Cisgenesis Is a Promising Approach for Fast, Acceptable and Safe Breeding of Pip Fruit

Introgression of traits from wild germplasm into pip fruit cultivars by means of classical breeding is painstakingly slow. Introgression of e.g., the apple scab resistance gene Vf from Malus floribunda 821 into marketable high quality apple cultivars took approximately 50 years. In the mean time the Vf resistance is being broken down in Europe. For durable resistance, different resistance genes should be accumulated. However, this may take another series of decades. This slow tempo is caused mainly by the long juvenile period of apple and the phenomenon that not only the allele of interest is inherited by the progeny, but also hundreds of unwanted alleles. The process would be much faster if only the allele of interest was inserted, without unwanted alleles. This can be achieved by cisgenesis. We defined cisgenesis as genetic modification of plants, inserting alleles of the plant itself or from crossable relatives. The allele should contain its native introns and should be flanked by its native promoter and terminator in sense-orientation. If the plant is equipped with foreign genes from outside the gene pool of the conventional breeder, the plant is named transgenic. Inquiries indicate that cisgenic plants are more acceptable to consumers than transgenic plants. As the phenotypic result of cisgenesis can, in principle, also be obtained by means of conventional breeding or translocation breeding, cisgenic plants are as safe as plants from conventional breeding or mutation breeding. Therefore we have proposed to treat cisgenic plants like conventionally bred plants, by exempting cisgenic plants from the GMO regulation. The number of isolated, functionally analysed genes and their alleles from fruit tree crops is increasing. Also technologies are available for introduction of these alleles without leaving selection genes behind. Cisgenesis is combining the knowledge of native alleles with marker free technologies. Cisgenesis is a promising path for utilizing the wealth of knowledge on plant genes to the benefit of the society in a fast, safe, and acceptable way. INTROGRESSION OF A MAJOR RESISTANCE GENE IN APPLE TOOK HALF A CENTURY In 1946, crosses were made for introduction of resistance to apple scab (Venturia inaequalis) into commercial apple varieties, using as source of resistance the crab apple Malus floribunda 821 (Hough et al., 1953). The progeny of the cross between M. floribunda 821 and susceptible cultivars segregated in a Mendelian fashion for resistance in a 1:1 ratio. The gene putatively underlying this resistance was named Vf-gene. However, the fruits of the resistant parent M. floribunda 821 were very small, approximately 1 cm. The apples of the progeny were also small, and did not have the fruit quality that was required for commercial cultivars. This was caused by linkage drag: not only the wanted resistance gene was inherited to part of the progeny, but also many unwanted alleles, leading to poor fruit quality and other unwanted traits. In order to get rid of the unwanted alleles, subsequent crosses had to be carried out between resistant 199 Proc. XII th Eucarpia Symp. on Fruit Breeding and Genetics Eds.: R. Socias i Company et al. Acta Hort. 814, ISHS 2009 progeny and susceptible high quality cultivars. About five generations were required to remove enough unwanted alleles from M. floribunda, yet keeping the desired Vf-gene for scab resistance. Approximately 50 years after the first cross, Vf-cultivars with a reasonable fruit quality were introduced onto the market (Anonymous, 1999). Therefore, it has taken half a century to introduce the Vf-gene and remove the linkage drag to an acceptable degree. MORE RESISTANCE GENES NEEDED In the mean time, Venturia inaequalis strains have been detected that are able to infect Vf-cultivars (Parisi et al., 1993). Especially in northwestern Europe, these strains are present and have spread (Parisi et al., 2006). As a result, several orchards that consist of Vf-cultivars have to be sprayed like orchards with susceptible cultivars (Trapman, 2006). Fifty years of breeding is fading away in ten years. Obviously, more individual resistance genes need to be accumulated for obtaining more durable resistance. Fortunately, many loci that confer resistance to apple scab have been discovered in Malus, both major genes and QTLs (Calenge et al., 2004; Gardiner et al., 2006; Gessler et al., 2006; Schmidt and Van de Weg, 2005). The increase in number of mapped resistance genes has even required a new system for nomenclature of all these genes (Bus et al., this symposium). Therefore, sufficient genes for resistance are present in the germplasm of apple. Introgression of one resistance gene took approximately 50 years. Introgression of four or more genes for durable resistance will require more time, when the breeding is performed in the classical way. Would this imply that we are left with another 50 years of intensive fungicide applications to apple scab in conventional apple production or sulphur in organic apple production, before the more durably resistant cultivars are introduced? We regard it as our challenge to shorten this period significantly, by introducing the resistance genes and preventing the linkage drag. ROUTES FOR INTROGRESSION OF MULTIPLE RESISTANCE GENES One way to speedup the breeding process is marker-assisted breeding. This method can be of tremendous use and we advocate this approach, but still this will be time-consuming, because linkage drag has to be removed through crosses and meiotic recombination. Accumulation of resistance genes from four sources of resistance and sufficient removal of unwanted alleles from these sources, will probably require at least another five generations of apple. As long as the juvenile period and additional evaluation time in apple is about eight years, this would require a minimum of 40 years of breeding. An alternative route is introduction of the resistance genes into susceptible elite cultivars in an asexual way without simultaneous introgression of unwanted alleles, so prevention of linkage drag, rather then removal of linkage drag. Then, durable resistance provided by several resistance genes is added to high quality cultivars in one step, preserving the proven fruit quality and other desired traits of these cultivars. We have named this process ‘cisgenesis’ (Schouten et al., 2006a). DEFINITION OF CISGENESIS A cisgenic plant is a crop plant that has been genetically modified with one or more genes isolated from an inter-fertile donor plant. A cisgene contains its native introns and flanking regions such as native promoter and terminator region in a sense orientation. We distinguish cisgenic plants from transgenic plants. Transgenic plants contain genes from non-inter-fertile organisms, synthetic genes or sequences, or artificial combinations of a coding gene with regulatory sequences, such as a promoter, from another gene (Schouten et al., 2006a; Schouten and Jacobsen, 2008). Cisgenic plants can, in principle, also be obtained by means of classical breeding, as far as the phenotype is concerned. This indicates clearly its limits regarding breeding possibilities, but also its limits regarding possible biosafety risks. Cisgenic plants are as safe as conventionally bred plants or safer (Jacobsen and Schouten, 2007; Schouten et al., 2006b).