featured image by the one and only @kandidkush

1. Introduction

In recent years, automation and standardization of the crop production process are becoming increasingly important. Especially in the production of cannabis, it is crucial to have uniform and disease-free planting material in order to reduce any plant protection measures to a minimum and to ensure a final product with consistent quality. For this reason, more and more growers decide to source their seedlings from in-vitro propagated or tissue cultured material. But what does In-Vitro production and tissue culture even mean? And how is it done with cannabis? That is what we want to explain in this Article.

In Vitro is Latin and means “in glass” or “in the glass” therefor when according to plant production, it is a technique that uses glass containers such as petri dishes and test tubes as a controlled artificial environment for the propagation of plantlets. This contrasts with in-vivo (“within the living”) and in-situ (“on site”) production, which are commonly used in horticulture.

Within in-vitro production there are various methods and ways to propagate the desired type of explant. Tissue culture and micropropagation are two terms that you will most likely come across while looking closer into that topic. But what exactly is the difference between those?

The main difference between micropropagation and tissue culture is that micropropagation is the production of a large number of plants from a small amount of plant material, whereas tissue culture is the first step of micropropagation, where plant cells are grown in an artificial medium to develop them into a large number of plantlets. In addition, micropropagation requires tissue culture for the propagation of plantlets.

As the global market advances to a greater reliance on plants for active ingredients, delivery of consistent and pathogen free plant material is crucial. Anyways, in the production of medicinal or recreational cannabis, the use of these techniques is not yet widespread, as cuttings can also be produced via “normal” vegetative propagation but, the higher the degree of automation and the producers’ demands for cleanliness become, the more this method comes into focus. In addition to the possibility of producing clean plants, in-vitro culture is also suitable for space-efficient growing, improved yields due to vigorous plants and for saving production costs.

However, the in vitro cultivation of plants is not only suitable for propagation but is also of great interest for research and especially for breeding. For example, pathogens can be eliminated from plants, mutations can be created and sterile backups of rare or hard-to-retain plants can be secured. There are many ways to utilize tissue culture and other biotechnological methods and there are even more to be discovered.

2. Basic Terms for In-Vitro production

2.1 Medium

2.1.1. What’s Agar Agar?

Some of you might know it as a plant derived gelatin substitute and that’s nearly the same purpose we will use it for. The thickener is based on a galactose containing extract taken from red-purple marine algae. This brings the typical nutrient solution to the right consistency that supports vigorous growth, but gets easily penetrated by the roots to insure the best development possible. In order to generate the perfect Agar mixture we have to add it in a ratio (depending on the source) 1-2:100. This leads to a nearly complete solid medium. If you want to cultivate in anaerobe/liquid conditions, you can simply half the concentration.

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But why not just use gelatin instead?

That’s a good question, but there’s a simple answer to it. It’s edible to bacteria and most other microbes so you would have to refill every plate after a short time. The nutrient dosing and validity of the experiment undertaken would be in danger as you would have to add the gelatin to the feeding chart of the microbes that are cultured.

It comes in different forms, but the most common are flakes, that need to be mixed with water und constant heat application until it’s fully homogenized. These flakes are easy to store and quite cheap to get. If you have the necessary budget, there are pre-mixed/pre-sterilised versions, mostly bottled up, that can be used instantly without waiting for the hot agar mix to cool down. The cooling process can take up to 30min per batch before use.
Not only the distribution form differs but also the additives. Many agar mixes have added ingredients for certain purposes as this is used for all types of cell cultures from human cells to the infectious disease causing Escherichia coli. 

The potato-dextrose agar for example is the most used form for fungi especially for varieties like Botrytis cinerea also known as the common grey mold. Another one is blood agar, which as the name already says, contains animal blood in order to study special microbes. For tissue culture the most common type is the standard galactose agar in combination with a nutrient matrix and a hormone, depending on the phase.

The standard nutrient matrix in nearly all cell culture work is the Murashige-Skoog-Medium, which contains simple amino acids, nutrient salts and photosynthesis products (saccharose) as the plant can’t photosynthesis in most cases. Inositol is also added in order to mitigate plant stress, strengthen cell walls and phosphate storage.

