Chilean Journal of Agricultural Research Instituto de Investigaciones Agropecuarias, INIA ISSN: 0718-5820 EISSN: 0718-5839 Vol. 70, Num. 2, 2010, pp. 297-308

Chilean Journal of Agricultural Research, Vol. 70, No. 2, April-June, 2010, pp. 297-308

Review

Advances in apple transformation technology to confer resistance to fungal diseases in apple crops: A Chilean perspective

Avances de la tecnología de transformación del manzano para conferir resistencia a enfermedades fungosas en su cultivo: Una perspectiva Chilena

Víctor Polanco¹, Mario Paredes², Viviana Becerra², Eduardo Pérez³

¹ Universidad Mayor, Laboratorio de Diagnóstico Molecular (DIAMOLAB), Casilla 234, Correo 35, Santiago, Chile
² Instituto de Investigaciones Agropecuarias INIA, Casilla 426, Chillán, Chile
³ Centro Regional de Estudios en Alimentos Saludables, General Cruz 34, Valparaíso, Chile

Correspondence Address: Víctor Polanco, Universidad Mayor, Laboratorio de Diagnóstico Molecular (DIAMOLAB), Casilla 234, Correo 35, Santiago, Chile, victor.polanco@umayor.cl

Date of Submission: 16-Jan-2009
Date of Acceptance: 25-Jun-2009

Code Number: cj10032

Abstract

Keywords: apple, fungal disease, transgenic, cisgenesis

Resumen

La manzana (Malus domestica Borkh.) es una de las frutas más consumidas en el mundo. La transformación genética es un proceso clave para sustentar esta demanda, permitiendo el mejoramiento potencial de los cultivares existentes, así como el desarrollo de nuevas variedades resistentes a plagas, enfermedades y problemas de almacenamiento que se originan en las zonas de producción más importantes. Esta revisión resume los avances de la ingeniería genética aplicada al desarrollo de variedades de manzana resistentes a enfermedades fungosas, con especial atención en la generación de manzanas resistentes a Venturia inaequalis (Cooke) G.Winter, el principal problema fitosanitario que afecta a los cultivos de manzana en Chile.

Palabras claves: manzana, enfermedades fungosas, transgénico, cisgénesis

Introduction

Apple belongs to the Pomoideae family, subfamily Rosaceae, along with other important fruit crops such as pear (Pyrus communis L.), prune (Prunus domestica L.) and cherry (Prunus avium L.). Domesticated apple probably originated in the area around of the Heavenly Mountains on the border Western of China, in the former USSR and in Central Asia, and it is putatively an interspecific hybrid complex, designated Malus domestica Borkh. (Korban and Skirvin, 1984; Phipps et al., 1990). In medieval times, monasteries were responsible for selection, propagation and perpetuation of hundreds of different cultivated types. These plantations became the major sources of breeding stock and selections for making controlled crosses to improve specific traits in the 1800′s (MacHardy, 1996).

During the late 19th and 20th centuries, M. domestica cultivars were genetically improved in Europe, Russia, North America, New Zealand, Japan and Australia and finally, apples were introduced to the rest of the world. These new introductions constituted the basis for most of the current commercial apple cultivars (Way et al., 1991; Janick et al., 1996). Nowadays, more than 7000 varieties have been described and breeders worldwide create new selections annually; nevertheless, only a few dozen cultivars are produced commercially (Janick et al., 1996).

In 2004, the apples were the third most cultivated fruit crop in the world (5280 M ha) and the third fruit crop in production (59 059 Mt), after Citrus sinensis Osbeck (orange) and Musa paradisiaca L. (banana) (FAOSTAT, 2004). The most recent statistical information published by the Department of Agriculture of the USA, locates Chile in the third place of the exporting countries of apple fruit in the world. However, we are behind the European Union (EU), and China, but above the USA, South Africa, New Zealand and Argentina. Chile provided about 18% of the total apple market supply and the EU represented the 22% and China a 19% (USDA, 2007).

