Centre of Plant Structural and
Functional Genomics
of the Institute of Experimental Botany AS CR

1. Harvest root tips (1 cm) and transfer them into deionized H₂O.
2. Immediately transfer root tips into 25 ml of 4 % formaldehyde fixative and fix at 5°C for 30 min.
3. Wash roots in 25 ml of Tris buffer three times for 5 min at 5°C.
4. Excise root meristems and transfer them into 5-ml polystyrene tube containing 1 ml of LB01 lysis buffer (pH 7.5).
5. Isolate chromosomes by homogenising at 15000 rpm for 15 sec.
6. Filter the suspension through a 50-µm nylon mesh into 5-ml polystyre

In this procedure, chromosomes are released from synchronized root tips mechanically after a mild fixation with formaldehyde. Chromosomes are released into a polyamine lysis buffer (LB01) which stabilises their structure. The method has been originally developed for chromosome isolation in field bean (Dolezel et al. 1992). Chromosome suspensions prepared according to this procedure are suitable for flow cytometric analysis and sorting.

Univariate Flow Karyotyping and Chromosome Sorting

This protocol describes the analysis and sorting of plant chromosomes stained with 4-,6-diamidino-2-phenylindole (DAPI). This method was originally developed by Lucretti et al. (1993) for field bean and requires a flow cytometer equipped with a light source providing excitation in UV. We use BD FACSVantage equipped with a Coherent Innova 305 argon-ion laser.

A general outline of the procedure for flow cytometric analysis and sorting of plant chromosomes consists of the following steps: 

1. accumulation of cells in metaphase 
2. preparation of chromosome suspensions 
3. flow analysis and sorting 
4. processing of flow-sorted chromosomes 

Although plant chromosome suspensions have also been prepared from cultured cells and mesophyll protoplasts, the most frequently used procedure, developed by Dolezel et al. (1992), involves the use of root tips.

Estimation of Purity of Sorted Chromosomes Using FISH

This protocol describes a procedure that allows precise estimation of the purity of a sorted fraction (Kubalakova et al. 2000). The procedure is based on specific fluorescent labelling of repetitive DNA sequences that show characteristic pattern of distribution among the chromosomes. The sequences are labelled using hybridization of specific probes labelled with biotin or digoxigenin and detected with avidin, streptavidin or anti-digoxigenin antibody conjugated with fluorochromes.

use deionized or double-distilled water in all recipes

Plant DNA Standards

It is generally agreed that flow cytometric estimation of nuclear DNA amount in absolute units should be performed using internal standard (the nuclei of a standard are isolated, stained and analysed simultaneously with the nuclei of a sample).