Proceedings of the XLV Italian Society of Agricultural Genetics - SIGA Annual Congress

Salsomaggiore Terme, Italy - 26/29 September, 2001

ISBN 88-900622-1-5

Poster Abstract

CELL CYCLE SYNCHRONIZATION AND FLOW KARYOTYPING OF THE ZEA MAYS VAR. INDURATA, LANDRACE “NOSTRANO DI STORO” ROOT TIP CHROMOSOMES

LUCRETTI S.*, VALVO S.*, NARDI L .*, PALLOTTINI L.**, BARCACCIA G.**

* ENEA C.R. Casaccia Divisione Biotecnologia e Agricoltura, Via Anguillarese 301, 00100 Roma

lucretti@casaccia.enea.it

** Dipartimento di Agronomia Ambientale e Produzioni Vegetali, Facoltà di Agraria, Università di Padova, Agripolis, Legnaro, 35020 Padova

corn, cell cycle synchronization, Plant chromosome and nuclei isolation, flow cytometric analysis and sorting

Chromosome separation by flow-sorting represents a powerful tool for fractionation of eukaryotic genomes into discrete parts which are individual chromosome types. The chromosomal DNA can be cloned into a suitable vector in order to obtain a chromosome specific library which can serve as a source of molecular markers for the region of interest. High density molecular maps are valuable tools for genome analysis, map-based gene cloning and marker-assisted breeding of crop plants. As a matter of fact, the large size and complexity of most of higher plant genomes make the map saturation a difficult and time-consuming task. The division of the genome to smaller parts which can be studied separately would simplify and accelerate its analysis.

Here we describe a highly effective methodology in the Zea mays var. indurata landrace “Nostrano di Storo” for the isolation of chromosomes and nuclei in suspension by homogenization, which is effective even from a single fixed root tip. The yield and quality of these suspensions are suitable for flow cytometric analysis and sorting. This procedure is based on a two-steps cell cycle synchronization of root tip meristems to obtain a high mitotic index, followed by formaldehyde fixation and mechanical isolation of chromosomes and nuclei by homogenization. In the explant, more than 60% of metaphases were induced through a synchronization of the cell cycle at G1/S interface with hydroxyurea (2.5 mM) followed, after a 1-2 hours release, by a block in metaphase with amiprophos-methyl (2.5-5.0 µM). The quality and quantity of nuclei and chromosomes were related to the extent of the fixation with formaldehyde.