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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    ABSTRACT: Fluorescence in situ hybridization (FISH) using T-DNA probes was applied to localize transgenes onto specific chromosomes and confirm the steady integration of transferred genes in three genetically modified (GM) rice lines, LS28 (event LS30-32-20-1), Cry1Ac1 (event C7-1-9-1) and LS28×Cry1Ac1 (event L/C1-1-3-1), which are a rice leaf blast-resistant single trait GM line, a leaf folder-resistant single trait GM line, and a rice leaf blast-resistant and leaf folder-resistant stacked GM hybrid line, respectively. The FISH signals were clearly detected on the arms of one homologous chromosome pair for LS28, and on the arms of another chromosome pair for Cry1Ac1 when using the transformation vector pSBM AtCK containing the rice leaf blast-resistant gene (LS28) and pMJ-RTB containing the leaf folder-resistant gene (mCry1Ac1) as a probe, respectively. As expected, we detected two pairs of FISH signals, each on the arms of different chromosome pairs in the stacked GM rice line LS28×Cry1Ac1 when using both pSBM AtCK and pMJ-RTB as probes. These results indicate that the transgenes are located at specific homologous loci and show position stability among generations in both single trait and stacked GM rice lines. The usefulness and the necessity of FISH to detect inserted genes in transformed plants will be discussed for the purpose of future studies to develop breeding programs and conduct risk assessment of GM plants.
    Genes & genomics 12/2010; 32:527-531. · 0.50 Impact Factor
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    ABSTRACT: Two barley transformation systems, Agrobacterium-mediated and particle bombardment, were compared in terms of transformation efficiency, transgene copy number, expression, inheritance and physical structure of the transgenic loci using fluorescence in situ hybridisation (FISH). The efficiency of Agrobacterium-mediated transformation was double that obtained with particle bombardment. While 100% of the Agrobacterium-derived lines integrated between one and three copies of the transgene, 60% of the transgenic lines derived by particle bombardment integrated more than eight copies of the transgene. In most of the Agrobacterium-derived lines, the integrated T-DNA was stable and inherited as a simple Mendelian trait. Transgene silencing was frequently observed in the T1 populations of the bombardment-derived lines. The FISH technique was able to reveal additional details of the transgene integration site. For the efficient production of transgenic barley plants, with stable transgene expression and reduced silencing, the Agrobacterium-mediated method appears to offer significant advantages over particle bombardment.
    Plant Cell Reports 04/2005; 23(12):780-9. · 2.94 Impact Factor
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    ABSTRACT: To estimate the possibility of plant genome mapping using human genome probes, the probes fluorescent in situ hybridization (FISH) of human 18S–28S rDNA (clon 22F9 from the LA-13NCO1 library) was carried out on chromosomes of the spring barleyHordeum vulgareL. As a control, wheat rDNA probe (clon pTa71) was taken. Hybridization of the wheat DNA probe revealed two major labelling sites on mitotic barley chromosomes 5I (7H) and 6I (6H), as well as several minor sites. With the human DNA probe, signals were detected in the major sites of the ribosomal genes on chromosomes 5I (7H) and 6I (6H) only when the chromosome preparations were obtained using an optimized technique with obligatory pepsin treatment followed by hybridization. Thus, this study demonstrates that physical mapping of plant chromosomes with human DNA probes that are 60 to 70% homologous to the plant genes is possible. It suggests principal opportunity for the FISH mapping of plant genomes using probes from human genome libraries, obtained in the course of the total sequencing of the human genomes and corresponding to the coding regions of genes with known functions.
    Russian Journal of Genetics 01/2001; 37(12):1452-1454. · 0.43 Impact Factor


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Oct 6, 2014