General Tropical Houseplant Tissue Culture Protocol#

Turn on BSC and allow to equalize and clean the space for at least 30 minutes before working. Thoroughly sterilize the work area inside the BSC and lab bench with 70% IPA and allow to sit wet before wiping clean.

Run a 15 minute sterilization cycle in the 2540E autoclave with any tools required in sterilization bags. Inlcude as many PP cups and lids required wrapped in oven bags. Allow to cool in the BSC under sterile flow. Optionally, sterilize tools and containers with the media below based on available space.

Mix media in a media bottle on magnetic stirrer according the ratios below. Add distilled water to mnake 1L of solution. CHeck and adjust pH as required to bring to goal pH.

Sterilize media in the autoclave with a liquid cycle along with a container of distilled water (sterile water to rinse the explants). Mix media on magnetic stirrer after removing to ensure even distribution of agar.

Move sterile water and media to the BSC under sterile flow. Pour media into sterile containers and loosely place lid to prevent contamination of media. Allow to cool and begin to solidify.

Warm up bead sterilizer in the BSC.

Use sterile scissors to remove nodes from plant. Sterilize with 70% IPA between cuts.

Wash nodal explants under running water with dish soap to clear dirt and external debris.

Run explants on orbital shaker at around 500RPM to sanitize with a 10% bleach solution (1 part bleach and 9 parts water) for 15 to 25 minutes. Best to tend longer to reduce contamination risk.

Place sterile scissors in bead sterilizer to heat.

Move bleached exlants to the BSC and rinse nodes 3 times under sterile water to remove bleach. Use scissors to off bleached, exposed portions. Sterilize with 70% IPA between cuts.

Press one or more explants into media containers with sterile forceps and secure the lid.

Maintain under a 16h/8h light cycle and subculture to Stage 2 media after around 6 weeks.