2.2 Hormones for plant tissue culture – is that even safe?

Many myths rank around plant hormones, which are mostly “feared” by the Cannabis community as it only has found real application in large scale agriculture. They get mixed up and thrown in the same bucket as GMO and Glyphosate. Not that those would be bad substances but the social stigma around them is strong.
The hormones on the other side are nearly identically to their natural/plant counterparts, which control nearly all the physiological responses of the plant to outside influences like the movement of the sun or the alignment of the plant to the gravity

2.2.1 Auxin

The first of these phytohormones (hormones in plants) is responsible for a multitude of responses to environmental stresses. Most auxin derivates in the plant and made by humans are based on the Indol-3- acetic acid. This compound is mostly synthesized in the apical region refering to the main growingtip of the plant. From there on it gets transported via the phloem and cell-to-cell by the PIN 1-9 transporterprotein.

The basic effects include wound response, apical dominance (the main branch is the tallest one), photo-/gravitropisms and it plays a important role in fruit development. But lets dive a bit deeper in the interaction of Auxin and the use cases of it.
The first practical application would be induction and promotion of root growth. This is the most used purpose and nearly all commercial rooting gels contain it. But not only in Cannabis, but also the floral industry uses it predominantly in their rooting SOP.
We also use this by giving our explants (plants in in-vitro conditions) a certain amount of it in order to achieve the highest rooting rate possible.

But as with everything in life, there is a limit to whats good for the plant. Excessive application of this substance can result in growth inhibition and plant death. Weedkillers like 2-4-D are based on the synthetic counterparts of auxin like indole-3-butyric acid. But not only damaging substances also “good” ones like common rooting gels are based on synthetic auxin.
“Natural” Auxin can be found in variety of plants shoots especially the ones of the willow tree.

2.2.2 Cytokinin

The next hormone in our repertoire is the counterpart to the previously mentioned Auxin. This compound focusses on the lateral growth of both roots and shoots. To be more exact the ratio of Cytokinin:Auxin is the driving factor in the morphology of the plant. They not only contradict each other, but also work together when influencing cells. When only Auxin is applied cells elongate and become big, but won’t expand or differentiate. Same takes place when the ratio is 1:1. This is used for Callus multiplication or expansion.
Complementary to Auxin it is synthesized in the roots and travels to the shoots via Symplast and Apoplast. Production of this chemical is regulated by two types of response regulators. One being the B-Type and the other being the A-Type. Both are regarded as transcription factors as the either activate or stop the production of Cytokinin via influencing the transcription of the corresponding gene loci.

Using this knowledge we can manage the morphology of the plant into growing more squat thus increasing internodal stacking and use of space.
But not only the space can be used more efficiently also the time saved by breaking seed dormancy can save time when germinating seeds for a phenohunt. The dormancy is induced by abscisic acid and by increasing metabolic activity the cytokinin decreases the level of abscisic acid in the seed. It’s commonly used in plants that have a hard time germinating.
The next effect might be especially interesting for the photographers and florist under you. Cytokinins can delay senescence in other words the decay of the plants. This is achieved by increasing synthesis and slowing decay of certain proteins. Also nutrients are drawn into the treated area from nearby tissue. It’s suspected that an enzyme is responsible for these actions, but no scientific consensus has been reached yet.
When using Cytokinins you have to differentiate between the plants own, adenine based, Cytokinins like Kinetin and Zeatin. Others have been found outside of plants, but they are based on Phenylurea. These exceed the effectivness of the Cytokinin in certain plants. Examples for this variety are TDZ and Diphenylurea, which are widely applied in agriculture

2.2.3 Gibberellic Acid

One of the most interesting phytohormones is the counterpart to abscisic acid. Exogenous GA3 (short version) is also responsible for breaking dormancy where it activates indigenous synthesis of more GA3, which adds to dormancy breaking factors. These are hypothesised to be a combination of growth promoting hormones (GA3, Cytokinin, Auxin etc) and reduced nutrient storage in the endosperm. Enzymes, primarily α-amylase, lead to the processing of sugars and other storage units. Another restricting factor is the Cuticula strength of the seed.
The application on an adult plant leads to a big stretch in all shoots. This is principle is also used in plant tissue culture and for virus cleansing. But this has to be done with the utmost focus on the concentration of GA3 as a high amount leads to different sex expression. This mechanic is commonly used in feminized seed breeding as it “reverses” a female plant into producing pollen with female Genome. In even bigger concentration it can lead to the complete sterilization of the specimen. Most fruit growers like in the citrus industry use the mechanic to suppress undesirable seed development

That said, when a trained horticulturist with a strict, thought through regime applies GA3 in flower, it can increase inflorescence mass and trichome development. For homegrowers and people that don’t have access to these chemicals, Kelp/Seaweedextract is a perfect allrounder as it contains a variety of growth hormones aswell as Macro-/Micronutrients.

The compound was discovered by japanese scientist while researching the Gibberella fujikuroi fungi. Cytokinin is a secondary metabolite to the pathogenic pathway of the fungi on rice plants.