Besides its economic importance, apple has become a woody perennial angiosperm model for genomic research due to its relatively small genome size (750 Mb/haploid), availability of genetic resources such as over 300 000 expressed sequence tags (EST), bacterial artificial chromosome (BAC) libraries, genetic maps, first-draft physical map, and a development of a robust genetic transformation system (Xu et al., 2001; Xu and Korban, 2002; Aldwinckle et al., 2003; Liebhard et al., 2003; Tatum et al., 2005; Newcomb et al., 2006; Han et al., 2007). Most of the cultivated apple lines are diploids (2n = 2x = 34), self-incompatible, open-pollinated, and display a juvenile period that ranges from 6 to 10 years or more (Korban and Chen, 1992).

Nowadays, the market demands apple cultivars with high productivity, uniformity, and good fruit quality. In addition, resistance to diseases, pests and storage disorders are also desired. Even though, the advantage of a resistant cultivar is evident, resistant cultivars still do not dominate the market. The reason is simple, the most successful commercial apple cultivars have lost the efficacy of their resistance genes toward to the two most frequent fungal pathogens, apple scab (Venturia inaequalis (Cooke) G. Winter), and apple mildew (Podosphaera leucotricha Ellis & Everh.). Then, almost all apple cultivars need constant fungicides protection, wherever the climatic conditions favors the development of these or other pathogens. For example, on average, 10 to 15 fungicide applications are necessary to produce apple scab-free fruits during a season, which increase the cost of apple production and the concerns from consumers and environmentalists over pesticide uses (MacHardy, 1996; Gessler and Patocchi, 2007). Therefore, most current apple breeding programs are oriented toward reducing the need for pesticides, without losing the fruit quality.

Unfortunately, because of the long juvenile period, high levels of heterozygocity and time required to evaluate hybrids, the process to release a new cultivars from a conventional apple breeding programs are slow, especially if the breeder transfers genes from non adapted genotypes like wild genotypes. For instance, a cross between domesticated and wild species needs several backcrosses to eliminate the unwanted traits inherited from the wild species. Releasing a new cultivar could take 10 or more years, and almost 40 years to introduce and establish the new cultivar on the market (Korban and Chen, 1992; Brown, 1992). The application of modem DNA analysis methods such as genetic maps, identification of DNA markers linked to traits of interest, and marker-assisted selection in breeding, could help to accelerate this process, and a new cultivar with resistance to diseases and pests could be developed in a shorter period. However, this new cultivar needs to be accepted by the consumers, who are very specific on fruit quality traits requirements. Under this situation, the genetic improvement of apple through the introduction of specific gene(s) onto current commercial cultivars, in a short run, is a very attractive strategy.

The objective of this work was to review the current knowledge on apple genetic transformation to improve its fungal resistance, with emphasis on Venturia inaequalis.

Biotechnological approaches to genetic improvement Genetic mapping and quantitative trait loci (QTL) analysis.

Molecular markers have been linked to a number of monogenic traits in apple (Tartarini and Sansavini, 2003). The most work has been done on the gene for scab resistance, where over 40 markers have been identified. Markers for the other scab resistance genes have also been developed by many groups and include Vh from Russian seedling R12740-7A of M. Sieversii (Hemmat et al., 2002), Vm (Cheng et al., 1998), and Vb (Hemmat et al., 2003; Erdin et al., 2006), Vd (Tartarini et al., 2004), Vbj (Gygax et al., 2004) and Vg (Durel et al., 2000; Calenge et al., 2004). Gessler et al. (2006) reviewed the literature in this area from type of resistance through gene pyramiding.

Vinatzer et al. (2004) used the inverse polymerase chain reaction and simple sequence repeats to identify BAC clones containing the apples scab resistance gene Vf and found the gene in scab-resistance accessions of Malus xmicromalus Makino and `Golden Gem′ of M. prunifolia (Willd.) Borkh., which were previously not known to carry this gene.

Markers have also been linked to the pest resistance genes Sdl for Dysaphis devecta, and Erl and Erg for E. lanigerum. A few markers have also been linked to genes regulating morfological traits including the columnar habit (Co), fruit color (Rj) and fruit acidity (Ma). Recently a cDNA/AFLP approach was used to identify a gene that contributes to lowering of fruit acidity (Yao et al., 2007).

