A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

Summary

A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

Abstract

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high quality plant chromosome spreads is challenging due to the rigid cell wall. One of the approved methods for the preparation of plant chromosomes is a so-called drop preparation, also known as drop-spreading or air-drying technique. Here, we present a protocol for the fast preparation of mitotic chromosome spreads suitable for the FISH detection of single and high copy DNA probes. This method is an improved variant of the air-dry drop method performed under a relative humidity of 50%-55%. This protocol comprises a reduced number of washing steps making its application easy, efficient and reproducible. Obvious benefits of this approach are well-spread, undamaged and numerous metaphase chromosomes serving as a perfect prerequisite for successful FISH analysis. Using this protocol we obtained high-quality chromosome spreads and reproducible FISH results for Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum and Secale cereale.

Introduction

Fluorescence in situ hybridization (FISH) is an effective tool for the physical mapping of single and high copy sequences at the chromosomal level. Prerequisite is the preparation of high quality chromosome spreads. There is no general chromosome preparation protocol that would be equally suitable for animal and plant cells. Preparation of plant chromosomes is particularly challenging due to the rigid cell wall and various cytoplasm consistency within different species. One of the favorable methods for the preparation of plant chromosomes is a so-called drop technique also known as drop-spreading technique and air-drying technique ^1,2. This method ....

Protocol

1. Chromosome Preparation

1. Seed germination and fixation of root tips
   1. Germinate 10-20 barley seeds on two layers of moist filter paper in a Petri dish under dark conditions for 2 days at 22-24 °C. Cut off vigorous roots with the length of 1-2 cm from the seed by using a razor blade.
   2. Prepare ice-cold water by placing a 500 ml glass bottle containing cold tap water into crushed ice-water. Aerate the ice-cold water and immerse root tips for 20 hr to increase the frequency of metaphase cells.
   3. Transfer roots from water to 50 ml of ethanol: acetic acid (3:1) fixative to fix them at RT for 2 days. Store roots in a freshl....

Results

Microscopic slides with the mitotic metaphase spreads were prepared by the fast air-dry dropping chromosome preparation method described above (Suppl. Figure 1). FISH analysis was carried out using both, repetitive and single-copy sequences. Images were obtained by a epifluorescence microscope with a set of filters enabling excitation of corresponding fluorophores and captured by a high-sensitivity CCD monochrome camera. For the image acquisition we used a computer with an image acquisition software. Res.......

Discussion

The chromosome preparation experiment has been carried out using young roots of cereals belonging to the grass family (Poaceae). All analyzed species have 14 relatively long mitotic metaphase chromosomes (11-15 µm) in the diploid genome set and belong to large-genome species (5.1-7.9 Gbp).

Length of germinated roots was not more than 2 cm to obtain a maximum of meristematic tissue. Synchronization of dividing cells was achieved by a 20 hr long ice-water treatment that improved the quantit.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Hot Plate	MEDAX GmbH	12603
Cellulase R10	Duchefa	C8001
Cellulase	CalBioChem	219466
Pectolyase	Sigma	P3026
Cytohelicase	Sigma	C8274
Texas Red-12-dUTP	Invitrogen	C3176	direct fluorochrome
Fluor488-5-dUTP	Invitrogen	C11397	direct fluorochrome
Fluorecsence microscope	Olympus BX61	BX61
CCD camera	Orca ER, Hamamatsu	C10600
4’,6-diamidino-2-phenylindole (DAPI)	Vector Laboratories	H-1200	fluorecsent dye