PREPARATION OF PEA (PISUM-SATIVUM L) CHROMOSOME AND NUCLEUS SUSPENSIONS FROM SINGLE ROOT-TIPS

A high-yield method for the isolation of intact nuclei and chromosomes in suspension from a variable number of pea root tips (1-10) has been developed. This procedure is based on a two-step cell-cycle synchroni zation of root-tip meristems to obtain a high mitotic index, followed by formaldehyde fixation and mechanical isolation of chromosomes and n uclei by homogenization. In the explant, up to 50% of metaphases were induced through a synchronization of the cell cycle at the G(1)/S inte rface with hydroxyurea (1.25 mM), followed, after a 3-h release, by a block in metaphase with amiprophos-methyl (10 mu M). The quality and q uantity of nuclei and chromosomes were related to the extent of the fi xation. Best results were obtained after a 30-min fixation with 2% and 4% formaldehyde for nuclei and chromosomes, respectively. The method described here allowed the isolation of nuclei and chromosomes, even f rom a single root tip, with a yield of 1 x 10(5)/root and 1.4 x 10(5)/ root, respectively. Isolated suspensions were suitable for flow cytome tric analysis and sorting and PRINS labelling with a rDNA probe.