Accurate Determination of Zygosity in Transgenic Rice by Real-time PCR Does Not Require Standard Curves or Efficiency Correction

Prior, F; Tackaberry, Eilleen S.; Aubin, Rémy A.; Casley, William L.

doi:10.1007/s11248-005-4024-3

Cited by 32 publications

(17 citation statements)

References 10 publications

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“…Traditionally, T 1 plants are screened for zygosity by a time-consuming segregation analysis of their T 2 progeny, which necessitates growing the T 1 plant lines to maturity for the collection of their seeds to be germinated on selection media for screening. Using quantitative real-time PCR allows relative quantification of gene copy number, and hence can be used to determine zygosity (Bubner and Baldwin 2004;Ji et al 2005;Prior et al 2006;Shitara et al 2004;Tesson et al 2002). In this investigation, the homozygous and hemizygous transgenic papaya lines were distinguishable by real-time PCR method for determining zygosity.…”

Section: Discussionmentioning

confidence: 89%

Transgene-specific and event-specific molecular markers for characterization of transgenic papaya lines resistant to Papaya ringspot virus

et al. 2009

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The commercially valuable transgenic papaya lines carrying the coat protein (CP) gene of Papaya ringspot virus (PRSV) and conferring virus resistance have been developed in Hawaii and Taiwan in the past decade. Prompt and sensitive protocols for transgene-specific and event-specific detections are essential for traceability of these lines to fulfill regulatory requirement in EU and some Asian countries. Here, based on polymerase chain reaction (PCR) approaches, we demonstrated different detection protocols for characterization of PRSV CP-transgenic papaya lines. Transgene-specific products were amplified using different specific primer pairs targeting the sequences of the promoter, the terminator, the selection marker, and the transgene, and the region across the promoter and transgene. Moreover, after cloning and sequencing the DNA fragments amplified by adaptor ligation-PCR, the junctions between plant genomic DNA and the T-DNA insert were elucidated. The event-specific method targeting the flanking sequences and the transgene was developed for identification of a specific transgenic line. The PCR patterns using primers designed from the left or the right flanking DNA sequence of the transgene insert in three selected transgenic papaya lines were specific and reproducible. Our results also verified that PRSV CP transgene is integrated into transgenic papaya genome in different loci. The copy number of inserted T-DNA was further confirmed by real-time PCR. The event-specific molecular markers developed in this investigation are crucial for regulatory requirement in some countries and intellectual protection. Also, these markers are helpful for prompt screening of a homozygote-transgenic progeny in the breeding program.

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Section: Discussionmentioning

confidence: 89%

Transgene-specific and event-specific molecular markers for characterization of transgenic papaya lines resistant to Papaya ringspot virus

et al. 2009

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“…The q-RT-PCR strategy has already proved useful for quantifying the transgene copy number (Ingham et al 2001;Batista et al 2008). However, in previous works a single copy gene was selected as reference gene in a relative q-RT-PCR (German et al 2003;Prior et al 2006). Bradeen et al (2009) showed a high correlation between the transgene quantification obtained using both a single copy gene (Urease) and a double copy gene (EF-1).…”

Section: Discussionmentioning

confidence: 97%

Potato R1 resistance gene confers resistance against Phytophthora infestans in transgenic tomato plants

Faino

Carli

Testa³

et al. 2010

Eur J Plant Pathol

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Tomato is challenged by several pathogens which cause loss of production. One such pathogen is the oomycete Phytophthora infestans which is able to attack all the aerial parts of the plant. Although a wide range of resistance sources are available, genetic control of this disease is not yet successful. Pyramiding R-genes through genetic transformation could be a straightforward way to produce tomato and potato lines carrying durable resistance to P. infestans. In this work the R1 potato gene was transferred into tomato lines. The tomato transgenic lines were analyzed by using q-RT-PCR and progeny segregation to determine the gene copy number. To test the hypothesis that R1 represents a specifically regulated R-gene, transgenic tomato plants were inoculated with P. infestans isolate 88133 and IPO. All the plants containing the R1 gene were resistant to the late blight isolate IPO-0 and susceptible to isolate 88133. These results provide evidence for specific activation of the R1 gene during pathogen challenge. Furthermore, evidence for enhancement of PR-1 gene expression during P. infestans resistance response was obtained.

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“…This adaptation of PCR allowed an easier quantification of target sequences [6,7]. Different techniques were developed and compared for these quantitative assays [8][9][10][11][12]. As more transgenic traits were commercialized, which in some cases contained the same gene constructs, the specificity of the assays had to be increased by the development of eventspecific detection assays [13,14].…”

Section: Introductionmentioning

confidence: 99%

Multiplex real-time PCR for the detection and quantification of DNA from four transgenic soy Mon89788, A5547-127, Roundup Ready, A2704-12 and lectin

Köppel¹,

Velsen²,

Felderer³

et al. 2012

Eur Food Res Technol

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In the past, the transgenic soybean Roundup Ready was the predominantly cultivated transgenic soybean. With the announcement of Monsanto Corp. that this trait will be replaced by Mon89788 and the release of other company's transgenic soy crops, this unique status of one trait has ended. Therefore, a multiplex quantitative realtime PCR system was developed and characterized for three additional transgenic traits Mon89788, A2704-12 and A5547-127. It showed amplification efficiency, correlation and sensitivity similar to the single PCR systems applied therein. This system allows relative multiplex quantification and/or delta-delta Ct method and therefore an efficient control of products during production, trade and sale.

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Accurate Determination of Zygosity in Transgenic Rice by Real-time PCR Does Not Require Standard Curves or Efficiency Correction

Cited by 32 publications

References 10 publications

Transgene-specific and event-specific molecular markers for characterization of transgenic papaya lines resistant to Papaya ringspot virus

Transgene-specific and event-specific molecular markers for characterization of transgenic papaya lines resistant to Papaya ringspot virus

Potato R1 resistance gene confers resistance against Phytophthora infestans in transgenic tomato plants

Multiplex real-time PCR for the detection and quantification of DNA from four transgenic soy Mon89788, A5547-127, Roundup Ready, A2704-12 and lectin

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