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Read our blog, and learn about marijuana growing

In-Vitro Cannabis explained step by step

The precess we know as in-vitro growing (from latin, inside crystal) is the practive of growing new plants from small pieces of a motherplant (organs, tissues, cells or protoplasts*) in a feeding medium, under sterile and controlled conditions.

This practice is not new. The huge potential this technology has multiplied the number of laboratories focussed in vegetable tissues grows, specially those dedicated to comercial fruit trees and ornamentals productions. Nowadays there are many home-growers who practice this technique to grow orchids at home with great results.

*Trying not to make from this text a tough-reading one, we attached at the bottom few descriptions and explanations to ease its understanding.

In-Vitro Marijuana

In-vitro cannabis situation today

Unlike the traditional cloning method, in wich we need a full branch of a motherplant, to take a single cutting, growing in-vitro we can take off the plant whatever to create new plants (leaves, new-shots, small pieces of the plant, so we can multiply the number of plants into many new plants, keeping them in vegetative stage for long time.

Nevertheless, in cannabis matter, this technique is too recent. The answer of plants to in-vitro growing conditions changes from strain to strain, and even from plant to plant, so the studies about techniques for in-vitro cannabis are still few, and of hard access. Today, some laboratories are working in describing protocols for in-vitro regeneration, valids for cannabis; and growers are drooling thinking in possible applications of these techniques.

In-vitro cannabis applications

What does this technique mean for cannabis industry? Now we will highlight applications for in-vitro growing, and why this can set a before and after for cannabis.

Micro-propagation

This term is usually unproperly used, and many times we end up talking about in-vitro when we actually refer to micro-propagation (or reverse). Micropropagation is the group of techniques and methods used to multiply plants asexually. From a small piece, taken from a mother plant, with micro-propagation, we'll get cuttings (genetically equal plants to it), that will be grown in-vitro in a growing chamber, where will continue developing with the target of being rooted and planted, or to be fragmented again to produce new plants.

Micropropagation could revolution classic techniques used to reproduce marijuana plants, because it allows a mass production, multiplying the number of lpants so quick, and exponentially. Let's start, in example, from a hypothetical mother-plant from wich we got 10 explants; let's say, after a month growing and, as the growing is exponential (every new explant will become two), in a year the total amount of new plants would be 40960, all of them got from the same motherplant, and genetically equal to it.

Preserving species

Plants produced in vitro are kept in a feeding growing medium, under conditions that allow to store them for long periods of time in germination state. This way, is possible to save in a fridge rare species, of a mother-plant that we would like to keep due its characteristics, until we need it again.

Sanitary applications

As it is done under asepsis conditions, in-vitro growing assures getting plants free from pathogens, what allows to produce certified plants.

Genetic engineering

In-vitro growth can be used to create "plants under demand", to get plants with desired characteristics, no matter if are for ludic, pharmaceutical, alimentary, or industrial interest.

Sanitary applications marijuana

Biological and farming studies

Advantages

These are the main advantages that in-vitro growth, and micro-propagation, present compared with classical cuttings, and seed propagation.

Reduced space: Growing-space needed is much more reduced, due plants are placed in small jars, that could be stored vertically, notoriously reducing the storage area needed compared with cutting propagation.

Low needings: This type of growth does not need excessive atention, there isn't time used for irrigation, nor cares, once the process is done. The medium needs to be changed every 4-6 weeks, and we only need to pay atention to the growth process.

Long-lasting: PLants can keep stored in limited vegetative stage for long period of time.

Massive production: The number of plants can be multiplied exponentially.

Quick process: The vegetable growth is quick, new shots start to appear in 15-20 days, what allows you to create new plants in not much more than a month.

Healthy plants: You skip pests and desease.

Uniform offspring: You'll get an exact copy from the mother plant.

Economic savings: At this point we only refer when growing large scales. The amounts of products used in in-vitro growing are way less than required for a classical growing operation, and the performance is way superior. Growing in-vitro does not invest in irrigation nor growing products (substrates, fertilizers, pesticides, amendments, etc), and savings in space and energy too.

Even the advantages of growing in-vitro cannabis, this method also presents some disadvantages difficult to skip.

Disadvantages

Difficult: The technology, methods, techniques, and materials needed for in-vitro growing are laboratory type, and are not easy to find by public. To get optimal results it is needed some knowledge in microbiology, and huge atention to details. The sale of growing kits and premixed growing mediums (what we guess is not far from today) would be a great help for home-growers. Even though, it is not impossible, and growers who like to experiment, with own mediums and sources, are not few.

Lack of protocols: The fact that every specie, and every strain responds different to in-vitro growth, and that there isn't yet an standard protocol for cannabis growing, makes it even more difficult.

Not worth it in small operations: Time invested, budget and resources required by this growing method in small operations, makes it not worth it for small producers and home-growers.

Industrialization: This type of massive production can represent a first step for cannabis industrialization, what tokers percieve with ambiguity and, why not to tell, with fear. Even cannabis industrialization could set an step forward in medical matter, it is also true that if big corporations and pharma aproppiate cannabis (like previously did tobacco companies) that would go against small growers, tokers, and against product quality.

