Article

Fluorescence in situ hybridization of single copy transgenes in rice chromosomes

In Vitro Cellular & Developmental Biology - Plant (Impact Factor: 1.14). 01/2001; 37(1):1-5. DOI: 10.1007/s11627-001-0001-6

ABSTRACT Fluorescence in situ hybridization (FISH) is a powerful tool for visualizing the chromosomal location of targeted sequences and has been applied
in many areas, including karyotyping, breeding and characterization of genes introduced into the plant genome. A simple, routine
and sensitive FISH procedure was developed for localizing single copy genes in rice (Oryza sativa L.) metaphase chromosomes. We used digoxygenin-labeled endogenous or T-DNA sequences as small as 5.6 kb to probe corresponding
endogenous sequences or the T-DNA insert in denatured rice metaphase chromosomes prepared from root meristem tissue. The hybridized
probe sequence was labeled with cy3-conjugated anti-mouse IgG and visualized using fluorescence microscopy. Single copy and
multiple copy introduced T-DNA sequences, as well as endogenous sequences, were localized on the chromosomes. The FISH protocol
was effectively used to sereen the chromosomal location of introduced T-DNA and number of integration loci in rice.

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