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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.
  • Inconsistent size of capsule; we are looking into a suitable fluid distribution system that can produce a consistent volume in each droplet.
  • Irregular shapes; we have found in our research that a surfactant can be added to the hardening bath to break the surface tension, which prevents the droplet from being deformed by the impact.
  • Nonconforming product placement; getting the explant into the center of the droplet consistently has been an issue. We are trying different strategies with promising results so far.
  • Lead time; this is an obstacle because the incubation time before sowing is 30 days with 75% success rate and 60 days for 100% success rate. In order to be successful explants also need to have come from a specimen already in culture because some varieties don’t take to culture. This could be the medium we are using or genetics we will know more in the future.

This technology has great promise if these obstacles are overcome. We are making headway with an automated capsule making machine design. We will keep you updated as this project progresses. Attached Photos

Primary Explants

Mature explants almost ready to tranfer

Divided and Tansfered cultures
Division
Division in Rooting medium

We now have the beginnings of a plant tissue culture lab and we are working on building back up our strain library. In the meantime check out the hand built laminar flow hood that I designed and built. It had to be built in the lab because there is no way it’s getting through either door. It has a 4’W X 2’T X 2’D workspace. It is made from ¾” melamine board and has a 1200 cfm fan inside. Two high quality pre-filters and a HEPA final filter. I didn’t take any construction pictures because it wasn’t a pretty sight until it was finished.  I got my HEPA filter from another pharmer for free and the rest it set me back around $500. The fan was $200 the Lumber around $150 I had to buy a brad nailer  for $50 and the tempered glass top with the rounded edge was $80, then all the edge tape and ¾ round plastic to secure the glass,  plus sealant’s, caulk and such ate the rest.  I will be getting some state paperwork finished up this week and will then start accepting donations of genetics for our reserve. All strains once established and multiplied will be shared free of charge with the local MMJ community. Once we finish the lab  and have some more eye candy;  we’ll be sure to share.

Laminar Flow Cabinet
Lab Bench
Sterilized Distilled Water
Multiplication Medium
Synthetic seeds rehydrating

Joe

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39 Comments

  1. Taylor

    You guys are so inspiring. I finally have got my flow hood and my lab setup. do you guys think you could send me a copy of the paper? cascademtnpainting@gmail.com Much thanks! Cheers!

    Reply       Edit
  2. sunset ltd

    The article about synthetic seeds that you reference above, is it also written by Lata and entitled "Propagation through alginate encapsulation of axillary buds of Cannabis sativa L. — an important medicinal plant?" just wan to make sure i'm reading the right thing.

    Reply       Edit
  3. ron

    GW, I ran into something several years ago that would be of interest to you possibly, I have the PDF of this if you don't have it in your library. I was going to send the PDF via this site but it wouldn't stick. Is there an email address? 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. Its a scholarly work on how to grow cannabis in vitro. Let me know if you would like me to send it or you could probably bring it up with Google using the above information. At the time of my interest I couldn't find anything like this, it the "recipe" that everyone was looking for at the time.

    Reply       Edit
    1. skunkpharmresearch

      Hi Ron, thanks for the heads up! That looks like something that Joe will be interested in too, as he is conducting our micropropagation experiments. I would love a copy at graywolf@skunkpharmresearch.com and I will forward Joe a copy, or you can also e-mail Joe attachments directly at joeoakes@skunkpharmresearch.com.

      Reply
      1. ron

        GW by now both you and Joe have the PDF, at the time when I found this I could find nothing like it, would be fun to contact the Chinese scientists and see how much further they have taken this. It apparently is the road map of application that so many people were looking for a couple of years ago.

        Reply
        1. skunkpharmresearch

          I look forward to learning more myself! After Joe has had time to absorb the contents, I plan to pick his keen alleged mind! He is consulting in CA land as we speak, but is scheduled to return soon. GW

          Reply
  4. One Sun

    Is this the paper on synthetic seeds? If not do you mind sharing it? http://www.medicinalgenomics.com/wp-content/uploads/2011/12/Synthetic-Seeds.pdf

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