In vitro cannabis

How to grow in-vitro cannabis

Probably, for many home-growers, the idea of crating an sterile ambient to develope an in-vitro growth could be discouraging. But these techniques should not have to be limited to laboratories. To be complicated is not a reason to don't try it. Following we will detail stages of in-vitro growth. If you are curious, and would like to experiment, here are some ideas and tricks for you to try it at home with your own cannabis plants.

Choosing a mother plant

New plants will be genetically equal to the mother plant, that means that will inherit its characteristics. So, mother plant should be chosen by its characteristics, wich we desire in new plants (strain, medical value, yield, taste, effect resistance against pests...), and should be in proper nutritional conditions, healthy and developement (a plant in vegetative stage will develope easily and healthier).

Introduction in growing-medium

Here is when the process of vegetables tissue regeneration begins. We should introduce explants in a growing-medium. As a medium we could try with a base of MS (a comercialized medium base) and then add agar-agar (10g/l), sugar (15g/l) and auxins (0,5 mg/l)* to enhance growth; it is convinient measure and adapt the medium pH to the strain likely one, with a product to increase or decrease the pH. The explant cut should be in contact with the surface of the medium.
Explants need to be kept in warm temperatures (21-23 ºC) and with much light. Ideal is to use a photoperiod of 18 hours of light and 6 hours of darkness.

Multiplying

At this point, plants (survivors) will develope callus. If the target is to reproduce, this is the right moment to brake the callus and repeat the process again to create more plants. If not we will step into the next stage.

Rooting

Explants should be moved to a medium containing a hormone concentration to induce them to root formation, keeping the same conditions of previous stage. To do so we could try adding cytokinins 83 mg/l) to the same blend used for previous medium. Once roots have developed, plants can be stored, or placed in new substrate.

- Conservation: Plants are transferred to a medium free from growth inductors ( a blend of MS in wich we add sugar and agar), and stored in the fridge (12-14 ºC). With this limited growth, plants should be kept in the fridge for some months.

- Planting: Resulting plants had been grown in ideal conditions, and should adapt to be able to survive. A high RH (80%), an sterile substrate and a photoperiod of 18h of light - 6 hours of darkness should help them to do so. (If plants have been kept in the fridge for some months, place them under the light, without changind the medium, until they recover a warm temperature).

*Growing mediums, and hormone concentrations are different for each strain. Our purpose is only an base to work with and start experiencing, and may not reach expected results).

Inspiring? Now you only need to get materials

In-vitro cannabis materials list

Mother plant

Ethanol

test tube

Ph meter

MS basal medium (or another premixed growing medium)

Agar-agar

Vegetable growth regulators: auxins and cytokinins

Sugar

Distilled water

Bleach

Preassure cooker

Tweezers

Scalpel

Scisors

Scale

Measuring cup

Measuring needle

CFL or low consumption lamp

Fridge

Sterilization

Medium contamination is the main problem in home in-vitro growing. All this process should be done inside an sterile enviroment. Build a glovebox is the easiest way, and cheapest, to be able to work at home in a sterile room.
In addition, you must be sure disinfection and sterilization of all material has been done (organic or not). Ethanol is useful to clean your hands and also to disinfect surfaces where we work on.

Disinfect the explant:

Submerge 1 minute in ethanol.
Dillute 5 ml of bleach in a liter of distilled water (sterilized). Submerge explants 10 minutes.
Proceed with 3-4 rinses (2 minutes) in sterilized distilled water.

To sterilize the growing medium

Place the medium in test tubes, place them in a preassure cooker with water and take them to boiling point.
Boil them for 30 minutes.
Let them cool down before placing the explants in.

Glossary

Agar-agar: Non-animal Jelly substance, with marine origine. The growing medium is got from cell walls of some seaweed species.

Callus: Vegetable cell mass no differenced that covers a wound.

Explant: Small piece of alive vegetable taken from a plant, and prepared in aseptic way to grow in an artificial growing medium, that works as new plants generator.

Growing medium: Liquid solution, or jelly, comound by a mix of mineral salts and vitamines, that contains needed nutrients to develope virus growth, microorganisms, cells, vegetable tissues or small plants. Its composition depends of de specie and stage of growing process.

Node: Plant's part where branches, leaves and flowers meet. In the nodes we'll find the vascular system that comunicates stems and leaves.

Mother plant: Is that one, chosen by its characteristis, to donate vegetable matter in an asexual propagation process.

Protoplasts: Cells from wich, within mechanical treatments, or enzymatic, cell walls are removed.

Growth regulators: Homones that act on plant's developement.

Sources

Aproximaciones biotecnológicas tendentes a la mejora del cáñamo

Plant tissue culture cloning of the future

Microprogramation system for cloning of cannabis sativa

Canna.es

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One thought on “In-Vitro Cannabis explained step by step”

  • Bortuzzo Patrick May 5, 2019 at 3:28 pm

    i have a question about multiplying, when i want to produce explants from the callus, i have to desinfect the plants before i put them into the rooting medium?

    Reply
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