Top Fruit

17.2.1.1 Malus Species (Apple)

The domesticated apple Malus domestica Borkh. is the most important pome-fruit worldwide. It ranked fourth within the fruit crops in 2006 behind bananas, grapes and citrus, with approximately 64 million t produced on 4.7 million ha (http://faostat.fao.org).

The genus Malus (apples) comprises approximately 55 species, including M. domestica Borkh. and numerous wild species (Phipps et al. 1990). Only the domesticated apple is economically important. All Malus species are grouped into infrageneric groups (section, series) and each species can be divided into intraspe-cific groups (cultivars; Harris et al. 2002; Qian et al. 2006). The taxonomy within the genus Malus is problematic, because of the intimate association that humans have with apples. Sometimes it is not as easy to distinct between wild and cultivated apples and hence between distinct categories (Harris et al. 2002).

Genetic transformation in apple and apple rootstocks was recently summarized by Bulley et al. (2007), Gessler and Patocchi (2007) and Dolgov and Hanke (2006). For general aspects of transformation see Chap. 1. The first report on apple transformation was published in 1989 by James et al. Since that time many protocols have been described for the transformation and regeneration of at least 24 apple scion cultivars, 12 rootstock cultivars and three apple wild species (Bulley et al. 2007; Dolgov and Hanke 2006; Szankowski et al. 2009). Initial studies were often focused on the improvement of the transformation efficiency by testing different transformation technologies using particle bombardment (Gercheva et al. 1994), Agrobacterium tumefaciens (James et al. 1989) and A. rhizogenes (Lambert and Tepfer 1991, 1992), respectively. Other studies were focused on the effect of different antibiotics on the regeneration, proliferation and morphogenesis of transgenic tissue (Norelli and Aldwinckle 1993; Yepes and Alwinckle 1994a, b). Different tissue, like leaf blades, stem internode explants or shoot apices were also tested to find out the best starting material for plant regeneration (Liu et al. 1998; Caboni et al. 2000). The most effective and reproducible method for apple regeneration has remained through adventitious shoot formation. In most cases in vitro plant leaves were inoculated with A. tumefaciens strains containing binary plasmid vectors with chimeric gene constructs. The transformation efficiency ranged between 0.02% and 20% (Bulley et al. 2007), depending on the apple genotype used for transformation, the type of tissue, the transformation method and the A. tumefaciens strain.

The selection of transgenic apple plants is still performed by using the nptII selectable marker gene and kanamycin as selective agent (see also Chap. 3). Only a few studies have been published that focus on alternative selection strategies. The bar gene of Streptomyces hygroscopicus, which confers resistance to phosphino-thricin, was used several times (de Bondt et al. 1996; Dolgov and Skryabin 2004; Lebedev et al. 2002; Szankowski et al. 2003). The manA gene of Escherichia coli was also tested on apple (Degenhardt et al. 2006, 2007; Flachowsky et al. 2004; Zhu et al. 2004). The manA gene encodes for a phosphomannose-isomerase that catalyses the conversion of mannose-6-phosphate to fructose-6-phosphate. Transgenic cells expressing the manA gene can utilize mannose as a carbon source. Although it is good to have alternative selectable marker genes, especially in view of future field trials, the ultimate aim is a marker-free transgenic plant (Gessler and Patocchi 2007). Marker-free transgenic plants can be produced by using clean vector technologies or by transformation without marker genes. A first study using a clean vector system was recently published on apple (Krens et al. 2004). The transformation without the use of marker genes has been reported twice (Flachowsky et al. 2004; Malnoy et al. 2007a), but with different results. However, both technologies offer the possibility to produce marker-free GM plants.

Beside the establishment of the transformation technology, work has also focused on the improvement of agronomical important traits like resistance to biotic and abiotic stress and herbicides. Other studies have been engaged with self fertility, dwarfing, rooting ability or precocity. Furthermore much work was done to improve traits, which are related to the fruit (Table 17.1).

