Preparing plant chromosomes for scanning electron microscopy

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Abstract
The scanning electron microscope may be useful in the analysis of plant chromosomes treated with in situ hybridization, especially when the probes and (or) chromosomes are near or beyond the resolution of the light microscope. Usual methods of plant chromosome preparation are unsuitable for scanning electron microscope observation as a result of cellular debris, which also interferes with probe hybridization. A method is described whereby protoplasts are obtained from fixed root tips by enzymatic digestion and applied to slides in a manner that produces little or no cellular debris overlying the chromosomes. The slides were examined by scanning electron microscopy and light microscopy after C-banding and in situ hybridization with a rye nucleolus organizer region spacer probe. This technique, which allows for scanning electron microscope visualization of bands and probes not easily identified with light microscopy, should prove useful in the physical mapping of low copy number or unique DNA sequences.Key words: protoplasts, rice, wheat, rye, physical maps, in situ hybridization.