Expression of a chimeric neomycin phosphotransferase II gene in first and second generation transgenic rice plants

Battraw, Michael J.; Hall, Timothy C.

doi:10.1016/0168-9452(92)90165-i

Cited by 39 publications

(12 citation statements)

References 25 publications

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“…1:l for RI progeny of JKA 52) imply that the mechanism by which gene inactivation is fully triggered may be complex and possibly affected by externa1 factors such as environmental conditions. In agreement with the concept that the presence of multiple homologous sequences targets the transgenic locus for silencing, progeny of rice lines containing a single copy (Battraw and Hall, 1992) or relatively few copies (JKA 58, JKA 60; Fig. 4) of the inserted gene have shown no evidence for silencing.…”

Section: Biotechnological Lmplications Of Homology-mediated Gene Silementioning

confidence: 61%

Epigenetic Transcriptional Silencing and 5-Azacytidine-Mediated Reactivation of a Complex Transgene in Rice

et al. 1997

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Despite a growing number of reports indicating non-Mendelian inheritance of transgene expression in monocots, no detailed description of the structure and stability of the transgene exists for transformants generated by direct DNA-transfer techniques, making the cause for these observations difficult to determine. In this paper we describe the complex organization of Btf crylllA and bar transgenes in rice (Oryza safiva 1.) that displayed aberrant segregation in R, progeny. Silencing rather than rearrangement of the bar gene was implicated because the herbicide-sensitive R, plants had a DNA hybridization profile identical to that of the resistant R, parent and R, siblings. Cenomic DNA analysis revealed substantial methylation of the Ubil/bar sequences in silenced plants and, to a lesser degree, in herbicide-resistant plants, suggesting that the transgene locus was potentiated for silencing. Nuclease protection and nuclear run-on assays confirmed that silencing was due to transcriptional inactivation. Treatment of R, progeny of silenced plants with 5-azacytidine resulted in demethylation of the Ubil promoter and reactivation of bar gene expression, demonstrating a functional relationship for methylation in gene silencing. These findings indicate that methylation-based silencing may be frequent in cereals transformed by direct DNA protocols that insert multiple, often rearranged sequences.

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Section: Biotechnological Lmplications Of Homology-mediated Gene Silementioning

confidence: 61%

Epigenetic Transcriptional Silencing and 5-Azacytidine-Mediated Reactivation of a Complex Transgene in Rice

et al. 1997

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“…This indicated that genomic co-integration of genes linked on a single construct occurs at very high eciency. A 100% co-integration frequency of two genes carried on one plasmid has been reported previously in transgenic rice plants (Battraw and Hall 1992;Cooley et al 1995). A 75% co-integration frequency has also been reported in transgenic soybean callus (Christou and Swain 1990) and Phaseolus vulgaris (Russell et al 1993).…”

Section: Discussionmentioning

confidence: 73%

Particle-bombardment-mediated co-transformation of elite Chinese rice cultivars with genes conferring resistance to bacterial blight and sap-sucking insect pests

Tang

Tinjuangjun

et al. 1999

Planta

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Transgenic rice plants were generated using particle bombardment to simultaneously introduce the rice Xa21 gene eective against bacterial blight and the Galanthus nivalis agglutinin (snowdrop lectin; gna) gene eective against sap-sucking insect pests, speci®cally the brown plant hopper. Using three plasmids, we cotransformed 5-to 10-d-old, mature seed-derived rice (Oryza sativa L.) callus of two elite Chinese rice cultivars, Eyi 105 and Ewan 5. The plasmids carried a total of four genes. The gna and Xa21 genes were carried on separate plasmids. The selectable marker hygromycin phosphotransferase (hpt) and the reporter gene b-glucuronidase (gusA) were linked on the same, co-integrate vector. We recovered over 160 independently derived transgenic rice plants. Over 70% of the transgenic plants carried all four genes, as con®rmed by polymerase chain reaction and/or Southern blot analysis. Furthermore, 70% of transgenic plants carrying all four genes also coexpressed all four genes, as con®rmed by growth on selection media (hpt), GUS histochemical assays (gusA), western blotting (gna) and reverse transcriptase-polymerase chain reaction (Xa21) analysis. The co-expression eciency reported for the four transgenes in our study is the highest ever found in any transgenic plant population generated through co-transformation. The linked genes (hpt and gusA) co-integrated with a frequency of near 100%, and we observed a co-integration frequency greater than 70% for the genes carried on separate plasmids. We observed no preferential integration of any particular gene(s). Genetic analysis con®rmed Mendelian segregation of the transgenes in subsequent generations. We report, for the ®rst time, generation and analysis of transgenic rice lines carrying genes eective against more than one taxa of pathogen or pest, substantiating that particle bombardment represents an eective way to introduce unlinked complex multiple traits into plants.Abbreviations: gna (GNA) = gene (protein) for Galanthus nivalis agglutinin; gusA (GUS) = gene (protein) for b-glucuronidase; hpt (HPT) = gene (protein) for hygromycin phosphotransferase; Xa21 (XA21) = gene (protein) for resistance to bacterial blight, RT-PCR = reverse transcriptase-polymerase chain reaction

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“…Rice has a well established classical genetic map [ 241, two high-density molecular linkage maps [ 12,261, a yeast artificial chromosome library [41], and a well developed genetic transformation system [ 6,11,21,221. The rice map of Causse etal.…”

Section: Introductionmentioning

confidence: 99%

Construction and characterization of two rice bacterial artificial chromosome libraries from the parents of a permanent recombinant inbred mapping population

et al. 1996

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Rice is a leading grain crop and the staple food for over half of the "world" population. Rice is also an ideal species for genetic and biological studies of cereal crops and other monocotyledonous plants because of its small genome and well developed genetic system. To facilitate rice genome analysis leading to physical mapping, the identification of molecular markers closely linked to economic traits, and map-based cloning, we have constructed two rice bacterial artificial chromosome (BAC) libraries from the parents of a permanent mapping population (Lemont and Teqing) consisting of 400 F9 recombinant inbred lines (RILs). Lemont Cjaponica) and Teqing (indica) represent the two major genomes of cultivated rice, both are leading commercial varieties and widely used germplasm in rice breeding programs. The Lemont library contains 7296 clones with an average insert size of 150 kb, which represents 2.6 rice haploid genome equivalents. The Teqing library contains 14208 clones with an average insert size of 130 kb, which represents 4.4 rice haploid genome equivalents. Three single-copy DNA probes were used to screen the libraries and at least two overlapping BAC clones were isolated with each probe from each library, ranging from 45 to 260 kb in insert size. Hybridization of BAC clones with chloroplast DNA probes and fluorescent in situ hybridization using BAC DNA as probes demonstrated that both libraries contain very few clones of chloroplast DNA origin and are likely free of chimeric clones. These data indicate that both BAC libraries should be suitable for map-based cloning of rice genes and physical mapping of the rice genome.

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Expression of a chimeric neomycin phosphotransferase II gene in first and second generation transgenic rice plants

Cited by 39 publications

References 25 publications

Epigenetic Transcriptional Silencing and 5-Azacytidine-Mediated Reactivation of a Complex Transgene in Rice

Epigenetic Transcriptional Silencing and 5-Azacytidine-Mediated Reactivation of a Complex Transgene in Rice

Particle-bombardment-mediated co-transformation of elite Chinese rice cultivars with genes conferring resistance to bacterial blight and sap-sucking insect pests

Construction and characterization of two rice bacterial artificial chromosome libraries from the parents of a permanent recombinant inbred mapping population

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