Much effort has been made to improve the resistance to fire blight caused by Erwinia amylovora. Initial studies were focused on transgenic expression of antimicrobial peptides (AMPs). In apple the AMPs attacin, lysozymes, and cecropin analogs were used. The results of these studies were summarized by Norelli et al. (1999). The best fire blight resistance was thereby observed with attacin E transgenic plants. However, the acceptance by growers and consumers of such GM plants is low, because of the bacterial and animal origin of the transferred genes (Norelli et al. 2003). Recent studies on GM apple plants were mainly focused on promoting plant defense reactions. The theory of this approach is based on the fact that E. amylovora secretes effector proteins into plant host cells, which are involved into suppressing host defense responses, redirecting normal host metabolism to facilitate pathogen multiplication and initiating cell necrosis. Different effector proteins (e.g. hrpN, eop1, hopCEa) were overexpressed in apple to simulate an infection and to induce defense mechanisms. Other strategies were based on silencing of apple genes (e.g. HIPM, DIPM), which interact with the effector proteins or on overexpression of apple genes (e.g. MdNPR1, mbr4) involved in the pathogen defense (for method, see Chap. 5). All strategies were successful, but most acceptable to consumers and growers are likely to be alterations in the expression of apple genes, resulting in enhanced resistance. No fire blight resistance gene has yet been isolated from resistant apple wild species until now, but several scientific groups are working on it.

Similar strategies as for fire blight were used to improve the resistance to apple scab. Beside genes of the biocontrol organism Trichoderma harzianum, AMPs from

Table 17.1 Summary on studies conducted on the transformation of apple and apple rootstocks for agronomically important traits. Overexpr. Overexpression

Selected trait

Genes used

Type of expression

Reference1

Insect resistance E. postvittana pPLA2 (chicken avidin) Overexpr.

pSAV2a (S. avidinii Overexpr.

  1. Na-PI (N. alata) Overexpr.
  2. pomonella

CpTI (V. unguiculata) Overexpr.

cryIA(c) Overexpr.

(B. thuringiensis) Fungal resistance V. inaequalis

MpNPR1 Overexpr.

(M. domestica) ech42 (T. harzianum) Overexpr.

Nag 70 (T. harzianum) Overexpr.

SB-37 (H. cecropia) Overexpr.

AttE (H. cecropia) Overexpr.

HEWL (hen egg white Overexpr.

lysozyme)

T4 lysozyme Overexpr.

(bacteriophage T4)

Rs AFP2 (R. sativus) Overexpr.

Ace AMP1 (A. cepa) Overexpr.

PinB (T. aestivum) Overexpr.

HcrVf1, 2 and 4 Overexpr.

  • M. floribunda) G. juniperi-virginianae MpNPR1 Overexpr.
  • M. domestica) Bacterial E. amylovora resistance SB-37 (H. cecropia) Overexpr.

Shiva 1 (H. cecropia) Overexpr. AttA, AttE (H. cecropia) Overexpr.

T4 lysozyme Overexpr.

(bacteriophage T4)

Gessler and Patocchi (2007),

Bulley et al. (2007) Gessler and Patocchi (2007),

Bulley et al. (2007) Maheswaran et al. (2007)

Gessler and Patocchi (2007),

Bulley et al. (2007) Gessler and Patocchi (2007), Bulley et al. (2007)

Bulley et al. (2007), Malnoy et al. (2007b) Gessler and Patocchi (2007),

Bulley et al. (2007) Gessler and Patocchi (2007),

Bulley et al. (2007) Gessler and Patocchi (2007),

Bulley et al. (2007) Gessler and Patocchi (2007) Gessler and Patocchi (2007)

Gessler and Patocchi (2007)

Gessler and Patocchi (2007), Bulley et al. (2007), Dolgov and Hanke (2006) Gessler and Patocchi (2007),

Bulley et al. (2007) Gessler and Patocchi (2007),

Bulley et al. (2007) Gessler and Patocchi 2007, Bulley et al. 2007

Gessler and Patocchi (2007), Bulley et al. (2007), Dolgov and Hanke 2006) Bulley et al. (2007), Dolgov and Hanke (2006) Gessler and Patocchi (2007), Bulley et al. (2007), Dolgov and Hanke (2006) Gessler and Patocchi (2007), Bulley et al. (2007), Dolgov and Hanke (2006) (continued)

Table 17.1 (continued)

Selected trait

Genes used

Type of expression

Reference1

HEWL (hen egg white

Overexpr.

Gessler and Patocchi 2007,

lysozyme)

Dolgov and Hanke 2006

Dpo (bacteriophage

Overexpr.

Gessler and Patocchi (2007),

FEalh)

Bulley et al. (2007),

Dolgov and Hanke

(2006), Flachowsky et al.

(2008b)

hrpN (E. amylovora)

Overexpr.

