Heat-shock-mediated elimination of the nptII marker gene in transgenic apple (Malus×domestica Borkh.)

Herzog, Katja; Flachowsky, Henryk; Deising, H. B.; Hanke, Magda‐Viola

doi:10.1016/j.gene.2012.01.074

Cited by 31 publications

(31 citation statements)

References 42 publications

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“…Several strategies were developed to create ''marker-free'' transgenic plants for their advantages for regulatory approval and public acceptance [44][45][46][47][48]. We therefore took advantage of our high rate of tomato transformation and efficiency of screening for resistance by whitefly inoculation for the development of antibiotic-resistance-free TYLCV-resistant plants.…”

Section: Size and Distribution Of T-sirna In Transgenic Rootstock Andmentioning

confidence: 99%

Immunity to tomato yellow leaf curl virus in transgenic tomato is associated with accumulation of transgene small RNA

et al. 2015

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Gene silencing is a natural defense response of plants against invading RNA and DNA viruses. The RNA post-transcriptional silencing system has been commonly utilized to generate transgenic crop plants that are "immune" to plant virus infection. Here, we applied this approach against the devastating DNA virus tomato yellow leaf curl virus (TYLCV) in its host tomato (Solanum lycopersicum L.). To generate broad resistance to a number of different TYLCV viruses, three conserved sequences (the intergenic region [NCR], V1-V2 and C1-C2 genes) from the genome of the severe virus (TYLCV) were synthesized as a single insert and cloned into a hairpin configuration in a binary vector, which was used to transform TYLCV-susceptible tomato plants. Eight of 28 independent transgenic tomato lines exhibited immunity to TYLCV-Is and to TYLCV-Mld, but not to tomato yellow leaf curl Sardinia virus, which shares relatively low sequence homology with the transgene. In addition, a marker-free (nptII-deleted) transgenic tomato line was generated for the first time by Agrobacterium-mediated transformation without antibiotic selection, followed by screening of 1180 regenerated shoots by whitefly-mediated TYLCV inoculation. Resistant lines showed a high level of transgene-siRNA (t-siRNA) accumulation (22% of total small RNA) with dominant sizes of 21 nt (73%) and 22 nt (22%). The t-siRNA displayed hot-spot distribution ("peaks") along the transgene, with different distribution patterns than the viral-siRNA peaks observed in TYLCV-infected tomato. A grafting experiment demonstrated the mobility of 0.04% of the t-siRNA from transgenic rootstock to non-transformed scion, even though scion resistance against TYLCV was not achieved.

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Section: Size and Distribution Of T-sirna In Transgenic Rootstock Andmentioning

confidence: 99%

Immunity to tomato yellow leaf curl virus in transgenic tomato is associated with accumulation of transgene small RNA

et al. 2015

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“…Further steps were performed as described by Herzog et al (2012). Number of T-DNA integration sites was detected with an nptII-specific probe, synthesized with the PCR DIG Probe Synthesis Kit (Roche Deutschland Holding GmbH, Grenzach-Wyhlen, Germany) using the primers NptIIF and NptIIR (Supplementary Table S1) and DNA of vector p9-Dao-FLPi-HcrVf2 as template.…”

Section: Southern Hybridizationmentioning

confidence: 99%

“…By definition, a cisgenic plant does not contain any foreign genes and is therefore quite close to traditionally bred plants. The ''clean vector technology'' was established for apple based on inducible recombinase systems (Herzog et al 2012;Vanblaere et al 2011;Würdig et al 2013) to realize the concept of cisgenesis. Recently, the first cisgenic 'Gala' apple plant carrying the Rvi6 scab resistance gene was produced (Vanblaere et al 2011) by exploiting a chemically inducible R/Rs system developed by Schaart et al (2004).…”

Section: Introductionmentioning

confidence: 99%

“…With the aim to minimize problems like multiple transgene integration and vector backbone insertion, which occurred using the pMF1 transformation vector (Vanblaere et al 2011), we employed a newly developed transformation vector based on previous studies (Herzog et al 2012;Würdig et al 2013). The obtained transgenic lines are described in detail, including the molecular state of the T-DNA, mRNA expression of the transferred scab resistance gene as well as scab resistance after artificial inoculation.…”

