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The Ins and Outs of Tissue Culture Production

Dr. Sma Zobayed, Chief Tissue Culture Officer at Segra International explores The Ins and Outs of Tissue Culture Production.

Dr. Zobayed has over 25 years of expertise in the tissue culture industry and he's the mastermind behind Segra Internationals plantlet production efforts in Canada. Today he'll be going through the overall process of tissue culture initiation, multiplication and acclimation of tissue culture cannabis plantlets. He'll also review the various methods of tissue culture production including explant multiplication methods which we use here at Segra.

Today we’ll discuss...

What plant tissue culture is. The challenge with the traditional vegetative propagation methods, specifically in the cannabis industry. Multiplication techniques that exist in different areas of tissue culture. The commercial tissue culture process in agriculture and horticulture process. And finally we’ll talk about, Photoautotrophic Tissue Culture system and its possibility in the cannabis industry for tissue culture.

Let's start with Plant Tissue Culture.

What is plant tissue culture?

It is a vegetative plant propagation method where rather than using the greenhouse or outer environment, we actually use this under a controlled sterile environment which is with artificial nutrients and growth regulator. We control the growth and the production efficiency of the plants so we can multiply them as we require rather than growing the plants to larger sizes. These plants are normally always true to type and pathogen-free/disease-free. Tissue culture is the only possible way of vegetative propagation where you can actually scale up the process.

What is tissue culture?

A method of mass vegetative plant propagation on an artificial nutrient medium under a controlled sterile environment, ensuring pathogen-free, true-to type plants.

So, if you require thousands or millions of plants through vegetative propagation, this is the only way you can do so.  Industries like agriculture and horticulture, both industries currently use this modern technology for their global supply of fruits.  For example: strawberries, raspberries, bananas, blackberries, are all through tissue culture. In the horticulture industry for example orchids, the rhododendron, azalea, ornamental grass farms, they're well established in tissue culture technology already worldwide.  We are using the same logic, process and technology for the cannabis industry. I'm going to explain how Cannabis is still in the adoption phase of this technology behind other industries and how we’re making progress in the right direction.

How the cannabis industry is propagating for their own demand?

The current methods are mostly using cuttings. That's the vegetative propagation. Almost 10% of their production facility is occupied by mother plants that are required to grow the plants and used to take cuttings.

Those cuttings are then turned into clones.  They go into a high and humid condition in a chamber where you induce the root formation.  Eventually those are rooted, transplanted, and are moved to the production facility for further growth. 

Now I'm going to explain what the issues are with these techniques, but before going there, let's discuss what is happening when you grow mother plants in a highly humid environment. There’s always the possibility of bacteria or fungal spores. If landed on the plants surface, they can cause pathogenic disease.  These bacteria or fungal spores can be carried on to the next group of clones produced.  When they get rooted in the main production facility where the environment is humid, the conditions are very good for these kind of microbes to multiply and result in crop loss. It's very common in the cannabis industry and we’ve seen a number of facilities shut down due to crop loss and other related issues.

Another industry challenge is the scaling up of cannabis propagation.

I'm going to use an example from California.  California is one of the largest cannabis production states in the U.S. and comparative with Canada in size. 

How the cannabis industry started in California? 

Let’s go back to 2005, at the time regulations restricted growers to a limit on the number of plants allowed to grow. So each grower was allowed only a hundred plants. To get around this limitation was they grew the plants to giant size with a low planting density. As a result they did not require a large number of plants. So with a hundred plants and their use of cuttings, there really wasn’t a practical reason or use for tissue culture at the time. However, the regulation had changed 10-15 years ago and now it's no longer regulated by the number of plants but rather by canopy size.

So today they have to work within the canopy area, this means the growers need to change their method of producing plants. They work with smaller plants at higher densities to produce more flowers.

So the previous technology that was used for 100 plants does not translate to the current needs and demand. Even though there is still cloning, the reality is that tissue culture technology needs to take over the cannabis industry, similar to the current agriculture and horticulture industries.

