Production of Transgenic Rice (Oryza sativa subspecies japonica cv. Taipei 309)

Buchholz, Wallace G.; Teng, Weimin; Wallace, Delphia; Ambler, J. R.; Hall, Timothy C.

doi:10.1385/0-89603-385-6:383

Cited by 6 publications

(9 citation statements)

References 14 publications

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“…The rice cultivar Taipei 309 ( Oryza sativa L., ssp. japonica , T309) was used for biolistics‐mediated transformation of rice as described (Buchholz et al . 1998b).…”

Section: Methodsmentioning

confidence: 99%

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Recurrent onset of epigenetic silencing in rice harboring a multi‐copy transgene

Kumpatla

Hall

1998

The Plant Journal

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SummaryExtensive methylation of the Ubi1 promoter has been shown to be associated with transcriptional silencing and bialaphos herbicide sensitivity in several R 1 progeny derived from a transgenic rice line, JKA 52, containing multiple copies of the introduced genes (Kumpatla et al., 1997; Plant Physiol. 115, 361-373). All of the progeny from silenced R 1 lines were silenced, and a proportion (24%) of the R 2 seedlings from JKA 52-6, a non-silenced homozygous R 1 line, were found to be silenced. Several nonsilenced (bialaphos-resistant) R 2 progeny derived from the selfed seed of JKA 52-6 were tested at various stages throughout the growth period and found to be resistant to bialaphos, indicating the absence of silencing. Five of these were investigated further to determine whether silencing could recur in subsequent generations. R 3 seedlings were raised from selfed R 2 seed and herbicide sensitivity was detected in 5-40% of the plants in three of the five lines. This silencing was accompanied by methylation of the Ubi1 promoter and the absence of steady-state bar mRNA. This recurrent onset of silencing and instability of expression associated with a transgene locus containing several repeated sequences is undesirable. The data strongly suggest that multi-generation molecular analysis should be undertaken for transgenic lines to be used for agricultural purposes and that lines containing methylated inserts should be carefully evaluated before being included in further breeding strategies.

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“…The rice cultivar Taipei 309 ( Oryza sativa L., ssp. japonica , T309) was used for biolistics‐mediated transformation of rice as described (Buchholz et al . 1998b).…”

Section: Methodsmentioning

confidence: 99%

“…particles (4 μg plasmid/2.4 mg particles). The day after bombardment, embryos were transferred to LS 2.5 medium (Buchholz et al . 1998b), supplemented with 4 mg l –1 bialaphos (Meiji Seika Kaisha, Japan).…”

Section: Methodsmentioning

confidence: 99%

Recurrent onset of epigenetic silencing in rice harboring a multi‐copy transgene

Kumpatla

Hall

1998

The Plant Journal

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“…The pir-Nt and pir-Ct constructs also include 35S-gfp-nos to express GFP as an easily detectable reporter of successful bombardment. Transient expression of RNAi corresponding to OsMET1-1 by particle bombardment (Buchholz et al 1998b) was predicted to inactivate maintenance methylation and thereby restore GUS activity.…”

Section: Genomic Organization Of Osmet1-1 and Osmet1-2mentioning

confidence: 99%

Characterization of two rice DNA methyltransferase genes and RNAi-mediated reactivation of a silenced transgene in rice callus