Several groups in Europe have been especially busy mapping the QTL associated with resistance to apple scab into various linkage groups (LGs). The cross of `Prima′ x `Fiesta′ and other related F, progenies have been used to identify major genes associated with resistance in the DARE project (Durable Apple Resistance in Europe) (Durel et al., 2003). The major genes for scab resistance were found on LG12. Several different nucleotide binding site (NBS)-type resistance gene analogues were clustered at bottom of LG5 and the top LG 17 for resistance to races 1 and 6. A major non-race-specific QTL was identified near an NBS-analog cluster on linkage group LG10. Three major genes for powdery-mildew resistance were also identified by bulked segregant approaches, and ones of them on LG2 was located in the same region as scab resistance.

Five apple progenies were used in the DARE project to identify QTL with broad spectrum of resistance towards a wide rande of strains of the fungus (Durel et al., 2004). It was verified that four major genomic regions exist that carry resistance to multiple strains of the fungus, with a QTL region on LG 17 carrying the widest spectrum of resistance. So, molecular markers and QTLs analyses are key for the identification of genes resistance to pathogens, and allows their application for the generation of news lines of apples genetically modified.

Genomic resources. Mining of existing apples EST information, such as the studies of Newcomb et al. (2006) and Park et al. (2006), and the use of microarrays (Pichler et al., 2007) promises to expand our knowledge of many genes important in the genetic improvement of apple. The development of public databases such as the GDR (Genome Database for the Rosaceae; Jung et al., 2004) and the European HIDRAS ApplesBreed (Antofie et al., 2007) also offer excellent prospects for enhanced collaboration amongst breeders, bioinformatics researchers and those involved in molecular biology. In the GDR database alone, over 50 000 ESTs are available from several species, tissues and developmental stages for use in genetic tranformation.

Apple transformation. Because of the high susceptibility to fungal diseases of the most important commercial apple cultivars and rootstocks, genetic transformation has been one good method for the development of resistant cultivars.

Genetic transformation of plants is the process where a defined fragment of DNA (a gene) is introduced and integrated into the genome of the plant, avoiding sexual reproduction. Genetic engineering enlarges the readiness of genes considerably, limited in conventional breeding programs, since genes isolated from other plants, animals or microorganisms can be transferred to plants (Brasileiro and Dusi, 1999).

Apple was an early target for the emerging recombinant DNA technology. Transformation of Malus is nowadays a common practice in several laboratories and the protocols have been constantly improved to enhance the transformation efficiency (James et al., 1993; Yepes and Aldwinclde, 1993; Yao et al., 1999; De Bondt et al., 1994; 1996; Norelli et al., 1996; Puite and Schaart, 1996; Hammerschlag et al., 1997; Liu et al., 1998; Sriskandarajah and Goodwin, 1998; Bolar et al., 1999).

The most widely used method for introducing foreign genes into dicotyledoneous plants is the Agrobacterium tumefaciens mediated transformation. In this process, A. tumefaciens, a disarmed Ti binary vector, and leaf fragments or callus cultures are the key component for an efficient transformation (James et al., 1989). Most studies started from wounded leaf sections (Norelli et al., 1996), but apical intemodal explants from etiolated `Royal Gala′ apple shoots has produced a higher efficiency in producing transgenic shoots (Liu et al., 1998).

Genes used in genetic transformation of apples

Transformations are mostly based on traditional cultivars and they have been carried out using genes isolated from apple (Belfanti et al., 2004b; Espley et al., 2007; Malnoy et al., 2007a; 2008) or from other organisms (Wong et al., 1999; Norelli et al., 1994; 2000; Bolar et al., 2000; 2001; Hanke et al., 2000; Liu et al., 2001; Szankowski et al., 2003; Markwick et al., 2003; Faize et al., 2004). Genes affecting some physiological or morphological characters like growth (Holefors et al., 2000), flowering (Yao et al., 1999) and self-fertility (Van Nerum et al., 2000) have also been incorporated into transgenic apples. Rootstock scions have also been used in transgenic assays to improve rooting rates and growth (Holefors et al., 1998; Welander et al., 1998; Sedira et al., 2001; Pawlicki-Jullian et al., 2002; Igarashi et al., 2002). The function of some genes like sorbitol-6-phosphate (Kanamaru et al., 2004; Cheng et al., 2005), stilbene synthase (Ruhmann et al., 2006), polygalacturonase (Atkinson et al., 2002) and from several promoters (Ko et al., 2000; Gittins et al., 2001; 2003; Szankowski et al., 2008) has also been studied using transgenic apple (see [Table - 1]).