Gessler and Patocchi (2007),

Bulley et al. (2007)

eop1 (E. amylovora)

Overexpr.

Lalli et al. (2008)

hopCEa (E. amylovora)

Overexpr.

Lalli et al. (2008)

DspF (E. amylovora)

Overexpr.

Malnoy et al. (2008a)

MpNPR1 (M.

Overexpr.

Gessler and Patocchi (2007),

domestica)

Bulley et al. (2007),

Malnoy et al. (2007b)

mbr4 (M. baccata)

Overexpr.

Flachowsky et al. (2008c)

DIPM (M. domestica)

Silencing

Gessler and Patocchi (2007),

Bulley et al. (2007)

HIPM (M. domestica)

Silencing

Malnoy et al. (2008b)

A. tumefaciens

iaaM, ipt

Silencing

Bulley et al. (2007)

(A. tumefaciens)

Stress resistance

Heat, drought, cold and

UV-B

Cytosolic ascorbate

Overexpr.

Bulley et al. (2007)

peroxidase (pea)

Osmyb4 (O. sativa)

Overexpr.

Pasquali et al. (2008)

Zinc deficiency

ZNT1

Overexpr.

Swietlik et al. (2007)

ZIP4

Overexpr.

Swietlik et al. (2007)

Iron deficiency

LeIRT2 (L. esculentum)

Overexpr.

Bulley et al. (2007)

Herbicide

Basta

resistance

bar (S. hygroscopicus)

Overexpr.

Gessler and Patocchi (2007),

Bulley et al. (2007),

Dolgov and Hanke (2006)

Self fertility

S3, S5 (S-alleles of

Silencing

Gessler and Patocchi (2007),

M. domestica)

Bulley et al. (2007)

Dwarfing and

PhyB (A. thaliana)

Overexpr.

Bulley et al. (2007), Dolgov

rooting

and Hanke (2006)

ability

rolA, rolB, rolC

Overexpr.

Gessler and Patocchi (2007),

(A. rhizogenes)

Bulley et al. (2007),

Dolgov and Hanke (2006)

GA 20-oxidase

Silencing

Bulley et al. (2007)

(M. domestica)

gai (A. thaliana)

Overexpr.

Dolgov and Hanke (2006),

Zhu et al. (2008)

Precocity

BpMADS4 (B. pendula)

Overexpr.

Bulley et al. (2007), Hanke

et al. (2007), Flachowsky

et al. (2009)

AP1 (A. thaliana)

Overexpr.

Hanke et al. (2007),

Flachowsky et al. (2009)

  • continued)
  • continued)

Table 17.1 (continued)

Selected trait Genes used Type of Reference1

expression

Table 17.1 (continued)

Selected trait Genes used Type of Reference1

expression

LFY (A. thaliana)

Overexpr.

Hanke et al. (2007),

Flachowsky et al. (2009)

MdMADS5

Overexpr.

Hanke et al. (2007),

(M. domestica)

Flachowsky et al. (2009)

MdAP1 (M. domestica)

Overexpr.

Hanke et al. (2007),

Flachowsky et al. (2009)

AFL1 and AFL2

Overexpr.

Hanke et al. (2007),

(M. domestica)

Flachowsky et al. (2009)

MdTFL1 (M. domestica)

Silencing

Bulley et al. (2007),

Flachowsky et al. (2009),

Hanke et al. (2007)

Traits related to Flavor

the fruit Thaumatin II protein

Overexpr.

Bulley et al. (2007), Gessler

(T. danielli)

and Patocchi (2007)

S6PDH (M. domestica)

Silencing

Bulley et al. (2007), Gessler

and Patocchi (2007)

Fruit ripening

ACS (M. domestica)

Silencing

Bulley et al. (2007), Gessler

and Patocchi (2007)

ACO (M. domestica)

Silencing

Bulley et al. (2007), Gessler

and Patocchi (2007)

MdPG (M. domestica)

Silencing

Bulley et al. (2007)

Reduced browning potential

PPO (M. domestica)

Silencing

Bulley et al. (2007), Gessler

and Patocchi (2007)

Color and health properties

Vst1 (V. vinifera)

Overexpr.

Bulley et al. (2007)

Lc (Z. mays)

Overexpr.

Li et al. (2007)

MdMYB10 (Malus ssp.)

Overexpr.

Bulley et al. (2007)

MdANS (Malus ssp.)

