What is Tissue Culture?

Tissue culture represents one of the most significant technological advances in modern plant breeding and propagation. At its core, the process involves isolating small sections of plant tissue and growing them in a sterile, nutrient-rich environment, allowing for precise control over plant development. For cannabis breeding programs, tissue culture offers extraordinary possibilities that extend far beyond traditional propagation methods.

The Stages of Plant Tissue Culture

Successful tissue culture involves a series of distinct stages, each with specific requirements:

1. Initiation Stage

• Selection of appropriate explant material (meristems, nodes, leaves)
• Surface sterilization to eliminate contaminants
• Placement on appropriate initiation media
• Establishment of viable, contamination-free cultures

2. Multiplication (Proliferation) Stage

• Rapid production of multiple shoots or somatic embryos
• Regular subculturing to fresh media
• Optimization of growth conditions for maximum multiplication
• Quality monitoring for genetic stability

3. Rooting (Pre-transplant) Stage

• Induction of root development in multiplied shoots
• Preparation of plantlets for ex vitro conditions
• Development of functional root systems
• Hardening begins in controlled environment

4. Acclimatization Stage

• Gradual adaptation to non-sterile conditions
• Transfer to soil or soilless media
• Humidity reduction protocols
• Transition to normal growing conditions

Understanding Culture Media

The composition of growth media is critical to tissue culture success. Standard media contains several key components:

Major Components

• Macronutrients: Nitrogen, phosphorus, potassium, calcium, magnesium, and sulfur
• Micronutrients: Iron, manganese, zinc, boron, copper, and molybdenum
• Carbon source: Usually sucrose (20-30 g/L), serving as an energy source
• Vitamins: B vitamins, especially thiamine, are essential for plant metabolism
• Solidifying agents: Agar or gellan gum (6-8 g/L) to create semi-solid substrate
• Plant growth regulators: Hormones controlling development (detailed below)

Common Media Formulations

Cannabis tissue culture typically relies on modified versions of these standard formulations:

1. Murashige and Skoog (MS): High salt concentration, suitable for many cannabis cultivars
2. Gamborg’s B5: Lower nitrogen content, beneficial for some cannabis strains
3. Woody Plant Medium (WPM): Lower salt concentration, useful for some recalcitrant cultivars
4. Driver-Kuniyuki Walnut (DKW): Developed for woody plants, balanced mineral composition that works well for certain cannabis genotypes

Each formulation can be prepared at full, half, or quarter strength depending on the stage and specific cultivar requirements.

The Role of Plant Hormones

Plant growth regulators (PGRs) are the primary directors of development in tissue culture. Understanding their roles is essential:

Auxins

• Primary functions: Root induction, callus formation, cell elongation
• Common types used in cannabis culture:
  • Indole-3-butyric acid (IBA): 0.1-2.0 mg/L for rooting
  • Naphthaleneacetic acid (NAA): 0.1-1.0 mg/L for callus induction
  • 2,4-Dichlorophenoxyacetic acid (2,4-D): 0.5-2.0 mg/L for embryogenesis
• Typical applications: Higher concentrations in rooting media, lower in multiplication

Cytokinins

• Primary functions: Shoot proliferation, axillary bud development, inhibition of root formation
• Common types used in cannabis culture:
  • 6-Benzylaminopurine (BAP): 0.5-2.0 mg/L for shoot multiplication
  • Kinetin: 0.5-1.0 mg/L for shoot development
  • Zeatin: 0.5-1.0 mg/L (more expensive but sometimes more effective)
• Typical applications: Higher concentrations in multiplication media, reduced or eliminated in rooting

Gibberellins

• Primary functions: Cell elongation, breaking dormancy
• Common type: Gibberellic acid (GA3)
• Typical applications: Generally used at low concentrations (0.1-0.5 mg/L) or omitted

Other Regulators

• Abscisic acid (ABA): Used for somatic embryo maturation and stress tolerance
• Ethylene inhibitors: Silver nitrate or aminoethoxyvinylglycine (AVG) to prevent ethylene effects
• Polyamines: Sometimes used to enhance morphogenesis and reduce browning

Hormone Balances for Different Stages

The ratio between auxins and cytokinins primarily determines developmental direction:

1. High auxin:cytokinin ratio: Promotes root formation and callus development
2. High cytokinin:auxin ratio: Encourages shoot formation and multiplication
3. Balanced ratio: Often leads to undifferentiated callus growth
4. Hormone-free media: Sometimes used for final rooting stage or to reduce somaclonal variation

Laboratory Requirements

Establishing a successful tissue culture operation requires specialized equipment and careful attention to sterility. Essential components include:

• Laminar flow hood: Creates a sterile workspace free from airborne contaminants
• Autoclave: Sterilizes media, instruments, and containers
• Growth chamber: Maintains precise temperature and lighting conditions
• pH meter: Critical for preparing media at correct pH (typically 5.7-5.8)
• Precision scale: For accurate measurement of media components

Next Steps

In the following articles, we’ll explore specific applications of tissue culture in cannabis breeding and cultivation, beginning with micropropagation techniques for mass clonal production. We’ll then examine advanced applications including pathogen elimination, germplasm preservation, polyploid induction, and integration of tissue culture into breeding programs.

For breeders interested in exploring tissue culture, I recommend starting with basic micropropagation before advancing to more complex techniques. Even a small-scale setup can provide significant advantages for maintaining clean stock plants and multiplying elite genetics efficiently.

Resources

1. Thorpe, T. A. (2007). History of plant tissue culture. Molecular Biotechnology, 37(2), 169-180. https://doi.org/10.1007/s12033-007-0031-3
2. Chandra, S., et al. (2017). Cannabis sativa L.: Botany and horticulture. In Cannabis sativa L.-Botany and Biotechnology (pp. 79-100). Springer, Cham. https://doi.org/10.1007/978-3-319-54564-6_4
3. Lata, H., et al. (2009). In vitro plant regeneration from leaf-derived callus of Cannabis sativa L. Planta Medica, 75(10), 1061-1064. https://doi.org/10.1055/s-0029-1185612
4. Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum, 15(3), 473-497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
5. George, E. F., Hall, M. A., & De Klerk, G. J. (Eds.). (2008). Plant propagation by tissue culture 3rd Edition. Springer, Dordrecht. https://doi.org/10.1007/978-1-4020-5005-3

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[This post assumes legal hemp/cannabis breeding in compliance with all applicable laws and regulations.]