Use of resistance genes. Apple scab is one of the most serious diseases affecting apple orchards causing weakness of trees and fruit damages. Six major scab resistance genes (Vf, Vm, Vb, Vbj, Vr, and Va) have been identified from wild small fruited Malus species (Williams and Kuc, 1969; Biggs, 1990). Up until now, only the Vf gene, originated from Malus floribunda 821 Siebold ex Van Houtte, has been widely introgressed into susceptible commercial apple cultivars (Crandall, 1926; Crosby et al., 1992; Korban, 1998). The Vf gene confers resistance to five out of seven known races of V. inaequalis and has held up quite well in the orchards for over 80 years.

A different approach to obtain scab resistant plants was attempted by the joint team of the department of Fruit Tree and Woody Plant Sciences of the Bologna University, Italy, and the Plant Pathology group at Swiss Federal Institute of Technology (ETH) Zurich, Switzerland (Sansavini et al., 2004). Starting from an European Union (EU) project which offered an excellent linkage map of apple and molecular markers mapped in the region of the scab resistance Vf, the positional cloning of Vf was initiated. A contig of BAC clones spanning the region between the two Vf molecular markers, M18 and AM19, one on each side of Vf was constructed and allowed the identification of four genes, named HcrVfl to HcrVf4. These genes codes for receptor-like proteins that have a high homology to the Cladosporium fulvum (CD resistance gene family of tomato. The genes have an extracellular leucine-rich repeat domain and a transmembrane domain (Vinatzer et al., 2001).

Using this information, the gene HcrVJ2 under the control of the CaMV 35S promoter was introduced into the susceptible cv. Gala using the nptll gene for selection. In the first step, the progression of the scab infection (penetration and stroma formation by the fungus) (Barbieri et al., 2003; Sansavini et al., 2003) was evaluated in vitro, followed by a greenhouse scab inoculations of the experimental lines containing a single functional copy of HcrVJ2. These evaluations demonstrated unambiguously that the four lines carrying HcrVJ2 were at least as resistant to scab (Belfanti et al., 2004a; 2004b) as the conventionally bred Vf resistant cv. `Florina′.

As the resistance Vf is known to be overcome by race 6 and 7, it was of interest to test if the introgressed gene really conferred the same type of resistance, i.e., recognition of the avirulent V. inaequalis genotypes and induction of the defense cascade, or through some artifact. Plants transformed with both HcrVJ2 and nptll, transformed with nptll only, and wild-type `Gala′ and ′Florina′ (Vf) were challenged with a field inoculum (mixture of genotypes) known to have the ability to cause scab on `Gala′, but not on ′Florina′, and with an inoculum derived from M. floribunda 821, the original donor of Vf. The results indicated that the HcrVJ2 line was, as expected, resistant to the field inoculum similarly to Florina, whereas all the other plant showed typical scab lesions with abundant sporulation. Inoculation with race 7 from M. floribunda 821 resulted in sporulating lesions on all plants; however, the inoculum was less aggressive and the sporulation was less abundant than the field inoculum. Even more, the HcrVJ2 line still retained some resistance, it is slightly more resistant than the line transformed with only nptll and the original `Gala′ (Silfverberg-Dilworth et al., 2005a; Gessler and Patocchi, 2007). This experiment demonstrated that resistance in HcrVJ2 transformed lines carried the Vf gene.

A further work identified the promoter sequence of the HcrVfl, 2 and 4 and demonstrated their functionality (Silfverberg-Dilworth et al., 2005b). Currently, it has been shown that Vfal (HcrVfl) and Vfa2 (HcrVJ2) under the control of their own promoter confer resistance to the V. inaequalis (Malnoy et al., 2008).

