# Preparing high-quality chromosome spreads from Crocus species for karyotyping and FISH

## TL;DR

This paper presents an effective method for preparing high-quality chromosomes in saffron and related crocus species, improving FISH analysis for genetic studies.

## Contribution

The study introduces ice water pretreatment as a novel and effective protocol for chromosome preparation in Crocus species.

## Key findings

- Ice water pretreatment yielded the highest metaphase index (2.05%) compared to other methods.
- Chromosomes prepared with ice water showed better morphology and suitability for FISH analysis.
- DAPI staining and FISH confirmed the effectiveness of ice water pretreatment in both C. sativus and C. cartwrightianus.

## Abstract

The saffron-producing Crocus sativus (L.) and its wild relative C. cartwrightianus (Herb.) are key species for understanding genetic evolution in this genus. Molecular-cytogenetic methods, especially fluorescent in situ hybridization (FISH), are essential for exploring the genetic relationships in this genus. Yet, preparing high-quality chromosomes for FISH analysis across Crocus species remains difficult. A standardized protocol for achieving clear and well-separated mitotic chromosomes is still lacking. This study aimed to assess the effectiveness of pretreatments with four chromosome synchronization methods for optimal chromosome spread preparation in Crocus. Root tips of different Crocus species were treated with four chromosome preparation methods namely hydroxyurea-colchicine (HC), nitrous oxide (NO), hydroxyquinoline (HQ), and ice water (IW) pretreatments to investigate their effectiveness in producing high-quality mitotic chromosome spreads. Metaphases obtained by the four methods were analyzed to assess their quality and metaphase index.

Evaluation of 22,507 cells allowed us to confidently recommend a protocol for Crocus chromosome preparation. Among the methods, ice water pretreatment yielded the highest metaphase index (2.05%), more than doubling the results of HC (1.08%), NO (1.15%), and HQ (1.16%). Ice water-treated chromosomes exhibited better chromosome morphology, with relatively proper size, and non-overlapping chromosomes that were optimal for FISH analysis. Ice water pretreatment was also applied to C. cartwrightianus, the diploid progenitor of C. sativus, where it demonstrated similar efficacy. DAPI staining of chromosomes in both species allowed for clear visualization of intercalary and terminal heterochromatin. FISH analysis using 18S-5.8S-25S and 5S rDNA probes confirmed the utility of IW-prepared chromosome spreads for cytogenetic studies.

We strongly recommend ice water pretreatment as a suitable and effective method for obtaining many metaphase spreads of high-quality in C. sativus and related species, particularly for applications involving a detailed cytogenetic analysis.