Small Fruit

17.2.2.1 Fragaria Species (Strawberry)

The genus Fragaria consists of approximately 20 species. The majority of these species are diploid. Commercially important is the octoploid species Fragaria x ananassa Duch. (Hadonou et al. 2004; Sargeant et al. 2003). In 2006 approximately 3.9 million of strawberry fruits were produced on 263 000 ha worldwide (http:// faostat.fao.org).

The cultivated strawberry (F. x ananassa) is a rapidly growing herbaceous perennial with a small genome, short reproductive cycle and facile vegetative and generative propagation for genetic transformation. The development of in vitro regeneration systems using a range of explants, including leaves, petioles, peduncle tissue, sepals, stipules, roots, runners, ovaries, protoplasts, stems and callus, and culture conditions has opened up the opportunity for strawberry improvement through genetic engineering (summarized by Mercado et al. 2007b).

Recently, the state of the art in strawberry transformation was reviewed by several authors (Debnath and Teixeira da Silva 2007; Folta and Davis 2006; Folta and Dhingra 2006; Graham 2005; Qin et al. 2008; Quesada et al. 2007). Enormous advances have been made in strawberry genetic transformation since the first transgenic plants were obtained in 1990 by two independent groups (James et al. 1990; Nehra et al. 1990). Besides F.x ananassa, transformation systems were also developed for related species, like F. vesca (Alsheikh et al. 2002; El-Mansouri et al. 1996; Haymes and Davis 1998; Oosumi et al. 2006; Zhao et al. 2004) and F. moschata (Mezzetti et al. 2002a).

The most important approach in strawberry relies on Agrobacterium tumefaciens-mediated leaf disk transformation (Barcelo et al. 1998; du Plessis and Brand 1997; Gruchala et al. 2004; Martinelli et al. 1996; Mathews et al. 1995a, b; Mezzetti 2003; Ricardo et al. 2000). Direct gene delivery into protoplast by electroporation was also reported (Nyman and Wallin 1988). A combined Agrobacterium-biolistic method was described later (Cordero de Mesa and Jimenez-Bermudez 2000). A new methodology to produce transgenic strawberries was developed using a temporary immersion bioreactor system (Hanhineva and Karenlampi 2007). Regardless of the sufficient regeneration levels achieved from leaf explants, the regeneration of transformed strawberry plants remains difficult and seems to be strongly genotype dependent. Since the 1990s, reliable protocols using Agrobacterium tumefaciens-mediated transformation were established for several commercial cultivars. Detailed surveys of literature are given by Mezzetti (2003) and Mezzetti and Constantini (2006). The effective production of markerfree transgenic strawberry plants using inducible site-specific recombination and a bifunctional marker gene was recently described by Schaart (2004). There are several promoter studies in strawberry. A tissue specific expression using the floral binding protein 7 promoter from Petunia was used by Schaart et al. (2002). Transgene expression driven by a heterologous phloem-specific promoter was published by Zhao et al. (2004). Agius et al. (2005) used a transient expression system to conduct a functional analysis of homologous and heterologous promoters in fruit. A near root-specific promoter was described recently (Vaughan et al. 2006). Transformation studies in strawberry are focused on modification of selected traits (Table 17.4).

There are a few studies related to environmental risk assessment of transgenic plants (see Chap. 27) in strawberry. The formation of chimeras during transformation has been reported in strawberry by several authors (Mathews et al. 1998; Monticelli et al. 2002) and is considered to be one of the major problems for strawberry transformation. Abdal-Aziz et al. (2006) described high frequencies of non-T-DNA sequence integrations in transgenic strawberry plants obtained through Agrobacterium transformation. An environmental risk evaluation of transgenic strawberry expressing a rice chitinase gene was performed in greenhouse, semi-greenhouse and field and revealed no effect on other plants, microflora, morphological characteristics and yield (Asao et al. 2003). The sexual transmission of transgenes to R1 generation progeny was reported for F. x ananassa (James et al. 1995) and for F. vesca (Haymes and Davis 1998).

The fact that the garden strawberry Fragaria x ananassa contains an octoploid genome made it difficult to use this species as a model for molecular studies and the interpretation of the transformation events. The wild strawberry F. vesca that contains a diploid genome represents an ideal model for functional genomics research in Rosaceae (Oosumi et al. 2006). However, recently transformation protocols were developed for a rapid-cycling genotype LF9 of F. x ananassa which allows high-throughput studies of gene function in the octoploid genetic background (Folta et al. 2006).

Transformation in strawberry is also used to study the function of genes, especially those related to fruit ripening (Hoffmann et al. 2006).