Use of defense-related gene. Currently, several research groups have demonstrated that the plants produce defense-related proteins, such as pathogenesis-related proteins (PR) and antimicrobial peptides. Constitutive expression of these molecules may enhance plant resistance. For instance, the puroindolines (PinA and PinB), antimicrobial peptides are antifungal cystein-rich proteins that are present in wheat seeds. Both genes are well characterized and they have already been used to transform rice. Transformant lines expressing these genes showed an increased resistance to major fungal pathogens in rice (Krishnamurthy et al., 2001). A similar experimental was set up to test the potential of PinB in apple. The results indicated that, the Strain 104, race 1 (which represents the common V. inaequalis population on the commercial cultivars), is not affected by the PinB at any expression level; however, the strain EU D42, race 6, is inhibited progressively with the increasing amount of PinB. The two strains exhibited differential tolerance to PinB (Faize et al., 2004).

On the other hand, other studies have demonstrated that constitutively high-level expression of PR proteins may protect susceptible cultivars from infection by different pathogens. Transcriptional analysis of apple susceptible and resistant to scab has demonstrated that in the resistant cv. Remo, the levels of transcripts encoding a number of proteins related to plant defense (such as β-1,3-glucanase, ribonuclease-like PR10, cysteine protease inhibitor, endochitinase, ferrochelatase, and ADP-ribosylation factor) or to detoxification in reactive oxygen species (such as superoxide dismutase) were highly up-regulated when compared their relative amounts with the susceptible cv. Elstar. Most surprising was the large number of clones derived from mRNAs for metallothioneins of type 3 found in the population of the resistant cv. Remo. However, the corresponding transcripts were only present in small amounts in young uninfected leaves of the susceptible cv. Elstar, but were up-regulated in this susceptible cultivar after inoculation with V. inaequalis (Degenhardt et al., 2005).

Other studies presented by the research group at Cornell University showed that over-expression of the apple MpNPR1 gene confers increased disease resistance in M. domestica. The NPR1 gene plays an important role in systemic acquired resistance in plants. An NPR1 homolog, MpNPR1-1, was cloned from M. domestica and overexpressed into two important apple genotypes, Galaxy and M26. Its over-expression in apple resulted in an increased disease resistance and elevated expression of pathogenesis-related (PR) genes. Transformed Galaxy lines over-expressing MpNPR1 had an increased resistance towards two important fungal pathogens of apple, V. inaequalis and Gymnosporangium juniperi-virginianae. Selected transformed lines have been propagated for evaluation in field trials for disease resistance and fruit quality (Malnoy et al., 2007a; 2007b).

Use of microbial genes. Since 1980s great efforts have been made in Europe, USA, and New Zealand to map the major scab resistance genes from V. inaequalis and mildew (P. leucotricha). These two fungal diseases adsorb the majority, if not the total of the fungicide treatments necessary to produce high-quality apples. However, until 1990s, once the apple transformation methodology was well established, no apple resistance gene had been cloned. Therefore, emphasis was put on foreign genes, with potentially toxic or inhibitory effects on V. inaequalis and mildew. This strategy includes the use of genes from other species encoding chitinases and glucanases, isolated from the fungus Trichoderma (a well-known biocontrol agent of fungal diseases), those encoding lytic enzymes from Lepidoptera, and some antimicrobial peptides (AMP) from phages.

In 1998, several studies reported that the constitutive expression of chitinolytic enzymes like endochitinase and chitobiosidase from the biological control agent Trichoderma harzianum, shown an antifungal activity and may increase the host resistance to scab (Wong et al., 1999). In this work, two out of three endochitinase (ech42) gene transgenic lines of `Royal Gala′ were more resistant than the untransformed `Royal Gala′ when micrografted shoots were spray-inoculated with scab. At the same time, it was also reported the transformation of the cv. McIntosh with the ech42 and Nag7O genes, and an exochitinase gene encoding N-acetyl-β-D-glucosaminidase, isolated from the biocontrol agent T. harzianum (Bolar et al., 1999). Transgenic lines with both genes (low each42 and high Nag7O expression) had a high level of scab resistance, while retaining a good vigor (Noreffi et al., 2000).

