topolin is believed to avoid disruption of the endogenous auxin pathway, negating the need for later application of IBA.

FYI to all, auxin (eg: IBA) and cytokinin are antagonistic to one another. It is preferential to use the least amount of any PGR at any time that induces the desired growth, as it is less likely to disrupt the plants natural systems, and result in aberrant variation or growth.