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 LB01 in a petri dish. It is preferable to include a DNA fluorochrome (DAPI or propidium iodide) 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, 2 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 LB01 to a concentration of 105 - 106/ml. The concentration of detergent (Triton X-100) in LB01 buffer should be increased to 0.5 % (v/v); this improves the release of the nuclei from the protoplasts.
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.