2.3 Basic Tools for Tissue Culture Propagation

2.3.1 Laminar Flow Hood

It’s one of the most important, if not the most important part of a Culturelab. The Laminar Flow Hood (LFH) insures that all steps are done in a steril environment. There is a differentiation between normal Flow hoods and special models for working with microbiological organisms. The first doesn’t filter the incoming and outgoing air, leaving the worker exposed to aerosols and microorganisms.

That’s why we use the second model in most cases. This LFH got a specialized HEPA-Filter before the air enters the Hood. After getting in it can be distributed in two ways. Vertical flow hoods and horizontal flow hoods can be differentiated easily as word explains itself

All work including the plant have to be done inside to keep out potential contaminants. It’s therefore of vital importance to wipe down all areas inside with ethanol (70%) and clean the outside of culture vessels aswell when introduced in the steril environment. Gloves and hair-protectants are a must, if mass-propagation is planed. At home you can easily get away with disinfecting your hands and arms up to the elbow. Another concern is your breath as it often contains bread-mold spores and a variety of microorganisms that thrive on tissue culture medium.
If a infection takes place and you have no copy/other option, you can add antibiotics to the medium, but be very careful. Most higher plants are more resistant to these compounds than the microorganisms, but the optimal balance is very hard to reach. Another factor is the difference between every plant genome/phenotype thus a cultivar-specific test would be necessary before adding antibiotics.

2.3.2 Autoclave

Speaking of sterilisation, we will subsequently be lead to autoclavs. These laboratory certified pressure cookers are an essential tool for cleaning glassware and used culture vessels. They come in different sizes and forms, which you can choose specific for your application.
Despite the different shapes, the mechanism is the same. The chamber is evacuated via a Vacuum pump in order to simplify the sterilisation progress. It gets filled with hot steam and thus put under a high pressure to increase the cleaning efficiency. After 15-20min the optimal cleansing effect is achieved, which means that the content can be removed. Be careful not to touch it with your bare hands as it’s still really hot.
You should also size the autoclave big enough for the operation, but not too big for your batchsizes as this will lead to unnecessary cost for energy.

2.3.3 Magnetic Stirrer

This device is rather simple and doesn’t need much explanation, but it’s still vitally important that we talk about it. You probably wonder why and we will will explain it shortly. Not the stirrer per se needs explanation, but rather how to use it and what for. It’s the base of a good tissue culture lab as it’s used for mixing and homogenizing of the medium components that we discussed earlier.
Each component has a specific boiling/inactivation point that you have to consider when mixing.
The goal is to find the right compromise between solubilityspeed and retention of effectiveness. Some compounds like hormones derivates have to be added after the mixing + autoclaving of the medium as they can’t even withstand the autoclaving procedure.
For these substances you have to consider other ways of sterilising them such as syringe filters or specific solvents.

It enables the production of large amounts of media as it’s fully adjustable in rotationspeed and temperature, if you have a heated version. Be careful to pick a appropriable sized stir bar as this determines the homogeny of your mixed substance.  But even with a large stir bar the stirrer has it’s limitations when the mixing materials are too viscous. If that’s the case, we would advise a mechanical alternative as they can plow through it better. Keeping that in mind, you have to pour your hot, mixed medium out of the vessel before it cools down as it gets really hard to remove the bar after.

2.3.3 Consumables

This chapter is for items that are important, but didn’t get a separate chapter as it would go beyond the scope of this article.
Firstly we have your PPE like gloves, masks, Labcoats and hair nets. These are especially important, if you have a large team and thus many vectors of contaminants/pests. But keep in mind that PPE doesn’t eliminate the necessity of disinfecting the whole work environment before and after you do explants in it.
Another vital point are the culture vessels. These are the new homes for your little clones and need to have a large enough volume to accommodate them. This refers especially to the amount of medium that a plant needs in its stage of growth. There are mayor differences from cultivar to cultivar so the best way to go about it, is to  experiment with it and keep tight journals in order to create SOPs. This takes a lot of time, energy and labour so be mindful of that, when trying to establish a plant tissue culture production.

Our last important item is the scalpel.  You can find many different forms of it for specific applications, but we recommend that you go for a more environmentally friendly version by using a scalpel holder with exchangeable blade. Using this will decrease your waste of material and money as the amount of cut plant material in an TC production puts a great stress on the blade thus making it dull. The alternatives would be to use whole single use scalpels, but the produce large quantities of plastic that we don’t want or high end versions. The later stays sharp for longer, but not long enough to make up for the huge price difference.

