Plant tissue culture (PTC) is a technique used to grow plant cells, tissues, or organs in an artificial, sterile environment under controlled physical conditions. This method allows for the rapid, asexual multiplication of plants, producing numerous genetically identical copies from a single parent. PTC is also used to obtain disease-free plants, often by culturing the meristematic tips which are naturally free of systemic pathogens. The process relies on providing the plant material with specialized nutrients and growth regulators to direct its development in vitro.
Establishing Aseptic Conditions and Necessary Tools
The foundation of successful plant tissue culture rests upon maintaining a completely sterile workspace, known as asepsis. Contamination by airborne fungi or bacteria is the most frequent cause of failure, as the nutrient-rich culture media provides an ideal environment for microbial growth. Therefore, the physical environment and all tools must be rigorously sterilized before starting the culture process.
The primary workspace is typically a laminar flow hood, which constantly pushes filtered, sterile air (HEPA-filtered) over the working area to prevent microorganisms from settling. If a specialized hood is unavailable, a clean box or highly sanitized enclosed space can serve as a substitute for simple procedures. The surface inside this sterile area must be wiped down with 70% ethanol or a similar disinfectant before use.
All instruments that will touch the plant material or the culture medium must also be sterilized, usually through heat. Tools like scalpels, forceps, and scissors are often sterilized using dry heat, such as dipping them in 95% ethanol and briefly flaming them, or by using a specialized glass bead sterilizer. For the culture vessels, such as test tubes or jars, moist heat sterilization is generally used, requiring an autoclave to apply high-pressure steam at 121 degrees Celsius for 15 to 20 minutes.
Formulating the Nutrient Media
The nutrient media serves as the artificial substrate for the plant material, and its formulation is a detailed biochemical recipe. The most widely used base formulation is Murashige and Skoog (MS) medium, which provides a balanced blend of components. The media consists of several categories of ingredients, beginning with inorganic mineral salts, which are divided into macronutrients (like nitrogen and potassium) and micronutrients (such as iron, boron, and zinc).
A carbon source, typically sucrose, is included because the cultured plantlets are unable to perform adequate photosynthesis in the low-light conditions of the culture vessel. Vitamins, particularly B vitamins like thiamine, are also added to support the metabolic pathways of the growing tissue. These components are dissolved in distilled water, and the solution’s pH is adjusted to a slightly acidic range, usually between 5.6 and 5.8.
A gelling agent, like agar, is added to solidify the liquid medium, providing a stable surface for the plant tissue to rest upon. Plant Growth Regulators (PGRs), often called plant hormones, are included to dictate the developmental outcome of the culture. Auxins generally promote root formation, while cytokinins stimulate cell division and shoot formation. By carefully adjusting the ratio and concentration of these PGRs, scientists can direct the explant to multiply shoots, form roots, or generate undifferentiated tissue known as callus. The completed media is then dispensed into the culture vessels and sterilized by autoclaving.
Preparing and Initiating the Culture
The first step involving the plant is the selection of the explant, which is the small piece of tissue chosen for culture, such as a nodal segment or a shoot tip. Since the exterior of the explant is covered in microorganisms, it must undergo surface sterilization before being placed into the sterile media. Preparing the mother plant beforehand by growing it in clean conditions can help reduce the initial microbial load.
The surface sterilization protocol typically involves a two-step chemical wash designed to kill external microbes without damaging the internal plant cells. The explant is first dipped briefly in 70% ethanol, which dissolves waxes and allows the subsequent disinfectant to penetrate the surface more effectively. This is followed by immersion in a solution of sodium hypochlorite, commonly known as household bleach, diluted to a working concentration of 0.5% to 1.0%.
The tissue is left in the hypochlorite solution for 10 to 30 minutes, depending on the species and tissue type, often with a wetting agent added to improve contact. After disinfectant treatment, the explant must be rinsed thoroughly, typically three to four times, with sterile distilled water to remove any residual chemicals. Working inside the sterile hood, the explant is trimmed to remove any chemically damaged edges and carefully transferred onto the prepared nutrient medium. The inoculated vessels are then sealed and placed in an incubation chamber, usually set between 22 and 25 degrees Celsius, under controlled lighting conditions.
Moving Plants Outside the Lab
The final stage is acclimatization, or “hardening off,” which transitions the lab-grown plantlets from the artificial environment to normal growing conditions ex vitro. Plantlets developed in culture are fragile, having grown in a highly humid, sterile environment with a constant supply of sugar, resulting in poor development of their waxy cuticle and root system.
The acclimatization process begins by gently removing the plantlets from the culture vessel and thoroughly washing the roots to remove all traces of the agar medium. They are then transplanted into a sterile, porous substrate, such as a mix of peat and vermiculite. The plantlets are initially maintained in a semi-controlled environment with extremely high humidity, often 80% to 90%, to prevent desiccation.
Over a period of several weeks, the humidity is gradually reduced, and the light intensity is slowly increased to stimulate the plantlet to develop a functional cuticle and begin proper photosynthesis. This gradual exposure allows the plant to develop the necessary resilience for survival outside of the laboratory setting. Once the plantlets show robust new growth and a functional root system, they are ready for transplanting into larger pots or the field.