Seed sterilization and plant growth
Bleach seed sterilization
To perform bleach seed sterilisation, firstly seeds should either be wrapped in a miracloth package (Cal-Biochem) or placed in an Eppendorf tube. Seeds are then immersed in 70% ethanol for two minutes, followed by a 5 minute soak in 5% bleach 0.5% SDS. Seeds should then be rinsed six times with sterile water. Seeds in te miracloth are air dried in the fume hood, and sprinkled onto GM plates; seeds in Eppendorf tubes can be pipetted onto GM plates.
Chlorine Gas Seed Sterilisation
When performing sterilization by using chlorine gas, firstly seeds in open Eppendorf tubes should be placed into a desiccator jar in a fume hood. A beaker containing 100ml of bleach is then placed in the desiccator next to the seeds. 3ml of concentrated HCL is added to the beaker to create chlorine gas. The desiccator jar should be quickly sealed and a vacuum applied. Seeds require to be exposed to the chlorine gas for six and a half hours. Following release of the gas, the tubes should be quickly sealed and transferred to a laminar flow hood, and transferred to GM growth plates.
Following transfer of seeds to GM plates, containing the relevant antibiotics, plates should be sealed with Micropore surgical tape (3M), then covered with tin foil and stored at 4°C for 3-5 days to imbibe seeds. After this period the Tin foil wrap can then be removed from the plates, and the plates transferred to Percival growth cabinets with the relevant growth conditions.