How Plant Tissue Culture Works

Let's talk a little bit about how plant tissue culture works.

In plant tissue culture, when we receive a plant, we usually take the cuttings from the mother plant and conduct the meristem culture from there. I'll explain the meristem culture further in this article.

The second step is we sterilize the surface. The surface sterilization cleans any of the bacteria and the fungal spores.  Also due to meristem cultures, we get rid of any virus. So it's a disease-free pathogen free plant.

At this stage we can either store the plant or the tissue in our storing media.  At that point we can go straight to the multiplication stages so we can multiply the plants according to the demand.  This can go to thousands of plants or millions of plants if required.

From there if we have enough stock, we can transfer to the rooting phase. We call it Invitro Rooting or Stage Three (3).

From stage three it can go to stage four where we acclimatize in an outer environment.

From either stage three or stage four we can deliver the plants to our clients, nurseries or licensed producers. 

Let’s discuss the standard operating procedure at Segra for tissue culture. 

At Segra we thoroughly validate the cannabis tissue culture process.

What do we do when we receive a plant from our clients?  We immediately initiate the plants upon arrival into our facility. We sample and perform a DNA Fingerprint in our lab. We then store that DNA fingerprint in our library.

Once again during initiation we are cleaning the plants to remove bacteria, virus and/or fungus.  So with clean plants they then go through physiological changes and onto the multiplication phase, we call this Stage two.

In the stage two phase, they multiply.   Once the required amount of stock is reached, we retain inventory to restock for the multiplication phase. For example if a client requires 10,000 plants monthly, we would need just two to three thousand plants to restock the inventory so we can transfer and multiply into 9000 or even 27,000. 

So we keep some inventory to stock the next batch and the rest go to stage three, also known as rooting.

From rooting, it requires three to four weeks for the plants to be rooted.  From there we can either ship it at the stage three phases or we can transfer them to stage four. 

Before shipping our plants to our clients, we perform a DNA fingerprinting to ensure it matches with our initial genetic fingerprinting data and phylogenetic tree data.  We make sure it's the exact same plant variety.  During the multiplication phase at stage two, depending on the size of the batch we're working with, it usually takes two to three months to reach it's quantities.  Every two to three months we do auditing.  Independent auditors come, collect the plants samples, go to the lab, do the DNA fingerprinting, and make sure it matches with the original samples.  The reason for this is to validate if there were any mix ups internally and to verify if there has been any somaclonal variation.

What is Somaclonal variation?

It's a kind of a mutation that sometimes happens in plant tissue culture. In our case at Segra, the way we manage the tissue culture process we technically shouldn’t see any somaclonal variation. However, in the rare occasion we encounter this we can actually track those plants which mutated, we use our DNA fingerprinting to separate the plants. We then isolate and destroy the somaclonal variant plants and we can reinitiate clean plants from the true genetics we store in our labs. So this is an established procedure where we can separate all kinds of mutation and any possible mixing.  We can ensure that clients are receiving the best plants possible and that are matched with the original genetic sample, true to type cultivar.

Let's discuss how, in general, plant tissue culture works.

The tissue culture technology that is used worldwide in agriculture and horticulture industry is generally regulated with four different stages.

Stage 0 is when we get the donor plants, at Segra we do the DNA fingerprinting at this stage.  

Stage one is the Invitro establishment where we actually do all the surface sterilization, clean the plants, take the meristem out of these tissues and then they are inoculated into the growing media (initiation media). This invitro environment called In vitro plants.  Then they're generally clean from any bacteria virus or fungus and they're pathogen-free.

The next step is the step, Stage Two or Multiplication, as explained earlier, they are multiplied here anywhere from two times up to twenty times. In agriculture we’ve seen grasses at twenty times multiplication and in horticulture some fruits can produce 15 to 20 times of multiplication in a month. This is that most important stage in tissue culture.

