Cannabis Tissue Culture

Until 2007 there were no published reports of highly successful methods for culturing Cannabis sativa. Hemp was successfully cultured in China but at a success rate of a mere 85%. Then Ole Miss published “Thidiazuron-induced high-frequency direct shoot organogenesis of Cannabis sativa L.” in The Society for In Vitro Biology which detailed how they achieved a 95% success rate.

We found this paper and started making the medium. We were able to get great results with the vegetative propagation medium which utilizes hormones TDZ and gibbrellic acid. We used this medium to multiply and expand our library. The rooting medium calls for half strength MS salts and the exchange of hormones to only IBA and with the addition of activated charcoal. We had problems initially with the medium being watery after sterilization but we have since linked the issue to our gelling agent and have moved forward.

Synthetic seeds

During our research we also came across another paper published by Ole Miss on synthetic seeds. These same scientists were able to develop a formulation that consistently would regenerate 100% of these synthetic seeds under greenhouse conditions. Impressive statistics, but not an original or groundbreaking discovery; synthetic seeds have been around a while and are underutilized technology.

We have also been tooling around in our spare time with synthetic seeds and have some preliminary observations on their implementation.

Our first batch of synthetic seeds.

For the rest of this article see:


Pharmer Joe

14 responses to this post.

  1. Posted by Aaron on October 13, 2013 at 5:34 PM

    Interesting that your rooting formula only contains IBA. The study I read (on hemp) said they had better results with 0.1ppm IBA and 0.05ppm NAA.


  2. Posted by western5 on October 8, 2013 at 6:19 AM

    marijuana from test tubes is now available on the kindle through amazon


  3. This information is invaluable. Where can I
    find out more?


  4. This is what I was referring to.

    Identification of candidate genes affecting Δ9-tetrahydrocannabinol biosynthesis in Cannabis sativa


  5. This is quite fantastic and right down my alley (im a senior year biotech major). I am also part of the largest NORML chapter (UCF), how should i go about getting funding for you all? also, cannabulent could you link me to the site discussing the mRNA profiles?


    • Hey Ryan,

      We are not a registered nonprofit nor are we looking to sell any ownership stake. So any funding would have to be an in kind gift. I believe the study that cannabulent referred to can be found at the site. There you can find the est’s related to your search.



  6. Yup, qRT-PCR would work really well, in a 96 well or 384 well format, you could look at transcript levels for every gene in the pathway at once! It would be super interesting to see how distinct chemotypes (CBD vs. THC rich for example) would differ. And as deep sequencing gets cheaper, it will get easier and more quantifiable to get this type of data.

    With regards to mutations that affect flower morphology, as a proof of principle for the genetic transformation, it could be interesting to make GFP flowers. As the marketplace saturates, one could make quite a splash with glow in the dark flowers. Also doing traditional forward genetics with chemical mutagens could be a lot of fun.


    • I will leave the genome sequence to Medicinal Genomics since they already have an Ion Torrent sequencer and I don’t have an extra 50k.
      Gfp is a fun marker to use and its not the only glowing gene. Check out Invitrogens living rainbow collection. You could use them in concert with different promoters for different localized expression, like green trichomes and red flowers.



  7. Yes, I am familiar with the latter study, but it was my understanding that once transformed, the researchers were unable to fully differentiate the plant into a vegetative state. To that point, do you have any pictures of older clones, ie. ones that look more well differentiated?

    In regards to the mRNA expression profiling. This has been done for cannabis trichomes, so the protocol and sequences are out there, using academic resources one could do this pretty easily. I like your idea of looking at this as a function of development, it would also be interesting to look across sub-types. These are experiments that would be foundational to any attempts to engineer and/or disrupt the cannabinoid biosynthetic pathway. One might also want to look at the GC-MS at the same time and then try to correlate the transcriptional changes with the chemical changes.

    until next time!


    • I know that they have done some Mrna studies and that they have found the specific genes I’m interested in. I was thinking120 clones and analyzing one at each change in the photoperiod. 120 because I would destroy each specimen to do the analysis. I would really like an rtpcr for Christmas but alas I’m not that good of a boy. That would be more interesting to me than the current studies. I’m also interested in mutations that affect flower morphology.

      I look forward to your comments.



  8. I am really impressed with the site and was very excited by this page. Are you mostly pursuing this type of work so that you can store genetic copies/clones of a given strain? It is my understanding that there are no publicly documented cases for the genetic transformation of cannabis. I fantasize about doing so. One could use trichome specific promoters to manipulate terpene and cannabinoid diversity and concentration. But what I would really like to do, is to play with the genes in the trichome development pathway. Work in Arabidopsis has led to the discovery of genes that dictate size, concentration and cellularity of trichome structure. Knocking out homologous repressors of trichome growth in cannabis could be very interesting and could serve as a model system for the use of a plant as a factory for the production of novel/difficult to synthesize natural products. Keep up the good work! You guys rock! (PS: I do not work for Monsanto, nor would I endorse the industrialization of said engineered strains)


    • Hello Cannabulent,

      In response to your knowlegeable comment:
      We are using TC to store clones for future genetic comparison. Our DNA project is currently on back burner status because it’s expensive. We aren’t currently doing any in vitro DNA assays; that doesn’t mean we aren’t paying attention to literature published that helps our on paper project outline. In the future we hope to be able link mutations in the cannabinoid biosynthetic pathway to GC analysis of the trichomes. I am particularly interested in gene expression analysis of the floral bracts and trichomes as they develop. Soon we will be adding a page about our DNA aspirations so keep an eye out for it. (Whole Genome Sequencing)

      There is one published report of transformation that I am aware of (see link) I think they would have been more successful obtaining transgenic plants with a floral dip while the seeds are still immature.




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