Structure and function of selectable and non-selectable transgenes in maize after introduction by particle bombardment

Register, James C.; Peterson, David J.; Bell, Philip; Bullock, W. Paul; Evans, Ian J.; Frame, Bronwyn; Greenland, Andrew J.; Higgs, Nicole S.; Jepson, Ian; Jiao, Shunshan; Lewnau, Carol J.; Sillick, Jeff M.; Wilson, Henrietta

doi:10.1007/bf00014669

Cited by 176 publications

(93 citation statements)

References 36 publications

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“…FISH screening of root tips of regenerated seedlings by using pWY96 (7) and pAHC25 probes separately revealed that both transgenes were present at the same locations in 84% of the transformants, as is often the case with cobombardment (14). There were 53 targeted B or miniB chromosomes that had both the pWY96 and pAHC25 transgenes (SI Table 2).…”

Section: Resultsmentioning

confidence: 96%

“…In the progeny of 76-10, only 2 of 12 individuals with the transgene showed GUS expression. This transgene might have undergone rearrangement or gene silencing, which occurs frequently in transformants recovered from biolistic-mediated gene transformation (14). In contrast, all individuals without a detectable transgene were negative for the GUS assay.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Construction and behavior of engineered minichromosomes in maize

Han

Gao

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

142

138

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Engineered minichromosomes were constructed in maize by modifying natural A and supernumerary B chromosomes. By using telomere-mediated chromosomal truncation, it was demonstrated that such an approach is feasible for the generation of minichromosomes of normal A chromosomes by selection of spontaneous polyploid events that compensate for the deficiencies produced. B chromosomes are readily fractionated by biolistic transformation of truncating plasmids. Foreign genes were faithfully expressed from integrations into normal B chromosomes and from truncated miniB chromosomes. Site-specific recombination between the terminal transgene on a miniA chromosome and a terminal site on a normal chromosome was demonstrated. It was also found that the miniA chromosome did not pair with its progenitor chromosomes during meiosis, indicating a useful property for such constructs. The miniB chromosomes are faithfully transmitted from one generation to the next but can be changed in dosage in the presence of normal B chromosomes. This approach for construction of engineered chromosomes can be easily extended to other plant species because it does not rely on cloned centromere sequences, which are species-specific. These platforms will provide avenues for studies on plant chromosome structure and function and for future developments in biotechnology and agriculture.artificial chromosomes ͉ FISH ͉ genetic engineering ͉ telomere truncation A rtificial chromosomes involving de novo centromere formation on an independently assembled unit and engineered minichromosomes produced by telomere truncation provide striking advantages over traditional methods of gene transformation in yeast and mammalian cells (1-5). The development of such chromosomes in plants would provide these advantages for many applications in basic studies, biotechnology, and agriculture. These chromosomes could be used as independent platforms for foreign gene expression without random integration into the normal chromosomes. Further additions of unlimited amounts of DNA could be added to these platforms in a sequential manner via different site-specific recombination cassettes. Genes introduced in this way would be present in a defined context and thus could be expressed at a more predictable level than through random integration (6). Hence, additional genes, multigene complexes, or even whole metabolic pathways could potentially be added to a genotype. Moreover, engineered or artificial chromosomes could be easily introduced or removed from a genotype by genetic crosses and would facilitate introgression of transgenes to different genetic backgrounds.To extend engineered chromosome technology to plants, we developed a method of telomere-mediated chromosomal truncation in maize by Agrobacterium-mediated transformation of constructs with multiple copies of the telomere sequence (7). Here, we report the use of this technology to produce minichromosome platforms by truncating both normal A and supernumerary B maize chromosomes and at the same time introducing site-spe...

