1a. Chop a small amount of plant material (typically 20 mg) with a new razor blade or a sharp scalpel in 1ml of ice-cold Tris-MgCl2 buffer in a petri dish. It is preferable to include a DNA fluorochrome (DAPI or propidium iodid) in the buffer prior to chopping. Alternatively, this compound may be added immediately after the filtration (step 2). The stains are used in the following concentrations: DAPI, 4 µg/ml; propidium iodide, 50 µg/ml + RNase, 50 µg/ml.
The actual quantity of plant material to be used for nuclei isolation depends both on the type of tissue and on the species, and must be determined experimentally (higher quantities are usually needed of callus or cultured cells).
1b. As an alternative, protoplasts can be prepared and resuspended in ice-cold Tris buffer to a concentration of 105 - 106/ml.
Nuclei cannot be released from "collapsed" protoplasts, hence protoplast viability is an important consideration. Typically the protoplasts should be 90-100% viable as determined using FDA.
2. Filter the suspension through a 42 µm nylon mesh.
3. Store on ice prior to analysis (a few minutes to one hour).
4. Analyse relative DNA content of isolated nuclei.