Several field trials with GM strawberries have been performed in the United States and in Europe. For the United States a total of 42 field test records were found within the Environmental Releases Database (http://www.isb.vt.edu/cfdocs/ fieldtests1.cfm). These field test records were focused on plants with improved resistance to herbicides and fungal diseases, respectively or on plants with altered agronomic properties.

In Europe, a total of eight summary notifications can be found for GM strawberries (http://bgmo.jrc.ec.europa.eu/deliberate/dbplants.asp). Summary notifications for the release of GM strawberries were submitted in Estonia (two), in Great Britain (one) and in Italy (five). GM strawberries are still quite a long way from commercial use in Europe. A commercial use of GM strawberries is not to be expected in the next years.

17.2.2.2 Grapevine

The genus Vitis comprises about 70 species, which are distributed over Southern Europe, Asia Minor, East Asia and North and Central America (Alleweldt and

Table 17.4 Summary of studies conducted on the transformation of strawberry for agronomically important traits

Selected trait

Genes used

Type of expression Reference

Insect resistance

Otiorhynchus spp.

Cowpea trypsin inhibitor Overexpr.

Virus resistance

Mild yellow edge Coat protein

Fungal resistance V. dahliae B. cinerea

S. humuli C. acutatum

Herbicide resistance Glyphosate Glufosinate

Abiotic stress Salt tolerance

Freezing tolerance

Fruit quality Reduced softening

Sugar content

Fruit color

Fruit flavor

Fruit size/ ripening Fruit size/yield

Plant morphology

Chitinase (L. chilense) Chitinase (P. vulgaris) Thaumatin II (T. danielli)

Chitinase (rice) Chitinase and glucanase (T. harzianum)

EPSP (A. tumefaciens) Phosphinothricin acetyl transferase

Late embryogenesis abundant protein (barley) Osmotin

CBF1 (Arabidopsis)

Acidic dehydrin (wheat) Type III antifreeze protein (fish)

Overexpr.

  1. Overexpr. Overexpr.
  2. Overexpr.
  3. Overexpr.

Overexpr.

Overexpr.

Overexpr.

Overexpr. Overexpr.

Strawberry pectate lyase Silencing

Strawberry glucanase cel1 Silencing S-adenosylmethionine Overexpr.

hydrolase (T3 bacteriophage) ADP-glucose Silencing pyrophosphorylase Strawberry chalcone Silencing synthase

Strawberry Silencing methyltransferase Strawberry GAST gene Overexpr.

defH9-iaaM (snapdragon/ Overexpr.

P. syringae) IAA-glucose synthase Overexpr.

(maize)

Graham et al. (1995, 1997, 2DD2), James et al. (1992), Watt et al. (1999)

Finstad and Martin (1995)

Chalavi et al. (2DD3) Vellicce et al. (2DD6) Schestibratov and Dolgov (2DD5) Asao et al. (1997) Mercado et al. (2007a)

Morgan et al. (2DD2) du Plessis and Brand (1997)

Husaini and Abdin (2008) Owens et al. (2002),

Owens (2005) Houde et al. (2004) Khammuang et al. (2005)

Jimenez-Bermudez et al. (2002) Palomer et al. (2006) Mathews et al. (1995)

Lunkenbein et al.

(2006a) Lunkenbein et al. (2006b) de la Fuente et al. (2006) Mezzetti et al. (2004)

Wawrzynczak et al. (2005)

Possingham 1988; Grando et al. 1996). The most renowed species is Vitis vinifera L., the European or bunch grape, which was domesticated 5000 years ago in Asia Minor or Armenia (Grando et al. 1996). In 2006 about 67 million t of grapes were produced on 7.5 million ha worldwide (http://faostat.fao.org).

