Preparation of chromosomes from plant leaf meristems for karyotype analysis and in situ hybridization

Kesara Anamthawat-Jónsson*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

31 Citations (Scopus)

Abstract

A reliable method for preparing metaphase chromosomes from plant leaf tissues is described. The chromosomes are suitable for karyotype analysis and gene mapping by fluorescence in situ hybridisation (FISH). The method is based on enzymatic digestion of young leaf tissues (shoot-tips) after which the resulting protoplasts are treated hypotonically before being dropped onto microscopic slides. Compared to root-tip chromosomes, leaf chromosomes tend to be longer, or less condensed, and hence more karyotypically differentiated. Metaphase index in young leaf tissues is also very high. Metaphase spread consists of evenly and well-distributed chromosomes and this allows accurate counting. The plant used to demonstrate this method is birch (Betula L.), a group of tree species that has extremely small chromosomes. Root-tip chromosomes of these plants are difficult to obtain, as cutting does not produce roots readily. Seedling chromosomes do not represent the same genomic constitution as their mother trees due to introgressive hybridisation. Furthermore, sample collection in the field is convenient and actively growing leaf buds are available throughout the growing season. FISH experiments with these leaf chromosomes also give good results comparable to those obtained with root-tip chromosomes or even better as mapping on long or extended chromosomes has high resolution in general. Mapping of the 16S-28S ribosomal genes on birch leaf chromosomes has been shown to differentiate between birch species and therefore can accurately confirm their interspecific hybrids.

Original languageEnglish
Pages (from-to)91-95
Number of pages5
JournalMethods in Cell Science
Volume25
Issue number3-4
DOIs
Publication statusPublished - Jan 2004

Bibliographical note

Funding Information:
I thank Ægir Thór Thórsson and Elina Salmela for their considerable help in testing this protocol. Funding from the Icelandic Research Council and the University of Iceland is appreciated.

Other keywords

  • Birch (Betula)
  • Chromosomes
  • Fluorescence in situ hybridization (FISH)
  • Karyotyping
  • Metaphases
  • Protoplasts

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