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Cannabis Tissue Culture Q&A with Propagation Specialist Mike Fujimoto

Here at Heavenly Buds we are passionate about being on the cutting edge with our grow processes and techniques. We are excited to be experimenting with some exciting new methods, like tissue culturing and synthetic seeds. To enlighten us on these, we sat down with Mike Fujimoto, our Grow Team Propagation Specialist. Mike holds double Bachelor’s degrees in Biology and Environmental Science.

Mike Fujimoto pictured in the Heavenly Buds clone room

Q. What is Tissue Culturing?

A. Tissue culture is a propagation tool where the cultivator would grow tissue or cells outside of the plant itself, commonly referred to as “Micropropagation” or “small cloning.” Basic requirements include macro and micro nutrients, carbs, and light, which is all provided in a test tube.

Master Yoda Tissue Culture Day 1 Master Yoda Tissue Culture Master Yoda Tissue Culture

Q. What are the Benefits of Tissue Culturing?

A. With a proper lab, the same amount of material that you would need for a few clones can make thousands of cultures. A prime example of tissue culturing is that it can be used to repopulate endangered species like the sequoia trees. It’s also more sustainable since you are using less of everything and turning a little bit into a lot.

Q. What are synthetic seeds?

A. Synthetic Seeds are a genre of tissue culturing, they allow the storage of genetic material if you need to hold off on a specimen to be used later and allows for the transfer of genetics. I take nodal segments and encapsulate them, and the gel hardens and forms a synthetic seed.

Q. How does it differ from using a traditional seed?

A. With traditional seeds you don’t know what you are going to get. There is a 50/50 chance of the seed being male or female (only the female cannabis plants produce buds). Synthetic seeds are a genetically identical to the mother plant so you know exactly what you are getting every time.

Q. What are the benefits of using synthetic seeds?

A. You know exactly what you are getting with regards to the genetics of the plant, and it enables the storage of genetic information for long periods of time. This is the future of tissue culture. We can now preserve elite phenotypes without fear of genetic degradation.

Q. Are there any downsides to tissue culturing and synthetic seeds?

A. It’s a complex process and can take up to 90 days to see results, compared to days or weeks with traditional seeds.

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"Synthetic" Cannabis Seeds – Explained

Axillary buds of Cannabis sativa isolated from aseptic multiple shoot cultures were successfully encapsulated in calcium alginate beads. The best gel complexation was achieved using 5 % sodium alginate with 50 mM CaCl2.2H2O. Regrowth and conversion after encapsulation was evaluated both under in vitro and in vivo conditions on different planting substrates. The addition of antimicrobial substance — Plant Preservative Mixture (PPM) had a positive effect on overall plantlet development. Encapsulated explants exhibited the best regrowth and conversion frequency on Murashige and Skoog medium supplemented with thidiazuron (TDZ 0.5 μM) and PPM (0.075 %) under in vitro conditions. Under in vivo conditions, 100% conversion of encapsulated explants was obtained on 1:1 potting mix- fertilome with coco natural growth medium, moistened with full strength MS medium without TDZ, supplemented with 3 % sucrose and 0.5 % PPM.

Sodium alginate was added in the range of 2-6 % (w/v) to full strength Murashigeand Skoog’s medium (MS) with or without 3 % sucrose. The solutions were supplemented with 0.5 μM Thidiazuron (TDZ) and 2.5 μM indole-3-butyric acid(IBA). A broad spectrum fungicide, Plant preservative mixture (PPM) in a range of 0.3-0.5 % was added to the gel matrix for in vivo experiments. For complexation, different concentrations (25-100 mM) of complexing agent (CaCl2.2H2O) were prepared in liquid MS medium containing the same adjuvents as the sodium alginate matrix but excluding the PPM. Both the solutions were autoclaved separately for 15 min at a pressure of 1.1 kg cm-2 and temperature of 121 ºC after adjusting the pH to 5.8.

Formation of beads:

The beads were formed by dropping explants mixed with sodium alginate solution into CaCl2.2H2O in a flask, placed on an orbital shaker at 80 rpm. The resulting beads (0.5-0.8 cm in diameter) containing the entrapped nodal segments were left in the calcium chloride solution for 30 min for complexation. These were retrieved using a nylon mesh and the traces of calcium chloride was removed by washing with sterilized distilled water.

Propagation through alginate encapsulation of axillary buds of Cannabis sativa L. — an important medicinal plant

Cannabis sativa L. (Cannabaceae) is an important medicinal plant well known for its pharmacologic and therapeutic potency. Because of allogamous nature of this species, it is difficult to maintain its potency and efficacy if grown from the seeds. Therefore, chemical profile-based screening, selection of high yielding elite clones and their propagation using biotechnological tools is the most suitable way to maintain their genetic lines. In this regard, we report a simple and efficient method for the in vitro propagation of a screened and selected high yielding drug type variety of Cannabis sativa, MX-1 using synthetic seed technology. Axillary buds of Cannabis sativa isolated from aseptic multiple shoot cultures were successfully encapsulated in calcium alginate beads. The best gel complexation was achieved using 5 % sodium alginate with 50 mM CaCl2.2H2O. Regrowth and conversion after encapsulation was evaluated both under in vitro and in vivo conditions on different planting substrates. The addition of antimicrobial substance — Plant Preservative Mixture (PPM) had a positive effect on overall plantlet development. Encapsulated explants exhibited the best regrowth and conversion frequency on Murashige and Skoog medium supplemented with thidiazuron (TDZ 0.5 μM) and PPM (0.075 %) under in vitro conditions. Under in vivo conditions, 100 % conversion of encapsulated explants was obtained on 1:1 potting mix- fertilome with coco natural growth medium, moistened with full strength MS medium without TDZ, supplemented with 3 % sucrose and 0.5 % PPM. Plantlets regenerated from the encapsulated explants were hardened off and successfully transferred to the soil. These plants are selected to be used in mass cultivation for the production of biomass as a starting material for the isolation of THC as a bulk active pharmaceutical.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.