Silencing

Szankowski et al. (2009)

Allergens

Mal d1 (M. domestica)

Silencing

Bulley et al. (2007), Gessler

and Patocchi (2007)

References include review articles (if available) and original research articles

References include review articles (if available) and original research articles insects and other plant species as well as genes involved in the pathogen defense were expressed in apple. In contrast to fire blight, the first scab resistance gene of apple (HcrVf2 of M. floribunda 821) has been isolated and transformed (Gessler et al. 2009). This gene should now be used in combination with a clean vector system to produce cisgenic scab-resistant apple varieties.

Another promising strategy was recently published by Flachowsky et al. (2009). A breeding program has been established at the Institute of Breeding Research on Horticultural and Fruit Crops (Dresden-Pillnitz, Germany), which uses transgenic early flowering apple plants (Fig. 17.1) to speed-up breeding cycles. Using this system one crossbred generation per year is feasible. This system can be used to introgress resistance genes from apple wild species into the cultivated apple by natural crossing, to realize several backcross generations in a few years and to

Bpmads4 Early Flowering Flachowsky
  1. 17.1 BpMADS4 transgenic apple seedling. First flowers were obtained approximately four months after seeding. The seedling was obtained after crossing a F1 plant of the cross T1190 (BpMADS4 transgenic line of the apple cv. 'Pinova'; Flachowsky et al. 2007) by M.fusca (fire blight resistant apple wild species) and the scab resistant apple cv. 'Topaz'
  2. 17.1 BpMADS4 transgenic apple seedling. First flowers were obtained approximately four months after seeding. The seedling was obtained after crossing a F1 plant of the cross T1190 (BpMADS4 transgenic line of the apple cv. 'Pinova'; Flachowsky et al. 2007) by M.fusca (fire blight resistant apple wild species) and the scab resistant apple cv. 'Topaz'

produce a new and highly resistant apple cultivar, free from any transgenic sequences, within a manageable amount of time.

With the focus on biosafety research several studies have been performed on transgene stability (Briviba et al. 2004; Flachowsky et al. 2008a; Reim and Hanke 2004; Zhu et al. 2007), inheritance of the transgenic trait (Flachowsky et al. 2009; James et al. 1995), pollen fertility (Du et al. 2007) and gene flow (Reim et al. 2006; Soejima et al. 2007).

Since the early 1990s, many field trials with GM apples have been performed worldwide, but no GM apple cultivar is yet on the market, neither in the United States nor in Europe.

A total of 47 field test records were found for the UnitedStates within the Environmental Releases Database (http://www.isb.vt.edu/cfdocs/fieldtests1.cfm).

Most of these were focused on GM apple plants with improved fruit quality (fruit ripening, low-browning, storability) or improved resistance to insects or bacterial and fungal diseases. Twelve out of the 47 field test records were focused on plants with altered polyphenol oxidase (PPO) levels. Especially Okanagan Specialty Fruits (Summerland, B.C., Canada) is working on the development of new apple varieties by genetic modification. This company has developed a low-browning GM apple for processing and apple chip production through silencing of PPO genes. The commercialization of the first PPO silenced low-browning apple cultivar is planned for 2009/2010 (http://www.okspecialtyfruits.com).

In Europe, releases of GM plants have to be notified according to Directive 2001/18/EC. A total of nine summary notifications can be found for apple (http:// bgmo.jrc.ec.europa.eu/deliberate/dbplants.asp): four from the Netherlands, two from Belgium, two from Sweden and one from Germany. GM apples are still quite a long way from commercial use in Europe.

However, scientific groups in Italy and in the Switzerland are working on the development of cisgenic (see Chaps. 4, 6) apple plants with improved resistance to apple scab using the HcrVf2 resistance gene from the crab-apple Malus floribunda 821. A nearly identical project was started in the Netherlands. The Plant Research International (PRI), in cooperation with the private fruit breeding company Inova Fruit BV and two further partners, is also working on the development of HcrVf2 cisgenic apple varieties. Both projects are promising, but the commercialization of the first cisgenic apple cultivar in Europe is expected in 2013 at the earliest. Further objectives of ongoing projects are the development of low allergenic GM apple cultivars by silencing the major apple allergen Mal d1 and cisgenic apple cultivars with an increased amount of healthy compounds through up-regulation of the flavonoid biosynthesis using the recently identified MYB10 transcription factor of apple.