Other studies reported by the research group at Cornell University showed that transfer of native (attacin E) and synthetic (SB-37) genes from the saturniid moth (Hyalophora cecropia), lysozyme genes from hen egg white, and T4 bacteriophage, all enhanced scab resistance to scab in a variable degree (Aldwinckle et al., 1999). A similar approach started to use the Fruit Breeding at Dresden Pillnitz, Germany, where they developed several apple transgenic lines transformed with the lysozyme, from the bacteriophage T4, and/or attacin E, from saturniid moth. However, this program concentrated its efforts on the subsequent effects on fire blight infections (Hanke et al., 2000).

Types of promoters. For gene expression, in many cases researchers relied on the well characterized constitutively expressed 35S promoter from Cauliflower mosaic virus (CaMV 35S). Some attempts have been made to find and use other promoters. For instance, promoters linked to targeted expression patterns have been identified such as the 940 extA promoter which is active in young tissue (Gittins et al., 2001), RBCS3C and SRS1, suitable for the expression of transgenes in green photosynthetic tissue of apple (Gittins et al., 2000), a native promoter for the apple scab resistance gene HcrVJ2 (Szankowski et al., 2008), and others promoters with particular expression patterns that are under development and characterization (Degenhardt and Szankowski, 2006).

Limitation of the technology and new development

Although considerable improvement has been gained in the process of transformation in apple, the use of antibiotics and herbicides as selectable markers still imposes a limitation according to the consumer acceptance (Penna et al., 2002; Degenhardt and Szankowski, 2006). Thus, a major problem for genetically modified (GM) apple is the use of the nptll as a selection gene-marker. Recently, some groups started to develop new selection systems that are more acceptable to the general public that include the elimination of the selectable markers from the final product, the transgenic plants.

Currently, one of the most promising alternative to antibiotic resistance and probably a more acceptable system is the use of the gene phosphomanose isomerase (PMI). PMI-transformed cells are able to use mannose as carbon source, which the untransformed apples cells cannot do and first successes using this technology have already been reported (Flachowsky et al., 2004; Zhu et al., 2004; Degenhardt and Szankowski, 2006).

Clearly the development of a "clean vector technology for marker-free transgenic" in apples and other crops is the ultimate goal. The Plant Research International in The Netherlands recently developed and proposed the use of such technology (Schaart, 2004; Schaart et al., 2004; Krens et al., 2004). This new technology allows obtaining transgenic apple plants free from the selection marker genes.

Acceptance and public perception of genetic modification

Transgenic plants. Almost all apple transformation reports reviewed, with few exceptions, relied on the selection of a foreign gene nptll, with a non-apple gene promoters (CaMV 35S, among others) and the utilization of A. tumefaciens. However, for experimental purposes, genes not influencing the target trait have been also tested, such as the gene producing β-glucuronidase (GUS).

To our knowledge, no environmental risk studies specific to transformed apple have been published. Probably researchers are still concerned with producing acceptable GM apple cultivars with commercial interest and having environmental benefits, such as reduction of pesticides use (James et al., 2003). Under these circumstances, the commercialization of transgenic apple carrying a DNA from different species or genera, in the near future is certain.

A recent multidisciplinary EU project entitled `Sustainable production of transgenic strawberry plants: Ethical consequences and potential effect on producers, environment and consumers′ was published (Iversen, 2003).

This project surveyed and gathered the opinion regarding the attitude of the consumers on the use or consumption of genetically transformed plants. The attitude of the consumers in Norway, Denmark and the UK towards genetic modification was rather negative, but for the type of genetically modified strawberry plants and the traits involved in the transformation process. For instance, consumer acceptance increased when the modified trait was perceived beneficial to them and when the own strawberry DNA was utilized to transform the plant. A recent consumer survey in the USA showed that a majority of the respondents would eat vegetables containing a gene from the same species (81%), or from another vegetable species (61%), in comparison to those from other sources like viral genes (14%) (Lusk and Sullivan, 2002).

Sociological studies (Iversen, 2003) showed that public perception of GM crop is driven by emotions, rather than an open discussion on the advantages or possible limitations in each case.