When the multiplication or stocks are established, we send it for elongation.  Some plants require more or less elongation and then we send them in the rooting or stage three. 

After stage three is Acclimation, Stage Four. Acclimation is where the tissue culture plants experience the Ex vitro environment.

What's the difference between an In vitro environment and Ex vitro environment?

In vitro is a Carbon source. We supply carbon, sugar or sucrose or any format carbon in the tissue culture media so plants do not require photosynthesis. There is some photosynthesis still happening however, it's minimal.   We call it for photoautotrophic plants.  Some of the plants are heterotrophic, some are autotrophic. So in this case they are not required to use their own energy to multiply.

In the Ex vitro condition, they are no longer carbon supplied.  They do their own photosynthesis, they update the CO2 from the air and do their own carbon production.  This is the first challenge they have to face to be acclimatized in the outer environment. 

In vitro is an aseptic environment with low light intensity and higher relative humidity. Where as in the Ex vitro environment they are exposed to outer elements, so it's not an aseptic environment anymore.  They have to face this fungal or microbial or pathogens or insects and fight back.  Higher light intensity is required for them to do the photosynthesis and relatively lower humidity, ambient humidity.  So this is the environment they need to acclimatize in this stage (4) and then go into the production facility.

Let's discuss some of the techniques currently developed in the area of plant tissue culture. 

Number one is Somatic Embryogenesis.

Number two is Artificial Embryos.

Number three is Organogenesis (direct and indirect) regeneration.

Number four is Meristem Culture.

Currently at Segra when plants arrive we do the Meristem culture. From there we do the direct Organogenesis.

First let’s take a look at what is Somatic Embryogenesis.

It's a somatic embryo developed from vegetative cells or somatic cells. The process starts with Globular small round shaped cells.  From there they go to the Heart shape and followed by a Torpedo shape. In it's final stage, Cotyledonary stage, they develop the roots and are considered as independent plants.

How is the process done in Somatic embryogenesis? 

There are two different ways. The first is indirect where an explant can induce a callus, so each cell can produce an embryo. From there they can develop the potential stage embryos and then finally the independent plants.

The other is direct embryogenesis - the leaf explant or any other like stem cuttings or a cotyledonary part or the root that can directly produce embryos, from the edge of the cuttings it will produce embryos. One of the examples I can give you is related to an Echinacea leaf for example, the leaf discs produce embryos from the edges. They're elongated and eventually produce roots. This is an example of root somatic embryogenesis direct regeneration.

Now let’s talk about Artificial Embryos.

It's a really interesting technique. It’s basically a somatic embryo. It's covered with a synthetic seed coat filled with endosperm that is artificial.

Endosperm is a food material. These reserved food materials give the longevity and shelf life for the embryos to survive when they go out and into the field or in a plug or anywhere they can germinate.

The real somatic chamber is sitting in the middle, that's the real plant.  Eventually they germinate like regular seeds.   There are some limitations that still exist and more research is required before there’s a fully commercialized version of this technology.

Next: Organogenesis, specifically non-meristematic.

Using a leaf as an example, which is a non-meristematic tissue or in any other explant, can produce plantlets indirectly.

For example they can produce a callus and from the callus they regenerate and produce plantlets or each explant can produce shootlets and then they become independent plants.

These are two different ways they can produce. 

If we can produce the callus or the shootlets from a meristematic tissue, we can call it organogenesis through meristematic tissue. Meristematic tissue are generally called virus free.

Organogenesis (non-meristematic) direct nodal culture technique

A process where for each plant you take a nodal cutting. You can take cuttings out of cuttings rather than multiplying. You can actually multiply in bunches from one shoot to 6 to 36. This is one of the techniques commercially used in the blueberry industry.  Millions of blueberries, here in the Lower Mainland of BC, are produced through this technology. However one thing to mention, we said it is non-meristematic, but if your first plants come through the meristematic tissue then it could be virus free again. In a lot of cases in order to cut down the wait time of six to 12 months, people just start with cuttings and then they multiply.  This is not recommended, in my view. 

