Introducing Protoplasts Part II

How do you isolate plant cells for high throughput analyses?

Under normal circumstances plant cells are held together in tissues by crosslinked pectin molecules. Enzymatic digestion of the cell wall releases individual cells, or “protoplasts” which can be genetically transformed and used for further analyses. Perhaps the most prominent proponent of protoplasts is Jen Sheen’s Lab at Harvard. Commendably on their website they provide detailed protocols about how to isolate protoplasts from Arabidopsis and maize, as well as answers to FAQs about preparing and transforming protoplasts and a very helpful video about how to isolate healthy Arabidopsis protoplasts.

In our initial trials (Figure 1) we have been relying heavily on these protocols; I am not going to repeat them here as I didn’t deviate from the Sheen Lab’s protocol for maize protoplast isolation. 

Figure 1: Protoplast isolation (A) Maize seed germination - 48 h in dH2O (B) Maize seedlings grown for 9 d under long day conditions (C) the middle 8 cm of the second leaves were cut into strips and incubated in enzyme mixture (D) vacuum infiltrate the enzyme mix into leaf sections (E) gentle shaking at 40 rpm for 90 minutes (F)  tissue was filtered through Miracloth and protoplasts harvested by centrifugation.   

For Marchantia, Christian has been using a different approach:

Marchantia polymorpha (Cam-strain) according to a protocol inspired by M. Bopp et al., 1988, Plant Cell Physiol.: 10 day old thalli were harvested and pre-incubated in 10 mL half strength Gamborg's B5 medium supplemented by 1 g/L casamino acids, 0.3 g/L L-glutamine, 20 g/L sucrose, and 60 g/L d-mannitol (Solution S1) for 30 min at room temperature. Subsequently, thalli were shaken in 10 mL S1 supplemented by 2% Driselase at room temperature for 3 h. Protoplast were then filtered through a nylon net of 80um mesh width, and washed four times in S1 (centrifugation: 100xg, 4 min). Protoplasts were shaken in S1 in light over night prior to processing.
 

As you can see in (Figure 2) the plant cell wall predominantly consists of cellulose microfibrils in a cross-linked polysaccharide network of pectins (a short review can be found here). To isolate Maize protoplasts we have been using a combination of Cellulase and Maceroenzyme R-10, a multi-enzyme mixture containing Cellulase, Pectinase and Hemicellulase from Rhizopus sp. (common saprophytic fungi). Likewise the Driselase used in Marchantia protoplast isolation is also a multienzyme mixture, this time from Basidiomycetes sp. (also fungi) that contains Cellulase, Pectinase and Hemicullase activity.

Figure 2: Diagram of the plant cell wall
Source: LadyofHats - Own work, Public Domain, https://commons.wikimedia.org/w/index.php?curid=2881078

Once digested for several hours protoplasts are harvested by filtering to remove residual tissue, followed by centrifugation. Our initial attempts were successful (Figure 3), although a fairly high amount of rupturing was observed in maize protoplasts, so the next few weeks will involve optimizing the procedure (if anyone has any tips we would love to hear from you), whilst trying to develop a transformation protocol.

Figure 3: Maize Protoplasts

PS: A tip for beginners – don’t use a glass slide with coverslip to view your protoplasts – they will pop leaving you with chloroplasts!