Fluorescence in situ hybridization of single copy transgenes in rice chromosomes

In Vitro Cellular & Developmental Biology - Plant (Impact Factor: 0.98). 01/2001; 37(1):1-5. DOI: 10.1007/s11627-001-0001-6


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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Available from: Timothy C Hall, Oct 06, 2014
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    • "Kato et al. 2004, Lim et al. 2005), and single-copy genes (e.g. Fransz et al. 1996, Kharb et al. 2001). Owing to their sequence conservation among eukaryotic genomes despite the repeating unit copy number, loci number, and distribution pattern variations, the multiple tandem repeats of the 5S and 45S rDNA are the most widely used probes in molecular cytogenetic analyses (e.g. "
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    ABSTRACT: While useful advances have been made using biolistic and other naked DNA uptake methods for rice transformation, high copy num- bers of rearranged sequences are common and lack of stable ex- pression due to silencing or other causes is proving to be a major problem. Attempts to transform rice and other monocots using Agrobacterium tumefaciens-based vectors have typically yielded re- sults that were promising, but inconclusive. Recently, an Agrobacterium-based system starting from scutellar tissue has been described in the literature. Using only minor modifications of this system, we were able to repeat their transformation of callus de- rived from the cultivar Koshihikari. We have also extended the work to include Gulfmont, a javonica variety widely used for commercial cultivation in Texas, and Taipei 309, a japonica line that has been widely used for transformation by direct DNA transfer. Transforma- tion using the Agrobacterium approach occurred at a reasonably high frequency and vigorous, phenotypically normal plants were recov- ered. Only one or two unrearranged copies of the T-DNA were present at a single locus; no silencing of the selectable marker or the gene of interest (gus) was observed and the introduced genes were trans- mitted in a normal Mendelian fashion.
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