Organogenesis (non-meristematic) Liquid culture

...is an indirect process using callus production through liquid culture. Using this technique, you can produce the callus through the liquid suspension culture to produce the solid callus culture and then you can regenerate the plants.  This is another type of organogenesis, however there are drawbacks. The callus production process has the possibility of mutation. This is a key reason why we avoid this kind of regeneration procedure in our facility, especially for the cannabis industry.

Our 4th example of tissue culture is Meristem Culture.

We've touched on this a few times now, so let’s discuss further.

What is meristem? 

It's the upper layer of the plants cell tissues where they are not connected with the vascular system like xylem or phloem. These are dome shaped in the upper layer of the tissue (cells). With this solution the cell division occurs faster than a virus can replicate, rendering this process as virus free.  This is the reason why you should always start with meristem culture.

Other techniques also include Microtubers.

This is a technique commercially used in different areas in the world. So this is true tissue culture through either liquid culture (bioreactor) or even solid culture.  This is a virus free process and often use for potatoes since they are susceptible to virus’.

Bioreactor is very common in the tissue culture industry nowadays for scaling up the multiplication phases.  Different types of bioreactors, is and has been used in the field of tissue culture.  Temporary immersion bioreactor is very common and very popular.  

Bioreactor Bamboo Tissue Culture production is also commercially used to create thousand plants for the Bamboo industry.

What is Photoautotrophic tissue culture?

This is the process of growing plants in sugar-free media.  This process is similar to growing the plants in an outer environment, still sterile pathogen-free but they're doing their own photosynthesis and not depending on the sugar supply.

Similar to what happens in an outer environment in the presence of light that chlorophyll produces, with the presence of CO2 and water, they produce their own carbon. This is a technique we are using in tissue culture. The reason for this is, if the sugar is the major source of contamination, then by avoiding the use of sugar you can scale up your production which at this stage and the acclimatization is faster because the plant is already adopting the up-take of CO2 and producing their own carbon source.

What is the difference between phototrophic and current conventional tissue culture system? 

The current conventional tissue culture system is low in CO2. It uses an artificial airtight system.  During dark periods, because the vessel is airtight, it's high in CO2 acclimation, it has low net photosynthetic rate and as a result has poor growth. It has the potential for microbial contamination. There is a possibility that as a result acclimatization generally have an issue.

In a Photoautotrophic system we increase the CO2 and then we don't add any sugar in the media, this way the plants are forced to develop their own  carbon, in essence they are practicing their own natural way of producing the carbon. When we reduce the humidity, we get a functional stomata that results in higher growth and higher survival in outer environment.

How we can enrich the CO2 at a smaller scale?

Filter membranes can be used and increase the room CO2 to 1000 to 2000 ppm. Then we can enter growing energy into the headspace of the cultural vessel and the plant can uptake the CO2 from there.

Coming back to Segra where we are using tissue culture. We developed a technique called "Stage 3.5 vessels".  Our plants are growing in a sterile vessel, sterile condition sugar-free media, and the plants can be shipped directly to our clients without any washing of roots or anything else. We simply transfer those plants & send them to our clients with the box.  We call it a "Micro Plug".

We use this for domestic shipping so plants are grown and shipped in the same box without any additional handling.  The vessel is designed to resist shipping damage or stress within the delivery.

For our international shipping as we cannot ship plants in this format. We use a Stage 3 system to get the supply to our clients. Domestic or International we can deliver our virus free tissue culture plants to our clients all around the world.

At Segra we do a lot of research and development that is vital to the cannabis industry.  There are many techniques and technology that will define the future of tissue culture in the cannabis industry.  Segra is a leader in Cannabis Tissue Culture in Canada and internationally.

Source: https://www.youtube.com/watch?v=4ABMYZ-E06Q

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