In plant tissue culture, surface sterilization, subculturing, and repeated transfer of explants are critical steps to ensure the successful growth of plant tissues in a controlled, sterile environment. Let's go over each process in detail:
1. Surface Sterilization
Definition: Surface sterilization is the process of removing or killing microorganisms (bacteria, fungi, etc.) present on the surface of plant tissues (explants) before they are cultured in a sterile medium.
Steps:
Selection of Explants: Choose the plant part you want to culture, such as a leaf, stem, root, or shoot tip.
Pre-washing: Clean the explants under running tap water to remove visible dirt, dust, and debris.
Treatment with Disinfectants:
Ethanol (70% solution): The explants are often dipped in 70% ethanol for about 30 seconds to 1 minute to kill surface microbes.
Sodium Hypochlorite or Bleach (2-10% solution): A common disinfectant used to kill microorganisms. The explants are submerged in a diluted bleach solution for about 5-10 minutes.
Mercuric Chloride (0.1-0.2% solution): This is a more toxic sterilant and is used in smaller amounts when dealing with very resistant microorganisms. The treatment usually lasts 1-5 minutes.
Rinsing: After disinfection, the explants are rinsed several times with sterile distilled water to remove traces of the sterilizing agent, which could otherwise damage the plant tissue.
Importance: This process ensures that no contaminants (fungi, bacteria) are introduced into the culture medium, which could outcompete the plant tissue and prevent successful growth.
2. Subculturing
Definition: Subculturing is the process of transferring plant tissues (explants or plantlets) from one culture medium to another, either to provide fresh nutrients, change hormone levels, or propagate new cultures.
When It Is Done:
When the culture medium depletes nutrients or dries out.
When the explants or plantlets outgrow the current vessel.
To induce further growth (e.g., rooting, shoot formation).
To remove explants from contaminated cultures.
Steps:
Prepare Fresh Culture Media: Make fresh media with the required nutrients and plant hormones depending on the growth stage (e.g., for rooting or shooting).
Sterile Environment: Under a sterile laminar air flow cabinet, the cultured tissue is carefully removed from its current vessel.
Cutting and Transferring: The explants are either divided into smaller sections or whole plantlets are transferred into new culture vessels containing fresh medium.
Sealing: The new vessels are sealed (e.g., with parafilm) to maintain sterility.
Incubation: The subcultured plant tissue is then placed back in the incubator or growth chamber under appropriate light and temperature conditions.
Importance: Subculturing provides fresh nutrients and helps the plant tissue continue its growth and development. It also allows for the multiplication of plant material in large quantities.
3. Repeated Transfer of Explants
Definition: Repeated transfer involves the frequent transfer of explants from one culture medium to another over a prolonged period. This process ensures the long-term survival and continuous growth of the plant tissue.
Why It’s Needed:
To maintain explant health and avoid nutrient depletion in the medium.
To maintain optimal hormone concentrations for different stages of growth (e.g., callus formation, shoot growth, or rooting).
To maintain sterility and avoid contamination buildup.
For cloning or propagating large numbers of plants (micropropagation).
Steps:
Monitoring: Regularly observe the growth of the explants in the culture medium. Check for contamination and overgrowth.
Transfer to Fresh Medium: As explants grow, transfer them to fresh culture media with specific nutrients or hormones to continue their development.
Repeat the Process: Depending on the growth phase (e.g., callus formation, shooting, rooting), transfer explants multiple times to optimize growth.
Importance: This repeated transfer allows for sustained plant development and helps produce large numbers of genetically identical plants (clones), a process widely used in agriculture and horticulture.
Key Points to Remember:
Sterility is critical: Even the smallest contamination can destroy your culture. Always use a sterile environment, tools, and media.
Media preparation is tailored: Depending on the growth stage of the explant, different media with varying nutrients and hormones are required.
Repeated transfers are strategic: As the explant grows or develops new structures, it needs to be transferred to media with suitable nutrients and conditions (e.g., from a shoot-inducing medium to a root-inducing one).
These three steps are fundamental to successful plant tissue culture, ensuring that the plant tissues are kept in a healthy, sterile environment while they grow and develop