Plant tissue culture, also known as micropropagation, is a laboratory technique for rapidly cloning plants in a sterile, controlled environment. This method allows for the production of numerous genetically identical plants from a single, small piece of tissue. Home enthusiasts can apply these principles to propagate rare or desirable plants, bypassing the slow process of conventional methods like cuttings or seeds. Success depends on maintaining a contamination-free workspace and providing the plant material with precise nutrients.
Essential Supplies and Medium Preparation
The foundation of successful micropropagation is the culture medium, which provides all the necessary elements for plant growth in the absence of soil. The medium is typically a modified Murashige and Skoog (MS) formulation, supplying macronutrients, micronutrients, and vitamins. Since the plant material cannot perform photosynthesis efficiently, the medium must also contain a carbon source, usually sucrose.
The medium must be solidified to support the tissue, achieved by adding a gelling agent like agar before sterilization. Plant growth regulators, such as auxins and cytokinins, are included to signal the plant cells to perform specific actions like shooting or rooting. These components are dissolved in distilled water, and the pH is adjusted to support growth, typically between 5.8 and 6.0.
Equipment includes glass jars or polypropylene containers and stainless steel tools like scalpels and forceps. A household pressure cooker serves as an affordable alternative to a laboratory autoclave, using high-pressure steam at 121°C for 15 to 20 minutes to sterilize the medium and tools. All equipment must be scrupulously cleaned prior to sterilization.
Establishing a Sterile Working Environment
The most significant challenge is preventing contamination from airborne fungi and bacteria, which rapidly outcompete the delicate plant tissue in the nutrient-rich medium. This requires adopting strict aseptic techniques. While professional labs use a laminar flow hood, a more practical solution for home use is a still air box (SAB).
A still air box is a simple, sealed plastic container with armholes that reduces airflow and airborne contaminants. Before starting, the inside of the SAB and all tools must be disinfected using a spray solution of 70% isopropyl alcohol or a 10% bleach solution. Tools can be sterilized within the SAB using a small glass bead sterilizer or by dipping them in 70% alcohol.
The work area should be draft-free, and the use of masks and gloves is advised to prevent contamination from human sources. Even a brief exposure of the open culture vessel to non-sterile air can introduce spores that will ruin the culture. Maintaining this sterile field is paramount for success.
Explant Selection and Inoculation
The explant is the small piece of plant tissue chosen to start the culture, and its selection influences the success rate. Young, actively growing tissues, such as shoot tips or nodal sections, are preferred because they contain meristematic cells that readily regenerate. Sourcing explants from plants grown indoors or in a greenhouse is better than using field-grown plants, as they harbor fewer surface microbes.
The explant must undergo surface sterilization to eliminate external pathogens before being placed into the sterile medium. This involves a two-step chemical wash: first, a brief dip in 70% ethanol to break surface tension. This is followed by submerging the tissue in a dilute sodium hypochlorite solution (0.5% to 1.0% final concentration) for 10 to 40 minutes.
After chemical treatment, the explant must be thoroughly rinsed three to four times with sterile distilled water to remove disinfectant traces that could damage the cells. Working within the still air box, the sterilized tissue is trimmed with sterile tools. The small explant is then quickly transferred onto the culture medium—a process known as inoculation—and the jar is immediately sealed.
Maintaining Cultures and Acclimating Plantlets
After inoculation, sealed culture vessels are moved to a growth area providing stable, controlled conditions. Cultures require a consistent temperature, ideally 20°C to 25°C (68°F to 77°F), and low-intensity light. A photoperiod of 16 hours of light followed by 8 hours of darkness is commonly used to encourage healthy growth.
Cultures must be monitored regularly for signs of contamination, such as fuzzy mold or bacterial slime, indicating a failed sterilization attempt. After several weeks, healthy explants will grow and multiply, allowing them to be removed for the next stage: acclimation, or hardening off. This stage is necessary because the plantlets developed in nearly 100% humidity and are highly susceptible to desiccation.
To begin acclimation, plantlets are gently removed from the medium, and all traces of the nutrient gel are rinsed from the roots with clean water to prevent rot. The fragile plantlets are then planted in a sterile, fine potting mix, such as peat, perlite, or coco coir. For the first one to two weeks, containers must be covered with a humidity dome or plastic bag. This high humidity is gradually reduced over several weeks by introducing ventilation, allowing the plantlets to adapt before final transplanting.