Sterilization of equipment and work space is critical throughout.
Initiation Phase (1 to 2 weeks)
Cutting of Explant – the tissue taken from the mother plant is known as the “explant”. The explant is disinfected, and then moved to a sterile vessel or test tube for insertion onto a sterile nutrient media. The tubes are then capped and moved to the grow room for 2 weeks.
Proliferation or Multiplication Phase (2 to 3 weeks)
Pathogens will typically grow in the media or express themselves in the plantlets within the first two weeks. By observing the traits of the plantlets, having the plant tissue tested at a third-party commercial viral testing lab, or performing a strip test, we can determine if elimination of the pathogen was successful or not. The successfully cleaned and healthy plantlets are then divided, added to a multiplication media and then moved to larger containers. During this stage, the plantlets must be divided and transferred to fresh nutrient media regularly. We typically get 5 generations of multiplication before the genetics start to degrade.
Pre-Transplant or Rooting Phase (2 to 4 weeks)
The goal of this phase is to make the plants stronger so they’ll be able to survive outside of the lab. During this phase nutrient media is used to initiate roots. We also increase the intensity of light. Until this phase, the plants have not been photosynthesizing and have been receiving all of their energy through the nutrient media. The success of this phase will depend on the following factors:
- Hormone blend
- Residual hormones from previous media
- Potential Contamination
- Environment (temperature, light, and humidity)
Establishment Phase (1 to 2 weeks)
Here the plantlet is transplanted to growing media in the clone room. A clone dome may be used during this phase to maintain humidity levels. Gradually reduce humidity level from 100% to 80% over the course of a week. Roots have been growing in a gel until this phase, so they are very fragile at this stage and the plants are more susceptible to infection.
Other Plant Tissue Culture methods
Germplasm storage allows preservation of plant material for extended periods of time. Refrigeration (above 40°) can preserve germplasm for a year or more. Encapsulation or Green Pod Sowing – the formation of embryos from single, identical cells in the lab. Artificial seeds are coated with a gel, containing various hormones and nutrients, by doing this you are able to cut months off of your growing time.
Embryo rescue involves removing tissue from the seed.
Mutagenesis involves inducing mutations to develop beneficial traits such as resistance to disease or pests. Mutagenesis can be accomplished two ways– one involves the use of a genetically modified agrobacterium containing a loop of non-chromosomal DNA that would normally induce tumors in the plant. However, the tumor-causing DNA is replaced with the new desirable genes prior to insertion so as not to make the plant sick. Another method involves the use of a “gene gun” to fire DNA-laced gold particles into the plant’s cells. Some of these particles pass through the plant cell wall and enter the cell nucleus, where they are integrated into the plant chromosome. Both methods of gene transfer are random in their efficacy and require extensive analysis to screen the plant cells for the modified DNA. We hope to eventually bring these techniques into the world of cannabis cultivation, but currently, the only genetic manipulation we do is through natural breeding methods. As previously stated, we use only non-GMO methods of PTC for cloning and eliminating pests and disease.