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Section: Resultsmentioning

confidence: 96%

Section: Resultsmentioning

confidence: 99%

Construction and behavior of engineered minichromosomes in maize

Han

Gao

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

142

138

View full text Add to dashboard Cite

show abstract

“…Although instability of transgene expression has been observed in maize (Klein et al, 1990) and rice (Register et al, 1994;Cooley et al, 1995), few published reports of gene silencing in transgenic monocots exist. Although an association between the presence of multiple copies of introduced gene sequences and lack of expression has been reported for maize (Register et al, 1994) and rice (Rathore et al, 1993;Cooley et al, 1995), no insight into the mechanism of silencing was obtained, other than the observation that low levels of GUS expression in stably transformed maize calli were correlated with methylation of the coding region (Klein et al, 1990).…”

Section: Transgene Silencing and Methylationmentioning

confidence: 99%

“…Although an association between the presence of multiple copies of introduced gene sequences and lack of expression has been reported for maize (Register et al, 1994) and rice (Rathore et al, 1993;Cooley et al, 1995), no insight into the mechanism of silencing was obtained, other than the observation that low levels of GUS expression in stably transformed maize calli were correlated with methylation of the coding region (Klein et al, 1990). Similarly, in a recent study a large number of transgenic rice plants produced by biolistics were analyzed and revealed both non-Mendelian inheritance of hygromycin resistance and suppression of GUS expression (Qu et al, 1996), although the molecular basis was not investigated.…”

Section: Transgene Silencing and Methylationmentioning

confidence: 99%

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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“…A number of plant transformation experiments have reported plants with a mixture of non-Mendelian and Mendelian inheritance of the transgene (Christou et al 1989;Tomes et al 1990;Spencer et al 1992;Walters et al 1992;Hiei et al 1994;Register et al 1994;Peng et al 1995). In our experiments, none of the four events we studied exhibited efficient pollen transmission.…”

Section: Discussionmentioning

confidence: 67%

Expression and inheritance of the wheat Glu-1DX5 gene in transgenic maize

et al. 2002

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We have produced transgenic maize plants containing a wheat Glu-1Dx5 gene encoding the high-molecularweight glutenin subunit 1Dx5. Analysis by SDS-PAGE showed that a protein similar in size to the wheat 1Dx5 subunit accumulates in the endosperm of transgenic maize from four independent transformation events. This protein reacts with a monoclonal antibody specific to the wheat 1Dx5 subunit and was not detected in nontransgenic controls or in pollen, anthers, leaves or embryos of plants grown from seeds expressing this protein in endosperm. Genomic Southern-blot analysis is consistent with results from SDS-PAGE and indicates that the transgene integration sites are complex and are different in the four events studied. Using the presence of this protein as a phenotypic marker, we studied the inheritance of this gene through three sexual generations. Reciprocal crosses with nontransgenic plants and self-pollinations were performed, and the resulting kernels were analyzed for the presence of the 1Dx5 subunit. These data, together with PCR analysis for the transgene, suggest that the transgene is inefficiently transmitted through pollen in all four events. Abstract We have produced transgenic maize plants containing a wheat Glu-1Dx5 gene encoding the highmolecular-weight glutenin subunit 1Dx5. Analysis by SDS-PAGE showed that a protein similar in size to the wheat 1Dx5 subunit accumulates in the endosperm of transgenic maize from four independent transformation events. This protein reacts with a monoclonal antibody specific to the wheat 1Dx5 subunit and was not detected in nontransgenic controls or in pollen, anthers, leaves or embryos of plants grown from seeds expressing this protein in endosperm. Genomic Southern-blot analysis is consistent with results from SDS-PAGE and indicates that the transgene integration sites are complex and are different in the four events studied. Using the presence of this protein as a phenotypic marker, we studied the inheritance of this gene through three sexual generations. Reciprocal crosses with nontransgenic plants and selfpollinations were performed, and the resulting kernels were analyzed for the presence of the 1Dx5 subunit. These data, together with PCR analysis for the transgene, suggest that the transgene is inefficiently transmitted through pollen in all four events.

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Structure and function of selectable and non-selectable transgenes in maize after introduction by particle bombardment

Cited by 176 publications

References 36 publications

Construction and behavior of engineered minichromosomes in maize

Construction and behavior of engineered minichromosomes in maize

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

Expression and inheritance of the wheat Glu-1DX5 gene in transgenic maize

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