In order to fully utilize the possibilities of genetic modifications for crop improvement, it would be interesting to discuss what is the minimal genetic distance required for the genome manipulation to ensure sufficient public acceptance. Until now, there is no public concern when breeders make crosses within o between genotypes that belong to the same species, including wild and domesticated germplasm. In several cases, conventional breeders utilize germplasm coming from gene pool I, genotypes closely related where crosses do not present any biological limitations), or from gene pool II; where breeders use some techniques such embryo culture to obtain fertile offspring when cross genotypes more distantly related or protoplast fusion when they need to obtain a progeny from different species.

Cisgenic plants. In order to increase acceptance of the genetically modified plants by consumers, a group of researchers at Plant Research International (Wageningen University) and at Research Centre in The Netherlands developed a new series of biotechnological strategies to reduce the limitations of the conventional genetic engineering tools available (Schaart, 2004; Schaart et al., 2004; Krens et al., 2004).

This innovation consisted in the use of genes from the same species or closely related species, along with their own native promoters and the markerless DNA transformation technology, where no selectable marker, like antibiotics and herbicide resistance genes are used to select the transformed lines (Schouten et al., 2006a; 2006b). All these innovations are expected to facilitate the acceptability and commercialization of genetically engineered plants by consumers, growers, and regulatory agencies. This new innovation was named as "Cisgenesis", and it is considered as a friendly technology and an excellent strategy to improve plant resistance and to complement conventional breeding programs (Schouten et al., 2006b; Jacobsen and Schouten, 2007; Haverkort et al., 2008; Schouthen and Jacobsen, 2008).

To date, the majority of established regulations on genetically modified organisms (GMOs) worldwide have not discriminated cisgenic from transgenic plants. This may be because until now, few cisgenic plants have been developed and submitted for approval. Canada is one of the countries that has a product-based regulation rather than a process-based regulations, therefore, it is possible that cisgenic plant could be treated less stringently than transgenic plants (Schouten et al., 2006b).

Cisgenic plants are fundamentally different from transgenic plants. In the case of transgenesis, a foreign gene, from different species or genera, is introduced into a plant. Therefore, it is postulated that a transgenic plant has a phenotypic trait that did not occur in the species (wild and domesticated) and could affect its fitness through the traits itself or by the gene flow from the domesticated transformed plant to its wild relatives (Schouten and Jacobsen, 2008).

In contrast, in cisgenesis the introduced gene of interest with its native promoter has already been present in the domesticated or wild species for centuries. Therefore, cisgenesis does not add an extra trait to the species. It does not invoke a fitness change that could not also occur through traditional breeding or in nature. The same holds true for other environmental risks, such as effects on non target organisms or soil ecosystems, and for usage in food or feed. As a result, deliberate release of cisgenic plants into the environment could be as safe as the deliberate release of traditionally bred plants (Jacobsen and Schouten, 2008).

Nowadays, modern fruit production and a higher consumer demand require cultivars with a better productivity, uniformity, long-term storage, resistance to diseases and pests and good quality to ensure a commercial success. In Chile, apple is one of the most important fruit exported to different markets and it plays a major economic and social role in the agricultural sector and in Chilean economy. Unfortunately, pests and diseases are one of the major factors that are limiting the potential growing of this industry.

Based on those facts, use of such cisgenic apples plants could aid to develop a new way of sustainable crop production practices. Recently, the Instituto de Investigaciones Agropecuarias INIA Quilamapu started to develop a platform to produce cisgenic Royal Gala and Granny Smith apple cultivars to improve their resistant to V. inaequalis.

Conclusions

Nowadays, a high market demand apple cultivars with high productivity and fruit quality, and a reduced pesticide application and recent advances in plant biotechnology could help to satisfy this consumer demand.

Transformation technology in apple has a long history but it practical application has been limited by the consumer acceptance. The development of a new "clean vector technology", the sequencing of gene controlling agronomic traits, under the control of their own promoters will allow to produce transformed plants with better consumer acceptance.

Applications of this technology could support and complement the apple breeding program in the development of a more sustainable crop apple production practices and to improve its productivity and competitiveness in the world market.^[110]

References

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