Advanced Biotechnological Techniques for Improving Cannabis Cultivars
Unleashing the power of state-of-the-art biotechnology to revolutionize cannabis breeding and cultivation, offering endless possibilities for medical, industrial, and recreational advancements.
Diverse Applications of Cannabis
Medical
Cannabis alleviates chronic pain, manages epilepsy, and treats multiple sclerosis.
Industrial
Hemp is used in textiles, paper, construction materials, cosmetics, and food products.
Bioactive Compounds
Cannabinoids and terpenoids determine the plant's various use cases.
Bioactive Compound Distribution
Inflorescences
High cannabinoid content, primarily found in trichomes on flower surfaces.
Leaves
Moderate levels of cannabinoids, terpenoids, and flavonoids distributed throughout leaf tissue.
Stem Bark and Roots
Concentrated with sterols and triterpenoids, important for plant structure and defense.
Cannabinoid Biosynthetic Pathway
1
Initial Synthesis
Tetraketide Synthase (TKS) and Olivetolic Acid Cyclase (OAC) produce olivetolic acid.
2
CBGA Formation
Prenyltransferase (PT) converts olivetolic acid to Cannabigerolic Acid (CBGA).
3
Major Cannabinoids
CBGA is converted to THC, CBD, and CBC through specific synthase enzymes.
Micropropagation Techniques in Cannabis
1
Stage 0-1
Select parent material and initiate sterile culture.
2
Stage 2
Multiply shoots through repeated subculturing.
3
Stage 3-4
Elongate shoots, induce rooting, and acclimatize to outdoor conditions.
Optimizing In Vitro Cannabis Cultures
1
Media Composition
Optimize nutrient balance and Plant Growth Regulators (PGRs) for each stage.
2
Environmental Factors
Control temperature, light intensity, and humidity for optimal growth.
3
Genotype-Specific Protocols
Tailor techniques to specific cultivars and tissue types for best results.
Unlocking the Genetic Potential of GPP Synthesis
The production of geranyl pyrophosphate (GPP), a crucial precursor in the cannabinoid biosynthetic pathway, is a complex process involving a series of strategic genetic modifications. This intricate engineering effort begins with the introduction of two key genes, EfmvaE and EfmvaS, from the bacterium Enterococcus faecalis.
Alongside this, the native mevalonate pathway genes - ERG12, ERG8, ERG19, and IDI1 - are overexpressed to further enhance the metabolic flux towards GPP synthesis. Additionally, a mutated version of the ERG20 gene, erg20*, is incorporated to optimize the enzymatic activity.
To produce the essential hexanoyl-CoA building block, a heterologous biosynthetic pathway is employed, drawing upon genes from various microbial sources. This includes contributions from Ralstonia eutropha (RebktB), Cupriavidus necator (CnpaaH1), Clostridium acetobutylicum (Cacrt), and Treponema denticola (Tdter). Alternatively, the process can utilize hexanoic acid as a substrate, with the CsAAE1 gene from cannabis facilitating this conversion.
The resulting olivetolic acid is then prenylated by the CsPT4 enzyme, leading to the production of cannabigerolic acid (CBGA). This critical intermediate is subsequently transformed into the cannabinoid acids THCA and CBDA through the action of specialized synthases, THCAS and CBDAS. Finally, the application of heat triggers the decarboxylation of these acids, yielding the coveted THC and CBD compounds.
Genetic Transformation in Cannabis
Agrobacterium-mediated
AMT is used to introduce foreign genes into cannabis genomes.
CRISPR/Cas9
Precise gene editing targets specific traits in cannabis plants.
Regeneration
Optimizing protocols for stable transformation and plant regeneration is crucial.