Plant tissue culture, often called micropropagation, is a laboratory technique for growing whole plants from tiny pieces of tissue in a controlled, sterile environment. This method relies on the principle of totipotency, the ability of a single plant cell to regenerate an entire organism. Micropropagation offers a way to rapidly produce thousands of genetically identical, disease-free plants from a small amount of starting material, especially for species difficult to propagate conventionally. Successfully performing this technique requires precise adherence to sterile conditions, careful preparation of nutrients, and proper handling of delicate plant material.
Establishing the Sterile Working Environment and Tools
The most frequent reason for failure in plant tissue culture is contamination from airborne microbes, fungi, or bacteria. Professionals typically use a laminar flow hood, which constantly pushes filtered, sterile air across the work surface. A still air box or glove box can serve as a functional, less expensive alternative, creating a contained environment that reduces contamination risk during transfer.
All tools that will touch the plant material or the medium, such as scalpels, tweezers, and Petri dishes, must be thoroughly sterilized. Metallic instruments are often sterilized using dry heat or flaming. Glassware and the culture medium itself are generally sterilized using moist heat, typically in an autoclave or pressure cooker for 15 to 20 minutes.
The work surface within the sterile area must be wiped down with a disinfectant, commonly \(70\%\) ethanol or a bleach solution, before and after use. Technicians should also wear gloves, often sterilizing them with \(70\%\) ethanol to prevent transferring microbes from their skin. Maintaining this aseptic technique throughout the entire procedure is foundational for preventing the growth of contaminants on the nutrient-rich culture medium.
Explant Selection and Nutrient Medium Preparation
The process begins with selecting the explant, the small piece of source plant tissue used to start the culture. Explants should be collected from healthy, vigorously growing mother plants. Meristematic tissues like shoot tips or nodal sections are often preferred due to their high regeneration capacity and lower incidence of contamination. Plant material collected from plants grown outdoors is generally more contaminated than those grown in a controlled greenhouse environment.
The explant must undergo surface sterilization to eliminate external microorganisms without destroying the plant cells. This chemical process involves a brief dip in \(70\%\) ethanol, followed by immersion in a diluted household bleach solution (sodium hypochlorite), typically at a \(0.5\%\) to \(1.0\%\) concentration. The explant is soaked for 10 to 20 minutes, depending on the species and tissue type.
Following the sterilization soak, the explant must be thoroughly rinsed three to four times using sterile distilled water to remove all traces of the toxic chemicals. Concurrently, the nutrient medium, which acts as the artificial soil, must be prepared. The most widely used formulation is Murashige and Skoog (MS) medium, a blend of macronutrients, micronutrients, and vitamins.
The MS medium provides high concentrations of elements like nitrogen and potassium, along with a carbon source, usually \(3\%\) sucrose, since the plantlets cannot photosynthesize effectively in the jar. A gelling agent, such as agar, is added to solidify the liquid medium. The mixture’s \(\text{pH}\) is adjusted to a range of \(5.7\) to \(5.8\) before the final sterilization by autoclaving. Plant growth regulators (auxins and cytokinins) are included at specific ratios to direct the explant to produce shoots, roots, or a mass of undifferentiated cells called callus.
Inoculation and Culture Initiation
The inoculation phase is the sterile transfer of the prepared explant onto the nutrient medium. This step must be performed entirely within the sterile working environment, such as a laminar flow hood, to maintain aseptic conditions. The sterilized explant is moved from the final sterile water rinse to the work surface using sterilized forceps.
Using a sterilized scalpel, the edges of the explant damaged by sterilization chemicals are carefully trimmed away to expose fresh, undamaged tissue. The explant is then cut into small segments, with the size depending on the tissue and the desired outcome. Speed and precision are important during this step to limit the explant’s exposure time to non-sterile air.
The trimmed explant piece is gently placed onto the solidified nutrient medium in the culture vessel, ensuring good contact with the gel. Immediately after the transfer, the culture vessel, which can be a jar, test tube, or Petri dish, must be securely sealed to prevent the entry of airborne contaminants. The seal also maintains the high humidity necessary for the explant’s initial survival.
Finally, the newly inoculated vessels are labeled with the plant name, date, and specific medium formulation used. The sealed containers are then moved to a dedicated growth area where controlled culturing conditions will be maintained, initiating the growth phase.
Culturing Conditions and Acclimation to Soil
After inoculation, the sealed culture vessels are placed in a growth room or chamber where environmental factors are strictly controlled to promote development. Temperature is typically maintained at a stable, moderate range, often between \(20^\circ \text{C}\) and \(25^\circ \text{C}\). Light is provided by fluorescent or \(\text{LED}\) fixtures, but usually at a lower intensity than natural sunlight, often for a photoperiod of 16 hours of light followed by 8 hours of darkness.
The sealing of the vessel creates a condition of nearly \(100\%\) relative humidity, which is necessary because the plantlets have not yet developed a fully functional cuticle layer to control water loss. Once the plantlets have multiplied and developed sufficient shoot and root growth, they are ready for the final, challenging stage: acclimation, or hardening off. Acclimation is the process of gradually adapting the lab-grown plants to the variable conditions of the external environment.
The plantlets are first removed from the culture vessels, a process called deflasking, and the nutrient gel is thoroughly washed from their roots with water. They are then transferred into a non-sterile growing medium, such as a mixture of peat, vermiculite, or perlite. The initial environment must replicate the high humidity of the culture vessel to prevent the plantlets from wilting.
To achieve this, the newly potted plantlets are often placed under a protective cover or in a high-humidity chamber, maintaining \(80\%\) to \(90\%\) humidity for the first week or two. The humidity is then slowly decreased over several weeks by gradually opening the cover or making small holes in the plastic to introduce ambient air. This slow reduction in humidity encourages the development of a functional cuticle, the waxy layer on leaves, and strengthens the plant’s ability to survive in a normal, non-sterile greenhouse or outdoor setting.