17.2.1.2 Pyrus Species (Pear)

The genus Pyrus belongs to the subfamily Maloideae of the Rosaceae and comprises at least 22 species, which are distributed in East and West Asia, Europe and Africa. Commercially used are mainly the two pear species P. communis L., the European pear, and P. pyrifolia (Burm.) Nakai (= P. serotina Rehder), the Japanese pear or 'nashi' (Katayama and Uematsu 2003; Monte-Corvo et al. 2000; Oliveira et al. 1999). Pears ranked at eight within the fruit crops in 2006 with about 20 million t produced on 1.7 million ha (http://faostat.fao.org). More than 80 countries produce pears, with China, Italy, USA, Spain and Argentina as the main producers. The lion's share (60%) of the world's production is produced in China.

Since the beginning of pear transformation many protocols have been published for Agrobacterium-mediated transformation and transgenic plant regeneration from in vitro leaves (Merkulov et al. 1998; Mourgues et al. 1996; Yancheva et al. 2006), axillary shoot-meristem explants (Matsuda et al. 2005) and cotyledons (Kaneyoshi et al. 2001).

The selection of transgenic pear plants is still performed by using the nptll selectable marker gene. A few other marker genes like the bar gene of Streptomyces hygroscopicus or the Vr-ERE of Vigna radiata have been tested on pear, but their efficiency was too low (Chevreau et al. 2007; Lebedev et al. 2002a). An increased transformation frequency compared to nptll was found in transformation experiments on a pear rootstock using the hpt gene for selection, which confers resistance to hygromycin (Lebedev and Dolgov 2000). However, the dream for the future is a GM pear plant which is free from any marker genes.

Equally important as the establishment of highly efficient transformation protocols on different pear species and cultivars are studies which were focused on the improvement of agronomical important traits (Table 17.2). As in apple, pears are affected by fire blight. Improvement of fire blight resistance is therefore one of the most important aims of research. Beside this, the resistance to abiotic stress and fungal diseases, the improvement of flavor and the delay of maturity are also traits of interest. Dwarfing and improvement of rooting ability are traits, which are of importance for pear rootstocks.

Since the early 1990s, several field trials with GM pears have been performed worldwide. A total of five field test records were found for the United States within the Environmental Releases Database (http://www.isb.vt.edu/cfdocs/fieldtests1. cfm). These field test records were focused on GM pears with altered fruit ripening and improved resistance to fire blight.

Table 17.2 Summary on studies conducted on the transformation of Pyrus for agronomically important traits

Selected trait

Genes used

Type of expression

Reference1

Fungal resistance

Rs-AFP2 (R. sativus)

Overexpr.

Lebedev et al. (2002b)

Fire blight

attE (H. cecropia)

Overexpr.

Petri and Burgos (2005)

resistance

SB-37 (H. cecropia)

Overexpr.

Reynoird et al. 1999

Shiva-1 (H. cecropia)

Overexpr.

Mourgues et al. 1999

bovine lactoferrin

Overexpr.

Malnoy et al. 2003

dpo (bacteriophages

Overexpr.

Malnoy et al. 2005a

FEalh) hrpN

Overexpr.

Malnoy et al. 2005b

(E. amylovora) hrpN

Overexpr.

Malnoy et al. 2008

(E. amylovora)

Traits related to the

Flavor: thaumatin II

Overexpr.

Lebedev et al. (2002c)

fruit

protein (T. danielli)

Fruit ripening: ACO

Overexpr.

Gao et al. (2007)

(pear)

Silencing

Murayama et al. (2003)

Health properties: Vst1

Overexpr.

Flaishman et al. (2005)

(V. vinifera)

Abiotic stress

MdPDS1 (M. domestica)

Overexpr.

Wen et al. (2008), He

resistance

et al. (2008)

Dwarfing and

rolB, rolC

Overexpr.

Petri and Burgos (2005)

rooting ability

(A. rhizogenes)

References include review articles (if available) and original research articles

References include review articles (if available) and original research articles

For Europe only one summary notification was found for pear, which was carried out in Sweden with plants with improved rooting ability (http://bgmo.jrc.ec.europa. eu/deliberate/dbplants.asp). GM pears are still quite a long way from commercial use. A commercial use of GM pears is not to be expected for the next years.

