Protocols for preparations of suspensions of intact nuclei sutiable for flow cytometric analysis of nuclear DNA content are described here. These protocols are routinely used in this laboratory. The procedure with LB01 buffer (Dolezel et al. 1989) works with most plants species and tissues. LB01 buffer is stored frozen at -20°C in aliquots for convenient use. For a very few species, the rezolution of DNA content histograms is not satiafactory using LB01. In those cases, a modification of a procedure originally developed by Otto (1990) can provide improved resolution (Dolezel and Göhde 1995). If neither of these buffers work, we recomend use of Tris-MgCl2 buffer (Pfosser et al. 1995). This buffer gives very good resolution with Arabidopsis thaliana tissues.
Nuclei can be released into cell homogenates by chopping or by lysis of protoplasts. Intact plant tissues should be disease- and stress-free. Young, rapidly growing leaves usually give the best results. Leaves may be transported or sent by post wrappedin moistened paper tissue and enclosed in a plastic bag. High temperatures should be avoided during transportation.