Since the first transgenic grape plant was reported in 1990 (Mullins et al. 1990), a lot of successful transformations have been reported (for a review, see Yamamoto et al. 2003). Early attempts to transform grape using Agrobacterium tumefaciens met with difficulties and a biolistic transformation using coated microprojectiles was established and improved (Hebert et al. 1993, 2005a; Kikkert et al. 1996; Scorza et al. 1995a, b, 1996). Presently, Agrobacterium-mediated methods are the predominantly employed protocols for grape transformation worldwide (Perl et al. 1996). The use of high-quality embryogenic cultures has allowed the transformation of grape to become routine. A range of methods was published to improve transformation efficiency, to optimize protocols for rootstock and scion cultivars and to avoid selectable marker systems (Dhekney et al. 2005, 2008; Dutt et al. 2007; Li et al. 2006; Lopez-Perez et al. 2008; Nakajima et al. 2006; Olah et al. 2003; Reustle et al. 2003; Xue et al. 1999). Agronomic genes introduced into grapevine (for a review, see Deng and Duan 2006) were focused on virus resistance (Barbier et al. 2000; Golles et al. 2000; Gribaudo et al. 2003; Jardak-Jamoussi et al. 2003; Krastanova et al. 2000; reviewed by Laimer 2006; Martinelli et al. 2000; Radian-Sade et al. 2000; Reustle et al. 2005; Spielmann et al. 2000), fungal resistance (Aguero et al. 2005; Hinrichsen et al. 2005; Kikkert et al. 2000, 2005b; Reisch et al. 2003; Vidal et al. 2003, 2006; Yamamoto et al. 2000), bacterial resistance (Aguero et al. 2005; Holden et al. 2003; Vidal et al. 2003,2006), herbicide resistance (Mulwa et al. 2007), stress tolerance (Gutaronov et al. 2001; Olah et al. 2004; Tsvetkov et al. 2000), seedlessness (Colova-Tsolova et al. 2003; Perl et al. 2000) and morphology (Geier et al. 2008). Most significant progress in grape genetic engineering was the obtaining of transgenic grape cultivars resistant to grapevine fanleaf virus (GFLV). Transgenic plants were tested under field conditions and assessment of the field safety has been performed (Vigne et al. 2004). Field evaluation was also reported for DefH9-iaaM plants, expressing an auxin synthesizing gene which influences fruitfulness and berry quality (Mezzetti et al. 2005). The state of the art in genetic transformation in viticulture was recently summarized by Perl and Eshdat (2007).

A total of 54 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 grape vine plants with improved resistance to bacterial, fungal and virus diseases or with improved tolerance to herbicides. Other field trials were focused on GM plants with improved traits related to product qualtiy.

In Europe, a total of six summary notifications (four for France, one for Italy, one for Germany) can be found for GM grapes (http://bgmo.jrc.ec.europa.eu/deliberate/ dbplants.asp). No commercial use of GM grape vine plants is expected for the next years.

17.2.2.3 Ribes Species (Blackcurrant, Redcurrant, Gooseberry)

The cultivated forms of currants and gooseberries which belong to a number of Ribes species have in recent years been the subject of increased interest due to the perceived health benefits. There are a few publications in Ribes on regeneration, but no information on transformation. First report was given by Graham and McNicol (1991) in black currant. Transformation methods were optimized and aimed on the development of virus resistant plants (Karjalainen et al. 2001).

17.2.2.4 Rubus Species (Raspberry, Blackberry)

Raspberry and blackberry are genetically diverse with several Rubus species of the Rosaceae in their background. Regeneration of adventitious shoots from different type of explants has been reported for several Rubus spp. (reviewed by Mezzetti 2003; Swartz and Stover 1996). Transformation methods were developed using the Agrobacterium-mediated system (de Faria et al. 1997; Hassan et al. 1993; Kokko and Karenlampi 1998; Mathews et al. 1995a, b). Transformation was aimed on the delay of fruit decay (Mathews et al. 1995a, b), resistance to raspberry bushy dwarf virus (Martin and Mathews 2001) and parthenocarpic fruit development (Mezzetti et al. 2002b, 2004). Transformed plants have been successfully field-trialed, although they were not commercialized (Finn and Hancock 2008). While regeneration systems have been developed for blackberries (Meng et al. 2004; Swartz and Stover 1996), no transgenic blackberries have been produced to date (Finn and Hancock 2008).

A total of 16 field test records were found for GM raspberries 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 raspberry plants with improved resistance to fungal and virus diseases and with a better product qualtiy. In Europe, only one summary notification (Italy) can be found for GM raspberry plants (http://bgmo.jrc.ec.europa.eu/deliberate/dbplants.asp).

17.2.2.5 Vaccinium Species (Blueberry, Cranberry)

Several species of Vaccinium are important commercially, like highbush, lowbush and rabbiteye blueberries as well as large cranberry. A number of studies were published to improve a regeneration system for blueberry using in vitro leaves as well as seedling explants as source material (summarized by Hancock et al. 2008). Transformation in blueberry was reported for the first time by Graham et al. (1996). Later Song and Sink (2004) published transformation in different highbush blueberry cultivars. Transformation in highbush blueberry was aimed on herbicide resistance, field trails were also performed (Song et al. 2006, 2008). An efficient regeneration system has been developed for cranberry (Qu et al. 2000) The first transgenic cranberry was obtained by particle bombardment by Serres et al. (1992). In these experiments the Bt gene from Bacillus thuringiensis (see Chap. 10) was used to obtain resistance to Rhopobota naevana. Zeldin et al. (2002) transformed cranberry for herbicide resistance. Polashock and Vorsa (2002) summarized knowledge on transformation and regeneration in cranberry.

Three and one field test records were found for GM blueberry and cranberry plants, respectively, 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 plants with improved resistance to insects and herbicides, respectively.

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