17.2.1.3 Prunus Species (Almond, Apricot, Sweet and Sour Cherry, Cherry Rootstocks, Peach, Plum)

The genus Prunus comprises several important stone fruit and nut species, including almond (P. dulcis Mill.), apricot (P. armeniaca L.), sweet and sour cherry (P. avium L. and P. cerasus L.), peach [P. persica (L.) Batsch] and plum (P. domestica L., P. salicina Lindl.). The economically most important stone fruit species are peach and plum, which ranked 10th and 12th within the fruit crops in 2006, with approximately 18 million t and 9.7 million t produced on 1.5 million ha and 2.3 million ha, respectively (http://faostat.fao.org).

Since the early 1990s many protocols have been published for the regeneration of adventitious shoots from various Prunus explants. Good overviews about this work are given by Burgos et al. (2007) and Canli and Tian (2008). Protocols for regeneration from leaf tissue are available for plum, almond, sour and sweet cherry as well as for wild cherry and black cherry. Regeneration of adventitious shoots from immature cotyledons was described for apricot, peach, plum, sour cherry and almond. Further studies have described the regeneration of peach, sweet and ornamental cherries using mature cotyledons or the regeneration of plum using hypocotyl slices (Burgos et al. 2007; Canli and Tian 2008).

Genetic transformation of Prunus species was recently summarized by Burgos et al. (2007), Petri and Burgos (2005) and Scorza and Ravelonandro (2006). Protocols for genetic transformation have been reported for plum (Mante et al. 1991; Petri et al. 2008a), almond (Miguel and Oliveira 1999), sour cherry (Dolgov and Firsov 1999; Song and Sink 2005), apricot (Laimer da Camara Machado et al. 1992; Petri et al. 2008b) and peach (Padilla et al 2006; Perez-Clemente et al. 2004; Wu et al. 2006). The selection of GM Prunus plants was mostly performed using the nptII or the hpt gene as selectable marker genes, which confer resistance to kanamycin and hygro-mycin, respectively (Burgos et al. 2007). Only a few studies have been published that focused on alternative selection strategies such as the bar gene of Streptomyces hygroscopicus (Druart et al. 1998) or the manA gene of Escherichia coli (Ramesh et al. 2006). The mannose/pmi selection system was tested on almond and compared to the traditionally used npt/7/kanamycin selection system (Ramesh et al. 2006). The transformation efficiency was higher with the mannose/pmi system (5.6% for kanamycin, 6.8% for mannose/pmi), which led to the conclusion that the mannose/ pmi system could be a usable tool to avoid the npt/7/kanamycin selection system.

Most studies on GM Prunus plants have been concerned with the establishment and improvement of regeneration and transformation protocols using Agrobacterium tumefaciens. A few studies have been performed on Prunus rootstocks using A. rhizogenes for transformation (Gutierrez-Pesce et al. 1998;

Table 17.3 Summary on selected studies conducted on the transformation of Prunus for agronomically important traits

Selected trait

Genes used

Species

Type of expression

Reference1

Virus

Sharka resistance (plum

Apricots

Overexpr.

Petri and Burgos (2005),

resistance

pox virus)

Scorza and Ravelonandro (2006)

cpPPV (plum pox virus)

Plum

Overexpr.

Petri and Burgos (2005)

PRSV (papaya ring-spot

Plum

Overexpr.

Petri and Burgos (2005)

virus)

Cold resistance

afp (P. americanus)

Sweet cherry

Overexpr.

Petri and Burgos (2005)

Herbicide

bar (S. hygroscopicus)

Cherry

Overexpr.

Petri and Burgos (2005)

resistance

rootstocks

Altered habit

ipt (A. tumefaciens)

Peach

Overexpr.

Petri and Burgos (2005)

Nematode

Meloidogyne incognita

Plum

Overexpr

Nagel et al. (2008)

resistance

gafp-1 (antifungal protein from G. elata)

Delayed

ACO (P. persica)

Peach

Silencing

Wu et al. (2006)

maturity

Functional

PDS (P. armeniaca)

Plum

Silencing

Petri et al. (2008a)

genomics

  • References include review articles (if available) and original research articles
  • References include review articles (if available) and original research articles

Gutierrez-Pesce and Rugini 2004). Nevertheless, several studies were published, which were focused on the production of GM plants with improved agronomically important traits using A. tumefaciens-mediated transformation. Traits of interest were virus, nematode or cold resistance, herbicide resistance and altered tree habit. GM plants were also produced with focus on studies of gene functions in the field of functional genomics (Table 17.3).

