TC Steps

Prep:

1. Turn on laminar flow hood to warm up (30 minutes at least)

2. Spray down flow hood with 70% EtOH, wipe with kimwipe

3. Light alcohol Bunsen burner flame or turn on Micro Bead Sterilizer

4. Put new blades on knives

5. Sterilize tools (knives and forceps), rest on bar to cool

If putting new plant material in culture:

1- Gather plant material from the field or greenhouse (4-6”) cuttings stripped of all leaves (leaving nodes in tact)

2. Make 10% (Greenhouse) to 20% (Field) bleach: TWEEN 20 (2-3 drops) solution

3. Gather appropriate number of empty clean flasks and put in flow hood

4. Write labels on tape and stick on flasks

5. Get a large beaker for dumping bleach/water into as waste

6. Get flask of autoclaved water

7. Place a small stack of autoclaved plates, clean Petri dishes, or autoclaved paper towels in the flow hood

8. Place a few cuttings in a flask (labeled for each cultivar) and cover with bleach solution

9. Agitate flask for 15 minutes

10. Decant the Bleach solution into waste beaker

11. Add sterile water to each flask covering the cuttings

12. Agitate for 3 minute

13. Decant the water into waste beaker

14. Repeat steps 12-13 for a total of 3 rinses

15. Place plant cuttings on plate/ Petri dish/ paper towel and cut into 3/4” pieces each containing a node (use different plate for different cultivars, don’t cut in same spot)

16. Place 1 cutting each in a 14 mL culture tube with the node oriented the correct direction (up)

17. Place under dark conditions at 25 土 1°C for 2 weeks

18. After 2 weeks, transfer to light conditions at 25 土 1°C under a 16-h photoperiod from cool white fluorescent lights. If using a grow room: Set up timer and AC for grow room (lights 7AM-7PM) and AC at 70 F.

19. After 4-6 weeks, shoots should be strong enough to transfer to Magenta boxes (5-10 shoots per box)

If transferring microcuttings in TC to new media – AKA refreshing media:

Note: Recommend sub-culturing every 2 weeks for for establishment and once per month for maintenance

1. Prepare necessary amount of new media and pour into Magenta boxes or culture tubes

2. Go to grow room/ incubator and get Magenta boxes or tubes that need to be transferred

3. Discard any contaminated boxes or tubes

4. Write new labels if need to or peel off old Magenta/ tube and stick on new one

5. Open Magenta/ tube with plant in it and use sterile forceps to gently remove the plant from the old receptacle and transfer it to the new one

6. Secure Magenta box lid or tube lid

7. Return to grow room/ incubator