Isolation of chromosomes from Pisum sati6um L. hairy root cultures and their analysis by flow cytometry

Continuously growing hairy-root cultures of pea (Pisum sati6um L.) line L-84 have been induced by transformation of seedlings by Agrobacterium rhizogenes A4-24. The cultures were then used for cell cycle synchronisation in vitro and for preparation of chromosome suspensions. Various concentrations of hydroxyurea (0.5–2.0 mM) and timing of amiprophos-methyl (APM, 2–4 h) treatment have been tested to achieve the highest mitotic synchrony, and to accumulate the highest number of synchronised cells in metaphase, respectively. Optimal conditions (2 mM hydroxyurea for 18 h, 10 mM APM for 2 h applied 6 h after a release from hydroxyurea block) resulted in a high frequency of cells in metaphase (43%). The synchronised root tips were fixed in formaldehyde and chromosomes were released into a lysis buffer by mechanical homogenisation. The chromosomes in suspension showed a well preserved morphology, and could be used for flow cytometric analysis. While only two chromosomes (5 and 7) were discriminated in a standard karyotype, four chromosomes (3, 5, 6 and 7) could be clearly discriminated in the line L-84 which contains a stable reciprocal translocation between chromosomes 3 and 6. The establishment of hairy root cultures and the development of an efficient chromosome isolation procedure from a translocation line L-84 represent a basic prerequisite for subsequent flow-sorting of selected chromosomes of pea. © 1998 Elsevier Science Ireland Ltd. All rights reserved.