An improved method for inducing prometaphase chromosomes in plants

Area of Science:

• Plant molecular cytogenetics
• Genomics and chromosome analysis

Background:

• Melon karyotyping is challenging due to small, poorly stainable metaphase chromosomes.
• Prometaphase chromosomes offer higher resolution for fluorescence in situ hybridization (FISH) but methods for obtaining them in melon are underdeveloped.

Purpose of the Study:

• To develop and validate a method for acquiring high-quality prometaphase and pachytene chromosomes in plants.
• To improve fluorescence in situ hybridization (FISH) analysis resolution in melon (Cucumis melo L.) and Abelia × grandiflora.

Main Methods:

• Utilized a modified Carnoy's solution II (MC II) for chromosome pretreatment.
• Combined MC II with a modified air-dry (ADI) method for enhanced chromosome spreads.
• Applied the developed methods to melon and Abelia × grandiflora for cytogenetic analysis.

Main Results:

• The MC II method successfully produced prometaphase chromosomes suitable for karyotyping and FISH.
• The combination of MC II and ADI yielded satisfactory meiotic pachytene chromosome preparations with reduced cytoplasmic background.
• Improved FISH images were obtained for both melon and Abelia × grandiflora using the new preparation techniques.
• Identified strong and weak 45S ribosomal DNA signals on Abelia × grandiflora metaphase chromosomes.

Conclusions:

• The MC II and ADI methods are simple and effective for preparing plant prometaphase and pachytene chromosomes.
• These methods reduce cytoplasmic background, leading to clearer chromatin spreads.
• The improved FISH imaging capabilities can accelerate molecular cytogenetic research in plants.