Cisgenic apple trees; development, characterization, and performance.

Frans A Krens,Cesare Gessler,Jan G Schaart,Linda P Kodde,Giovanni A L Broggini,Aranka M Van Der Burgh,Henk J Schouten,Remmelt Groenwold,Iris E M Tinnenbroek-Capel

doi:10.3389/fpls.2015.00286

Frans A Krens, Cesare Gessler + Show 7 more

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https://doi.org/10.3389/fpls.2015.00286

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Abstract

Two methods were developed for the generation of cisgenic apples. Both have been successfully applied producing trees. The first method avoids the use of any foreign selectable marker genes; only the gene-of-interest is integrated between the T-DNA border sequences. The second method makes use of recombinase-based marker excision. For the first method we used the MdMYB10 gene from a red-fleshed apple coding for a transcription factor involved in regulating anthocyanin biosynthesis. Red plantlets were obtained and presence of the cisgene was confirmed. Plantlets were grafted and grown in a greenhouse. After 3 years, the first flowers appeared, showing red petals. Pollination led to production of red-fleshed cisgenic apples. The second method used the pM(arker)F(ree) vector system, introducing the scab resistance gene Rvi6, derived from apple. Agrobacterium-mediated transformation, followed by selection on kanamycin, produced genetically modified apple lines. Next, leaves from in vitro material were treated to activate the recombinase leading to excision of selection genes. Subsequently, the leaf explants were subjected to negative selection for marker-free plantlets by inducing regeneration on medium containing 5-fluorocytosine. After verification of the marker-free nature, the obtained plants were grafted onto rootstocks. Young trees from four cisgenic lines and one intragenic line, all containing Rvi6, were planted in an orchard. Appropriate controls were incorporated in this trial. We scored scab incidence for three consecutive years on leaves after inoculations with Rvi6-avirulent strains. One cisgenic line and the intragenic line performed as well as the resistant control. In 2014 trees started to overcome their juvenile character and formed flowers and fruits. The first results of scoring scab symptoms on apple fruits were obtained. Apple fruits from susceptible controls showed scab symptoms, while fruits from cisgenic and intragenic lines were free of scab.

Highlights

IntroductionIn 2006, the concept of cisgenesis was brought to the attention of a large group of scientists and opened for debate by publications in Nature Biotechnology and EMBO ReportsCisgenic apple trees (Schouten et al, 2006a,b)
In 2006, the concept of cisgenesis was brought to the attention of a large group of scientists and opened for debate by publications in Nature Biotechnology and EMBO ReportsAbbreviations: SIM, shoot induction medium; nptII(I), neomycinphosphotransferaseII(I); LBD, ligand-binding domain; trfA, gene encoding a replication initiation protein of the broad host-range plasmid RK2.Cisgenic apple trees (Schouten et al, 2006a,b)
The European Food Safety Authority (EFSA Panel on Genetically Modified Organisms (GMO), 2012) compared the hazards associated with cisgenic, intragenic and conventionally bred plants and concluded that hazards associated with cisgenic plants are similar to the ones associated with conventionally bred plants

Summary

Introduction

In 2006, the concept of cisgenesis was brought to the attention of a large group of scientists and opened for debate by publications in Nature Biotechnology and EMBO ReportsCisgenic apple trees (Schouten et al, 2006a,b). The main reason for this was to explore possibilities to apply genetic modification (GM) as a tool to speed up breeding and to open up novel ways of breeding in crops such as apple in a way that would be acceptable to a wide public. Once a high quality, elite variety has been bred after many years, a new crossing for the introduction of a single trait will make it disappear without a chance of ever being able to breed a similar variety again (Gardiner et al, 2007). No foreign genes are present in the final product, no antibiotic resistance genes of bacterial origin. Such crops were found to be more acceptable for consumers (Lusk and Sullivan, 2002; Gaskell et al, 2011). The European Food Safety Authority (EFSA Panel on Genetically Modified Organisms (GMO), 2012) compared the hazards associated with cisgenic, intragenic and conventionally bred plants and concluded that hazards associated with cisgenic plants are similar to the ones associated with conventionally bred plants

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