Cisgenic apple trees; development, characterization, and performance

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.

Effect of Organic Potato Farming on Human and Environmental Health and Benefits from New Plant Breeding Techniques. Is It Only a Matter of Public Acceptance?

“…However, the presence of a selectable marker gene of bacterial origin makes it not substantially equivalent to the original one. To avoid this issue, bacterial marker genes and marker genes from non-crossable plant species should be eliminated by recombination or avoided in favor of selectable cisgenes, as already reported for the apple Myb10 gene [66,67]. Potato is advantaged with respect to other species because cisgenesis can be obtained without the use of any selectable marker gene, due the high transformation and regeneration rates that are necessary for this strategy.…”

Back into the wild—Apply untapped genetic diversity of wild relatives for crop improvement

“…This technique has been successfully used to confer resistance to late blight in potatoes (Haverkort, Struik, Visser, & Jacobsen, 2009) and scab resistance in apples (Vanblaere et al, 2011). It is important to note that because cisgenesis only transfers a gene from a native or cross-compatible species, this results in plants with a performance comparable to that possible from conventional breeding (Krens et al, 2015). However, transfer of a single desired gene by cisgenesis avoids any linkage drag and reduces the time involved compared with traditional breeding strategies.…”

New developments in green biotechnology - an inventory for RIVM

“…Examples published comprise stacking R genes, for instance against late blight caused by Phytophthora infestans in potato (Haesaert et al 2015;Haverkort et al 2016) or against scab caused by Venturia inaequalis in apple (Krens et al 2015). Possible applications in intragenesis may vary widely, including disease resistance, e.g.…”