Evaluation of an alternative D-amino acid/DAAO selection system for transformation in apple (<i>Malus</i>×<i>domestica</i>Borkh.)

Hättasch, C.; Flachowsky, Henryk; Hanke, Magda-Viola

doi:10.1080/14620316.2009.11512619

Cited by 9 publications

(7 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…About 3,200 explants were subjected to heat shock activation of the Flp recombinase. The negative D-amino acid / dao1 selection system proposed by Hättasch et al [ 42 ] could not be applied, as it was recently found that the selective medium containing D-Ile hinders the regeneration [ 24 ]. On this account PCR was used to screen the obtained putative cisgenic regenerants.…”

Section: Resultsmentioning

confidence: 99%

Development of the First Cisgenic Apple with Increased Resistance to Fire Blight

Kost¹,

Gessler²,

Jänsch³

et al. 2015

PLoS ONE

View full text Add to dashboard Cite

The generation and selection of novel fire blight resistant apple genotypes would greatly improve the management of this devastating disease, caused by Erwinia amylovora. Such resistant genotypes are currently developed by conventional breeding, but novel breeding technologies including cisgenesis could be an alternative approach. A cisgenic apple line C44.4.146 was regenerated using the cisgene FB_MR5 from wild apple Malus ×robusta 5 (Mr5), and the previously established method involving A. tumefaciens-mediated transformation of the fire blight susceptible cultivar ‘Gala Galaxy’ using the binary vector p9-Dao-FLPi. The line C44.4.146 was shown to carry only the cisgene FB_MR5, controlled by its native regulatory sequences and no transgenes were detected by PCR or Southern blot following heat induced recombinase-mediated elimination of the selectable markers. Although this line contains up to 452 bp of vector sequences, it still matches the original definition of cisgenesis. A single insertion of T-DNA into the genome of 'Gala Galaxy' in chromosome 16 was identified. Transcription of FB_MR5 in line C44.4.146 was similar to the transcription in classically bred descendants of Mr5. Three independent shoot inoculation experiments with a Mr5 avirulent strain of Erwinia amylovora were performed using scissors or syringe. Significantly lower disease symptoms were detected on shoots of the cisgenic line compared to those of untransformed 'Gala Galaxy'. Despite the fact that the pathogen can overcome this resistance by a single nucleotide mutation, this is, to our knowledge, the first prototype of a cisgenic apple with increased resistance to fire blight.

show abstract

Section: Resultsmentioning

confidence: 99%

Development of the First Cisgenic Apple with Increased Resistance to Fire Blight

Kost¹,

Gessler²,

Jänsch³

et al. 2015

PLoS ONE

View full text Add to dashboard Cite

show abstract

“…Using this system, two cisgenic lines were obtained, one originating from 'Pinova' and one from 'Brookfield Baigent'. The D-amino acid/DAAO selection system, which has been suggested by Hättasch et al (2009) for the selection of shoots without recombination cassette based on the dao1 gene, did not give any advantage. Apple plantlets were not able to produce shoots under pressure of D-isoleucine and transgenic cells survived for months.…”

Section: Discussionmentioning

confidence: 96%

“…The selection of shoots harboring nptII and dao1 was realized by adding 100 mg/l kanamycin (Duchefa, Haarlem, the Netherlands) and 20 mM D-serine (Sigma-Aldrich, St. Louis, MO, USA) (Hättasch et al 2009) to shoot proliferation medium, respectively. The extent of selection was assessed after 4 weeks.…”

Section: Application Of Selective Agents During Plant Regeneration Anmentioning

confidence: 99%

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

View full text Add to dashboard Cite

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 (

show abstract

“…In future studies, the efficiency of the recombination system could additionally be enhanced by using other tissues (e.g. leaves instead of entire shoots) for flp gene induction or by using the nptII gene fused to a negative selection marker such as the dao1 gene from Rhodotorula gracilis, as suggested by Hättasch et al (2009).…”

Section: Discussionmentioning

confidence: 98%

“…After induction of the FLP recombinase, the plants/ explants could be transferred to medium containing D-isoleucine to confirm removal of the dao1/nptII cassette by recombination (Hättasch et al, 2009).…”

Section: Discussionmentioning

confidence: 99%

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

Herzog

Flachowsky

Deising

et al. 2012

Gene

View full text Add to dashboard Cite

Evaluation of an alternative D-amino acid/DAAO selection system for transformation in apple (Malus×domesticaBorkh.)

Cited by 9 publications

References 28 publications

Development of the First Cisgenic Apple with Increased Resistance to Fire Blight

Development of the First Cisgenic Apple with Increased Resistance to Fire Blight

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

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

Contact Info

Product

Resources

About