Transgenic tobacco plants and their progeny derived by microprojectile bombardment of tobacco leaves

Tomes, D. T.; Weissinger, Arthur K.; Ross, Margit; Higgins, Regina; Drummond, Bruce J.; Schaaf, Steve; Malone-Schoneberg, JoBeth; Staebell, Mark; Flynn, Pam; Anderson, John W.; Howard, John A.

doi:10.1007/bf00018566

Cited by 86 publications

(34 citation statements)

References 22 publications

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“…Such assays are used widely in studies of transcriptional enhancers. However, transiently transfected DNA is poorly organized into nucleosomes (Weintraub, 1985;Archer et al, 1992), and the fact that only a small minority of expressing cells go on to become stably transformed suggests that most transient expression occurs without chromosomal Direct gene transfer procedures can result in complex integration patterns (e.g., Paszkowski et al, 1984;Tomes et al, 1990;Mittelsten Scheid et al, 1991;Christou, 1992;Koziel et al, 1993;Wan and Lemaux, 1994). Therefore, each cell line was compared by using DNA gel blot analysis after digestion of isolated genomic DNA with EcoRl and Hindlll, which cut on either side of the 35S::GUS::NOScassette ( Figure 3).…”

Section: Transient Expressionmentioning

confidence: 99%

High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco.

et al. 1996

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We have previously shown that yeast scaffold attachment regions (SARs) flanking a chimeric 0-glucuronidase (GUS) reporter gene increased per-copy expression levels by 24-fold in tobacco suspension cell lines stably transformed by microprojectile bombardment. In this study, we examined the effect of a DNA fragment originally identified in a tobacco genomic clone by its activity in an in vitro binding assay. The tobacco SAR has much greater scaffold binding affinity than does the yeast SAR, and tobacco cell lines stably transformed with constructs containing the tobacco SAR accumulated greater than fivefold more GUS enzyme activity than did lines transformed with the yeast SAR construct. Relative to the control construct, flanking the GUS gene with plant SARs increased overall expression per transgene copy by almost 140-fold.In transient expression assays, the same construct increased expression only approximately threefold relative to a control without SARs, indicating that the full SAR effect requires integration into chromosomal DNA. GUS activity in individual stable transformants was not simply proportional to transgene copy number, and the SAR effect was maximal in cell lines with fewer than 4 0 transgene copies per tobacco genome. Lines with significantly higher copy numbers showed greatly reduced expression relative to the low-copy-number lines. Our results indicate that strong SARs flanking a transgene greatly increase expression without eliminating variation between transformants. We propose that SARs dramatically reduce the severity or likelihood of homology-dependent gene silencing in cells with small numbers of transgenes but do not prevent silencing of transgenes present in many copies.

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Section: Transient Expressionmentioning

confidence: 99%

High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco.

et al. 1996

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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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“…This explanation has been widely used to interpret the common observation of similar integration patterns between individually transformed plant cell lines or plants, including those from the protoplast approach (Czernilofsky eta/., 1986;Fromm et a/., 1986;Hain et a/., 1985;Hayashimoto et a/., 1990;Kohler eta/., 1987;Meijer eta/., 1991;Paszkowski etal., 1984;Rhodes et a/., 1988;Tada eta/., 1991 ;Toriyama et a/., 1988;Z.Y. Wang et a/., 1992), from the biolistic methods (Christou etal., 1988;Finer and McMullen, 1990;Gordon-Kamm etal., 1990;Hagio etal., 1991 ;Klein eta/., 1988;Spencer eta/,, 1990;Tomes eta/., 1990), and other DNA delivery methods (Hess eta/., 1990;Kaeppler eta/., 1992;Luo and Wu, 1988;Schnorf eta/., 1991).…”

Section: Discussionmentioning

confidence: 99%

Stability of transgenes and presence of N⁶ methyladenine DNA in transformed wheat cells

et al. 1994

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Summary A number of stably transformed wheat cell lines were obtained using two different direct gene transfer techniques. When integration of the foreign genes was investigated in DNA samples taken at 10 months post‐selection, complicated profiles of transgene bands were observed in Southern blot analysis. Among these, a number of common bands were identified showing similar hybridization patterns between independently transformed cell lines. This type of hybridization pattern has been a common observation and is usually interpreted as concatameric rearrangement of integrated DNA. However, when integration was reinvestigated with DNA samples taken at 30 months postselection, the hybridization pattern changed and most of the common bands had disappeared. Further analysis using a set of methylation‐sensitive enzymes revealed that the DNA represented by the common bands was N6‐adenine methylated (m6A DNA), and there was even m6A DNA in some 30 month samples. Although the source of m6A DNA in the wheat cultures was not clearly established, the data indicate that transformation of an endophyte (e.g. a mycoplasma‐like organism) may have occurred at the same time as the transformation of wheat cells. The integration pattern of undermethylated transgenes in the transformed cells became simpler and clearer after treating the DNA preparations with Dpnl, which only cuts m6A DNA. The implications of these data for other methods of inoculating cereals with DNA are discussed.

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Transgenic tobacco plants and their progeny derived by microprojectile bombardment of tobacco leaves

Cited by 86 publications

References 22 publications

High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco.

High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco.

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

Stability of transgenes and presence of N⁶ methyladenine DNA in transformed wheat cells

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Transgenic tobacco plants and their progeny derived by microprojectile bombardment of tobacco leaves

Cited by 86 publications

References 22 publications

High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco.

High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco.

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

Stability of transgenes and presence of N6 methyladenine DNA in transformed wheat cells

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Stability of transgenes and presence of N⁶ methyladenine DNA in transformed wheat cells