The agronomically most important trait in Prunus is virus resistance. Especially the Plum pox virus (PPV), the etiological agent of sharka, is one of the most devastating diseases of stone fruits. The sharka disease is responsible for extensive economic losses (Nemeth 1994; Roy and Smith 1994) and the PPV virus has quarantine status in many countries (Scorza and Ravelonandro 2006). Alone in Europe there are about 100 million stone fruit trees currently infected with the virus (Kegler and Hartmann 1998). The breeding of sharka resistant Prunus trees is not as easy because of the polygenic and strain-specific nature of the PPV resistance and the long juvenile period of seedlings. Genetic engineering offers an exciting tool to overcome these problems and during the past decade several studies have been published in which researchers have tried to introduce resistance to PPV into apricots and plums via direct gene transfer.

Previous studies were performed on GM apricot and plum plants overexpressing coat proteins of the Plum pox virus (Laimer da Camara Machado et al. 1992; Scorza et al. 1994) or the papaya ring-spot virus (Scorza et al. 1995a, b). From GM plum plants overexpressing the PPV coat protein, the line C5 (today named 'Honey Sweet') was selected, because of the high level of resistance. This line contains a multicopy insert of the cpPPV gene. The expression level of this gene is reduced in

C5 as a follow of post-transcriptional gene silencing (PTGS, reviewed by Scorza and Ravelonandro 2006; see also Chap. 5). Based on inoculation studies, it was found that C5 is highly resistant to the major serotypes of PPV. The stability and durability of the PTGS-based PPV resistance of C5 was tested in field trials in different countries for several years (Fuchs et al. 2007; Hily et al. 2004; Malinowski et al. 2006). Based on the results obtained from the numerous studies on C5, Scorza and Ravelonandro (2006) concluded that PTGS-based strategies could be used in future approaches to produce PPV-resistant stone fruits.

First PTGS-based strategies for resistance to PPV were recently tested in heterologous systems (Nicola-Negri et al. 2005; Zhang et al. 2006). Different PPV-specific hairpin constructs were developed and evaluated for their effect on the PPV resistance in transgenic N. benthamiana plants. The majority of the transgenic lines were significantly less susceptible than control plants. The silencing constructs will now be tested on different Prunus species. A similar strategy to induce multivirus resistance in Prunus was recently published by Liu et al. (2007). The authors created a chimeric gene (PTRAP6) by fusion of gene fragments (400-500 bp) from six major Prunus fruit viruses (American plum line pattern virus, peach mosaic virus, plum pox virus, prune dwarf virus, prunus necrotic ringspot virus, tomato ringspot virus). Using this chimeric gene, a hairpin construct (PTRAP6i) was developed and constitutively overexpressed in N. benthamiana plants. Tests on transgenic plants of homozygous R3 generation lines with three out of the six viruses presented evidence that transgenic expression of PTRAP6i could be a powerful tool to produce virus-resistant Prunus fruit trees.

Since the early 1990s several field trials with GM plums and cherries have been performed. For the United States a total of seven field test records were found for GM plums within the Environmental Releases Database (http://www.isb.vt.edu/ cfdocs/fieldtestsl.cfm). These field tests were focused on plants with a reduced juvenile stage, delayed maturity, improved resistance to nematodes or fungal and virus diseases. A first petition for deregulation of a GM fruit tree was approved in the United States in June 2007. The Animal and Plant Health Inspection Service (APHIS) of the United States Department of Agriculture (USDA) excluded the GM plum line C5 'Honey Sweet' from the regulations at 7 CFR part 340 (http://www. isb.vt.edu/cfdocs/fieldtests1.cfm).

In Canada two field trials have been performed with GM cherries, which were focused on trees with improved fruit quality (http://www.gmo-compass.org/eng/ home/).

In Europe, a total of five and three summary notifications can be found for GM plums and cherries, respectively (http://bgmo.jrc.ec.europa.eu/deliberate/dbplants. asp). Field trials with GM plums were performed in Spain (two), in Poland (one), in the Czech Republic (one) and in Romania (one). All plum field trials were focused on PPV resistant plants. Three field trials with GM cherries have been performed in Italy. These field trials were carried out with plants with a better rooting ability. However, GM plums and GM cherries are still quite a long way from commercial use in Europe. A commercial use of GM plums and GM cherries in Europe is not to be expected in the long run.

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