Teerawanichpan

Chandrasekharan

Jiang

et al. 2004

Planta

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Two genomic clones ( OsMET1-1, AF 462029 and OsMET1-2, TPA BK001405), each encoding a cytosine-5 DNA methyltransferase (MTase), were isolated from rice ( Oryza sativa L.) BAC libraries. OsMET1-1 has an open reading frame of 4,566 nucleotides with 12 exons and 11 introns while OsMET1-2 has an open reading frame of 4,491 nucleotides with 11 exons and 10 introns. Although OsMET1-1 and OsMET1-2 have high sequence similarity overall, they share only 24% identity in exon 1, and intron 3 of OsMET1-1 is absent from OsMET1-2. As for other eukaryotic DNA MTases of the Dnmt1/MET l class, the derived amino acid sequences of OsMET1-1 and OsMET1-2 suggest that they are comprised of two-thirds regulatory domain and one-third catalytic domain. Most functional domains identified for other MTases were present in the rice MET1 sequences. Amino acid sequence comparison indicated high similarity (56-75% identity) of rice MET1 proteins to other plant MET1 sequences but limited similarity (approx. 24% identity) to animal Dnmt1 proteins. Genomic blot and database analysis indicated the presence of a single copy of OsMET1-1 (on chromosome 3) and single copy of OsMET1-2 (on chromosome 7). Ribonuclease protection assays revealed expression of both OsMET1-1 and OsMET1-2 in highly dividing cells, but the steady-state level of OsMET1-2 was 7- to 12-fold higher than that for OsMET1-1 in callus, root and inflorescence. The functional involvement of the rice DNA MTases in gene silencing was investigated using an RNAi strategy. Inverted repeat constructs of either the N- or C-terminal regions of OsMET1-1 were supertransformed into calli derived from a rice line bearing a silenced 35S-uidA-nos transgene. Restoration of uidA expression in the bombarded calli was consistent with the inactivation of maintenance methylation and with previous evidence for the involvement of methylation in silencing of this line.

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“…japonica, cv. Taipei 309) (T309) was as described (20). Embryos were isolated from seeds close to the end of the milky stage (« 2 wk after anthesis) and subjected to bombardment (Bio-Rad PDS 1000/He biolistics system) using pJKA and pUbi1-bar plasmids at a 5:1 ratio on 1 l M (o.d.)…”

Section: Biolistics-mediated Plant Transformationmentioning

confidence: 99%

“…particles (4 l g plasmid/2.4 mg particles). The day after bombardment, embryos were transferred to LS (Linsmaier and Skoog) 2.5 medium (20) supplemented with 4 mg/L bialaphos (Meiji Seika Kaisha, Tokyo, Japan). Selection of resistant calli, regeneration, polymerase chain reaction (PCR) screening of plantlets for the Btt cryIIIA gene, and culture of fertile plants were as detailed in (20,21).…”

Section: Biolistics-mediated Plant Transformationmentioning

confidence: 99%

Organizational Complexity of a Rice Transgene Locus Susceptible to Methylation-Based Silencing

Kumpatla¹,

Hall²

1999

International Union of Biochemistry and Molecular Biology: Life

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Molecular analyses of a rice (Oryza sativa L.) transgene locus introduced using biolistic techniques revealed the presence of multiple copies of rearranged fragments, as well as an intact copy of the supplied constructs. Both the gene of interest (35S-Btt cryIIIA) and the selectable marker used (Ubi1-bar) were methylated and silenced. Additionally, vector sequences were present in great abundance and were also highly methylated, indicating that the entire transgene insert was marked for methylation. The rearrangement of input DNA resulted in interspersion of plasmid backbone regions with the gene of interest. Permutation of segments encoding the gene of interest and the selectable marker was also detected, perhaps explaining why sequences introduced on separate plasmids are frequently found to be inserted at the same locus. The 35S promoter contained several hotspots for fragmentation. These observations strongly support the concept that intrusive DNA is recognized by host surveillance systems and that transgene loci with anomalous structural organization are subjected to inactivation by processes such as methylation.

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Production of Transgenic Rice (Oryza sativa subspecies japonica cv. Taipei 309)

Cited by 6 publications

References 14 publications

Recurrent onset of epigenetic silencing in rice harboring a multi‐copy transgene

Recurrent onset of epigenetic silencing in rice harboring a multi‐copy transgene

Characterization of two rice DNA methyltransferase genes and RNAi-mediated reactivation of a silenced transgene in rice callus

Organizational Complexity of a Rice Transgene Locus Susceptible to Methylation-Based Silencing

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