Section: Introductionmentioning

confidence: 99%

“…The obtained transgenic lines are described in detail, including the molecular state of the T-DNA, mRNA expression of the transferred scab resistance gene as well as scab resistance after artificial inoculation. Based on these results, selected transgenic lines were used for the production of cisgenic plants by exploiting a recently established heat-inducible Flp/FRT recombinase system (Herzog et al 2012;Würdig et al 2013). Obtained cisgenic plants were analyzed in the same manner as the transgenic lines.…”

Section: Introductionmentioning

confidence: 99%

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Improving resistance of different apple cultivars using the Rvi6 scab resistance gene in a cisgenic approach based on the Flp/FRT recombinase system

et al. 2015

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Cisgenic apple plants of two different cultivars were developed by transferring the Rvi6 scab resistance gene of Malus floribunda 821, using a new transformation vector based on the Flp/FRT recombinase system. Transformation experiments on seven different cultivars resulted in 22 transgenic lines for the cultivars 'Brookfield Baigent', 'Mitchgla', 'Novajo', and 'Pinova', whereby 16 lines thereof were resistant to Venturia inaequalis strain 104 (race 1).Analysis of the transgenic lines revealed Rvi6 mRNA expression levels comparable to several traditional bred Rvi6 containing cultivars and identified four transgenic lines, harboring a single T-DNA insertion, as suitable for the production of cisgenic lines. The T-DNA insertion site of these lines was determined, and lines were subject to induction of the recombinase system. Two cisgenic lines originating from the cultivars 'Brookfield Baigent' and 'Pinova' were obtained for which the exact excision of the recombinase cassette was confirmed by sequencing the previously determined T-DNA integration site. Further investigations revealed both cisgenic lines as fully resistant to V. inaequalis race 1. Rvi6 mRNA expression of the cisgenic lines and traditionally bred Rvi6 harboring cultivars was still comparable. The transformation vector developed is useable for the production of cisgenic apple plants to a certain extent.Keywords Rvi6 expression Á Cisgenesis Á Flp/FRT recombinase system Á Fruit tree Á Malus9 domestica Á Biotechnology Electronic supplementary material The online version of this article (

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Cisgenesis and Intragenesis as New Strategies for Crop Improvement

Moradpour

Abdullah

2017

Crop Improvement

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Cisgenesis and intragenesis are emerging plant breeding technologies which offer great promise for future acceptance of genetically engineered crops. The techniques employ traditional genetic engineering methods but are confined to transferring of genes and genetic elements between sexually compatible species that can breed naturally. One of the main requirements is the absence of selectable marker genes (such as antibiotic resistance genes) in the genome. Hence the sensitive issues with regard to transfer of foreign genes and antibiotic resistance are overcome. It is a targeted technique involving specific locus; therefore, linkage drag that prolongs the time for crop improvement in traditional breeding does not occur. It has great potential for crop improvement using superior alleles that exist in the untapped germplasm or wild species. Cisgenic and intragenic plants may not face the same stringent regulatory assessment for field release as transgenic plants which is a clear added advantage that would save time. In this chapter, the concepts of cis/intragenesis and the prerequisites for the development of cis/intragenesis plants are elaborated. Strategies for marker gene removal after selection of transformants are discussed based on the few recent reports from various plant species.

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Heat-shock-mediated elimination of the nptII marker gene in transgenic apple (Malus×domestica Borkh.)

Cited by 31 publications

References 42 publications

Immunity to tomato yellow leaf curl virus in transgenic tomato is associated with accumulation of transgene small RNA

Immunity to tomato yellow leaf curl virus in transgenic tomato is associated with accumulation of transgene small RNA

Improving resistance of different apple cultivars using the Rvi6 scab resistance gene in a cisgenic approach based on the Flp/FRT recombinase system

Cisgenesis and Intragenesis as New Strategies for Crop Improvement

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