I am interested in starting a liquid suspension arabidopsis culture to provide a source of protoplasts. I have previously only used callus' for protoplast isolation. If anyone has a method of establishing a cell suspension culture it would be appreciated.
- To start a liquid suspension culture, you need to first obtain callus from Arabidopsis seeds or leaves. You can sterilize the seeds or leaves with bleach and then germinate them on solid MS0 medium. After a few weeks, you can transfer the plants to a sterile tile and cut the roots or leaves into small pieces. Then, you can place them on callus induction medium, which contains Gamborg's B5 basal medium, glucose, agar, MES, 2,4-D, and kinetin. You should keep the Petri dishes in a constant temperature room with or without light.
- After callus formation, you can transfer a small piece of callus, about the size of a pea, to a 100 ml conical flask with 20 ml of liquid MSA medium. This medium contains Murashige and Skoog basal medium, sucrose, 2,4-D, and kinetin. You should replace the lid with aluminum foil and shake the flask at 40 rpm in a constant temperature room. After 7-10 days, you can subculture the flask by decanting the liquid and adding fresh MSA medium. You should use 100 ml of medium per 250 ml flask and subculture at weekly intervals.
- To obtain protoplasts from the liquid suspension culture, you can use cellulase and pectinase enzymes to digest the cell walls. You can filter the protoplasts through a nylon mesh and wash them with a suitable buffer. You can then use the protoplasts for various experiments, such as transformation, fusion, or electrophysiology
- Badges
- Science topic
- Similar topics
- Computer Science and Engineering
- Computer Graphics and Animation
- 3D