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

Abstract

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 (

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

“…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.…”

“…The first cisgenic apple lines with scab resistance were developed by Vanblaere et al [ 23 ]. Würdig et al [ 24 ] and Krens et al [ 25 ] later developed further cisgenic apple lines with Rvi6 .…”

“…In the present study the generation of a cisgenic 'Gala Galaxy' line carrying the FB_MR5 gene showing increased resistance to fire blight is presented. In vitro shoot cultures of 'Gala Galaxy' were transformed with the novel binary transformation vector p9-Dao-FLPi [ 24 ], allowing the post-transformation heat induced flippase ( Flp ) based removal of the excisable cassette containing three transgenes ( NptII , Flp and dao1 ). Although it was not possible to use the selectable marker dao1 to increase the number of regenerants of lines without transgenes, a cisgenic line C44.4.146 was identified.…”

Cisgenesis and genome editing: combining concepts and efforts for a smarter use of genetic resources in crop breeding

“…, Würdig et al. ). Additional effects of the terminator type and length were discussed by the same authors.…”

“…), while new cisgenic apples with the same trait have been developed using an alternative recombinase system (Würdig et al. ). Furthermore, marker‐free cisgenic potato ( Solanum tuberosum ) plants expressing late blight resistance genes from S. stoloniferum ( Rpi‐sto1 ) and S. venturii ( Rpi‐vnt1.1 ) have been produced by Agrobacterium‐ mediated transformation without using any marker gene but PCR for the selection of transformed plants.…”