2.4 Standard terms for plant meristem tissue culture

In order to understand the basic mechanics of saving genetic material and growing callus cultures we have to take a look at the structure of a growing plant tip. This is the location we’re using to explant a meristem from. But what is a meristem?

The term comes from the ancient greek word merisein, which means divide. It was used by the swiss scientist Carl Wilhelm von Nägeli to describe the undifferentiated, multiplying Cells that made up the growing tips of the plant. The apical meristem is the highest growing tip in the plant and sits on top. The counterpart of this region is the basal meristem, which sits of the tip of the roots as you might have guessed.

The plant builds new meristemcells in the top middle parts as the lower parts differentiate into the predetermined functional parts. Some go to be Parchenym cells, some are going to be part of the vascular system of the plant. Knowing this, we can extract these cells before they’re differentiated in order to get totipotent cells (cells that can be any part of the plant). These are full of potential and are kept in a cryofreezer to be saved for later usage, as backup or as basis for mass cell production in a bioreactor.

3. Basic mechanics of mass micropropagation

To get an idea of how micropropagation could benefit your growing operation we’re gonna draw a little plan of how it could look like.
First of all we need a small motherstock from which we can take our microcuttings. These are about 2,5cm/1 inch long and at best, taken from a meristematic region.
Then we plant it in a multiplication medium under sterile conditions in order to avoid contamination and the subsequent demise of our plant matter. After 2-3 weeks we should see 2-4 new shoots coming from our tissue, which we can also cut up into separate tissue samples. The we put them in individual containers with the same medium mix again and repeat the previously mentioned cycle. Even thought there will be loses due to contamination, the replication-rate is extremely high due to each new tissue replicates itself 2-4 times. If we calculate this through, we can see that with minimal space use, an enormous plantcount can be achieved in a short period of time.
After we finished the replication cycle, we can move onto the rooting phase. This uses a special medium containing auxin derivates, which induce the production of roots on our previously made shoots. After 2-4 weeks again, we have to inspect the plants accordingly and if they’re sufficiently developed, we beginn the hardening phase.
For this part of the productioncycle the plants get translated in the medium of choice that will be used later on in production. Most growers will use rockwool as it has excellent water holding capacities, but also coco or small soil pots can be used. Light plays a huge role now and has to be increased in order to acclimate the plants to the later vegetative conditions.
The humidity also has to go from the comfy 80-90% RH in the boxes down to the values of your vegroom.
This was a quick overview of the process and as you could see, there are many obstacles along the way, but in the end it’s not that hard to achieve at home. For this exact purpose we will compare DIY kits that can be bought online with a home made microlab.

4. What are ready-made home kits?

There are some suppliers on the internet who offer hobby kits for micropropagation. Even if these kits are suitable for experimenting and for first experiences, they cannot replace a professional laboratory, because similar to mushroom cultivation, a sterile working method and environment must be guaranteed to avoid failures. In addition to all the materials and equipment, professional application requires trained personnel and constant quality control of the work process.

5. Future vision

As cannabis production becomes more and more automated and the costs of labor, electricity and rent continue to rise, we at research gardens see in-vitro production as a key advantage for companies that want to remain sustainable in a rapidly evolving industry. Due to the prospect of a long-term reduction in production costs as well as a simultaneous increase in product quality your company will get an advantage against your competitors. Particularly in pharmaceutical operations, a consistent quality of the plants and ingredients is indispensable. For the recreational market, however, this is just as important since similar requirements exist here as well.

Interested? Then let us start working out a concept for an in-vitro laboratory in your company today! Send us an e-mail to info@research-gardens.com

Sources:

 

1. “Communication by Plant Growth Regulators in Roots and Shoots of Horticultural Crops” by Anish Mallad; Jacqueline K. Burns; August 2007; HortScience: a publication of the American Society for Horticultural Science 42(5) (https://www.researchgate.net/publication/237379591_Communication_by_Plant_Growth_Regulators_in_Roots_and_Shoots_of_Horticultural_Crops/figures?lo=1)
2. “Hot Plate with Magnetic Stirring: 6.7″x6.7″ SS Plate Max.300C – SH3”; MTI Cooperation; (https://www.mtixtl.com/hotplateswithmagnetcstirring67x67stainlesssteelsurfaceupto300c-eq-sh-3.aspx)
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7. “[Tuesday Scoop] Puzzlement Between Agar-Agar And Gelatin”; Namrataa Mahalley; (https://bulbandkey.com/blog/tuesday-scoop/puzzlement-between-agar-agar-and-gelatin/)