Plant tissue culture is a technique involving growing plant cells, tissues, or organs in a controlled, sterile environment. This method allows for the propagation of plants, genetic modification, and the study of plant growth and development under precise conditions. The success of any plant tissue culture endeavor depends on maintaining a sterile environment throughout the process. Contamination by microorganisms, such as bacteria, fungi, or yeasts, poses a significant threat to plant tissue cultures. These contaminants can rapidly outcompete the plant tissues for nutrients, leading to the death of the culture and the failure of the experiment.
Sterilizing Plant Material
Sterilizing the plant material, often referred to as explants, is a fundamental step before introducing them into a sterile culture medium. This process, known as surface sterilization, aims to eliminate surface-dwelling microorganisms without causing damage to the delicate internal plant tissues. The effectiveness of sterilization depends on the type of explant, its initial microbial load, and the chosen sterilizing agents.
Sodium hypochlorite, commonly found in household bleach, is a frequently used sterilizing agent due to its broad-spectrum antimicrobial activity. Explants are typically submerged in a solution ranging from 0.5% to 2.5% active chlorine for durations spanning 5 to 30 minutes, depending on the tissue type and level of contamination. After treatment, multiple rinses with sterile distilled water remove residual hypochlorite, which can be detrimental to plant cells.
Calcium hypochlorite offers an alternative to sodium hypochlorite, often preferred for its slightly milder action on plant tissues. Solutions are generally prepared at concentrations between 5% and 10% (w/v), with explants exposed for 10 to 20 minutes. Similar to sodium hypochlorite, thorough rinsing with sterile water is imperative to wash away any remaining sterilant.
Mercuric chloride is another highly effective sterilant, typically used at very low concentrations, often between 0.01% and 0.1% (w/v). Despite its effectiveness, its high toxicity and environmental concerns have led to a decrease in its general use, though it may still be employed for recalcitrant explants with high contamination rates. Regardless of the agent used, the duration of exposure is carefully balanced to ensure microbial elimination without compromising explant viability.
The type of plant material significantly influences the sterilization protocol. For instance, seeds, which often carry a high microbial load on their surfaces, may require longer exposure times or stronger sterilants compared to young, tender shoot tips. Robust stem segments might tolerate more aggressive treatments than delicate leaf tissues. Optimizing the sterilization procedure involves careful consideration of the explant source and its inherent susceptibility to both contamination and chemical damage.
Sterilizing Growth Media
The nutrient-rich growth media used in plant tissue culture provide an ideal environment for microbial proliferation, making their sterilization a necessity. Autoclaving, a process of steam sterilization, is the primary and most reliable method for sterilizing these media. This method uses saturated steam under pressure to achieve temperatures high enough to kill all microorganisms, including bacterial spores.
Typically, culture media are autoclaved at a temperature of 121°C (250°F) and a pressure of 15 pounds per square inch (psi). The duration of autoclaving varies based on the volume of the medium, with smaller volumes (e.g., 100 mL) requiring approximately 15 minutes, while larger volumes (e.g., 1 liter) may need 20-30 minutes. Prior to autoclaving, the medium’s pH is adjusted to the desired range, usually between 5.6 and 5.8, as pH can affect the stability of certain components during heat treatment.
Preparation of the media before autoclaving involves ensuring appropriate container size. Bottles or flasks should not be overfilled, typically leaving sufficient headspace to prevent boiling over during the sterilization cycle. Loose caps or vented closures are also used to allow steam penetration and prevent pressure buildup within the containers.
For heat-sensitive components, such as certain vitamins, plant hormones like cytokinins or auxins, or antibiotics, filtration sterilization is employed as an alternative. These components are prepared separately and passed through sterile filters with a pore size typically of 0.22 micrometers. This physical removal of microorganisms is performed after the main media has been autoclaved and cooled, ensuring that the integrity of these delicate substances is maintained.
Sterilizing Equipment and Workspace
Beyond the plant material and growth media, all equipment and the working environment must also be meticulously sterilized to prevent contamination. Glassware, including petri dishes, culture vessels, and measuring cylinders, is commonly sterilized using either autoclaving or dry heat sterilization. Dry heat ovens operate at higher temperatures, typically 160-180°C (320-356°F) for 2-4 hours, which is effective for heat-stable items and ensures complete dryness.
Dissecting tools such as scalpels, forceps, and spatulas are routinely sterilized by autoclaving or by flaming. Flaming involves dipping the metal parts of the tools in 70% ethanol and then briefly passing them through a Bunsen burner flame until the alcohol burns off. This rapid method effectively sterilizes the surfaces of the tools, and it is commonly performed between handling different explants to prevent cross-contamination.
The working environment itself plays a paramount role in maintaining sterility. Laminar flow hoods, also known as clean benches, are indispensable for providing a sterile working area. These specialized workstations direct a continuous, filtered stream of air over the work surface, creating a barrier that prevents airborne contaminants from reaching the culture materials. The air is typically passed through a High-Efficiency Particulate Air (HEPA) filter, which removes particles as small as 0.3 micrometers, including most microbial spores and dust.
Before initiating any work, the interior surfaces of the laminar flow hood, including the work surface and side walls, are thoroughly wiped down with a disinfectant, most commonly 70% ethanol. This alcohol solution effectively denatures proteins and dissolves lipids in microbial cell membranes, leading to their inactivation. Hands are also disinfected with 70% ethanol or a similar antiseptic solution before beginning work within the sterile environment.