Embryo Rescue Protocol for Old Seeds Using Tissue Culture:
Materials:
- Old seeds with low germination potential
- Sterile distilled water
- Disinfectant (e.g., bleach)
- Sterile culture medium (e.g., Murashige and Skoog medium)
- Sterile containers (Petri dishes, culture tubes)
- Scalpel or sterile razor blade
- Plant growth regulators (e.g., auxins and cytokinins)
- Incubator with controlled temperature and light conditions
- Laminar flow hood for aseptic work
Procedure:
- Seed Surface Sterilization:
- Immerse seeds in a solution of 10% bleach for 10-15 minutes.
- Rinse seeds thoroughly with sterile distilled water.
- Isolation of Embryos:
- Work under a laminar flow hood.
- Using a sterile scalpel or razor blade, excise embryos from the surface-sterilized seeds.
- Initiation of Culture:
- Place isolated embryos onto the sterile culture medium in Petri dishes or culture tubes.
- Ensure the medium contains appropriate plant growth regulators to stimulate embryo development.
- Incubation:
- Seal containers to maintain sterility.
- Place containers in the incubator at a controlled temperature (typically 25°C) with appropriate light conditions.
- Sub-culturing:
- Monitor embryo development regularly.
- Sub-culture embryos onto fresh medium as needed, adjusting plant growth regulators accordingly.
- Rooting and Acclimatization:
- Once embryos develop into plantlets, induce rooting by adjusting growth regulator concentrations.
- Transfer rooted plantlets to soil or another suitable substrate for acclimatization.
- Hardening Off:
- Gradually expose acclimatized plants to ambient environmental conditions for successful adaptation.
- Documentation:
- Record all steps, observations, and any modifications made during the process.
Notes:
- Maintain aseptic conditions throughout the procedure to prevent contamination.
- Adjust plant growth regulator concentrations based on the specific needs of the plant species.
