Volume 5 Special Issue 1 2011
Genomics, Transgenics, Molecular Breeding and Biotechnology of Strawberry

ISBN 978-4-903313-81-8

How to reference: Husaini AM, Mercado JA, Teixeira da Silva JA , Schaart JG (2011) Review of Factors Affecting Organogenesis, Somatic Embryogenesis and Agrobacterium tumefaciens-Mediated Transformation of Strawberry. In: Husaini AM, Mercado JA (Eds) Genomics, Transgenics, Molecular Breeding and Biotechnology of Strawberry. Genes, Genomes and Genomics 5 (Special Issue 1), 1-11

Guest Editors

Amjad M. Husaini

Sher-e-Kashmir University of Agricultural Sciences & Technology of Kashmir

http://skuastkashmir.ac.in/

José A. Mercado

Departamento de Biología Vegetal, Universidad de Málaga, Spain

www.biolveg.uma.es/

CONTENTS AND ABSTRACTS

Amjad Masood Husaini (India), José A. Mercado (Spain), Jaime A. Teixeira da Silva (Japan), Jan G. Schaart (The Netherlands) Review of Factors Affecting Organogenesis, Somatic Embryogenesis and Agrobacterium tumefaciens-Mediated Transformation of Strawberry (pp 1-11)

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Invited Review: Standardization of an efficient regeneration system for each strawberry genotype is generally an indispensible pre-requisite for the successful development of transgenic plants. In this paper, we review some key factors affecting the regeneration of strawberry plants via adventitious organogenesis or somatic embryogenesis, such as type of explant, growth regulators or dark/light treatments. Since Agrobacterium tumefaciens-mediated transformation is the method of choice for strawberry transformation, we review the strategies adopted by different scientists to achieve higher transformation efficiencies and recovery of marker-free transgenic plants. Sufficient Agrobacterium cells during cocultivation, an adequate cocultivation period, the use of vir gene inducers like acetosyringone, introduction of a pre-selection phase between co-cultivation and selection, and optimum selection pressure, are all important factors to obtain stable transformants. For effective transformation, the antibiotic regime should control bacterial growth without inhibiting the regeneration of plant cells. A general protocol for the Agrobacterium transformation of strawberry leaf discs is also described. Finally, we discuss the metrics employed by different researchers for measuring the success of transformation, and highlight the difference between transformation efficiency and transformation percentage.

Francesca Massetani, Ramesh Gangatharan, Davide Neri (Italy) Plant Architecture of Strawberry in Relation to Abiotic Stress, Nutrient Application and Type of Propagation System (pp 12-23)

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Invited Review: Vegetative growth of strawberry plants turns to generative growth (flower induction and differentiation) under specific thermo-photoperiods, but agronomic and nutritional factors may establish which kind of growth (vegetative or reproductive) to be strengthened. As a consequence, environmental stressing conditions assume a key function in determining plant quality during plant propagation and culture, in relation to different physiological internal factors. The result is a significant modification of plant architecture that influences the timing of forcing conditions for out of season production, the quantity of chilling to be applied and the effect of day length. Plant architecture describes the spatial distribution of vegetative and reproductive organs and their developmental phase and thus it is useful to evaluate plant quality and also to study the results of a few different propagation techniques in the nursery. Time of transplanting, regulated nutrient deficit, water application and limited substrate soil volume (smaller pot) in tray plant production are some possible stress manipulations to control plant quality. Important differences in plant architecture are also possible between cultivars, within the same cultivar grown in different environments and between farms (growers) in the same area, because growing conditions affect plant vigour, induce the formation of a very different number of lateral flower buds and modify the ability of the plant to form new lateral shoots along the main shoot. The developmental phase of the flowers is also affected. Depending on the desired production system, the plant architecture and its potential fruit production can be strongly modified, but in a predictable way, by changing and modulating the growing techniques in the nursery.

Carlos Garrido, María Carbú, Francisco Javier Fernández-Acero, Victoria E. González-Rodríguez, Jesús M. Cantoral (Spain) New Insights in the Study of Strawberry Fungal Pathogens (pp 24-39)

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Invited Review: Strawberry (Fragaria ananassa) is one of the world’s most commercially important fruit crops, and is grown in many countries The commercial viability of the crop is continually subject to various risks, one of the most serious of which is the diseases caused by phytopathogenic organisms. More than 50 different genera of fungi can affect this cultivar, including Botrytis spp., Colletotrichum spp., Verticillium spp, and Phytophthora spp. The development of new molecular biology technologies, based on genomics, transcriptomics and proteomics approaches, is revealing new insights on the diverse pathogenicity factors causing fungal invasion, degradation and destruction of the fruit (in planta and during storage and transport). Researchers have focused attention on the plant’s own defence mechanisms against these pathogens. In this review, advances in the study and detection of fungal plant pathogens, new biocontrol methods, and proteomic approaches are described and the natural defence mechanisms recently discovered are reported.

Sara Posé, Juan A. García-Gago, Nieves Santiago-Doménech, F. Pliego-Alfaro, Miguel A. Quesada, José A. Mercado (Spain) Strawberry Fruit Softening: Role of Cell Wall Disassembly and its Manipulation in Transgenic Plants (pp 40-48)

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Invited Review: Strawberry is the most economically important soft fruit characterized by its delicious flavour, intense colour and soft texture. The rapid loss of firmness during strawberry ripening is the main determining factor of its short shelf life, and great losses occur by bruising, oversoftening and subsequent fungal infections during postharvest transportation and storage. Cell wall disassembly and the reduction of cell to cell adhesion due to middle lamella dissolution have been recognized as the main causes of fruit softening. In the case of strawberry fruit, cell wall disassembly during ripening is characterized by a solubilization of pectins, a slight depolymerization of covalently bound pectins, a loss of galactose and arabinose, as well as a reduction in the hemicellulosic content. The genetic manipulation of genes encoding proteins involved in wall disassembly has been explored as a biotechnological approach to reduce fruit softening. Thus, the silencing of fruit specific pectate lyase or polygalacturonase genes, both acting on demethylated pectins, by antisense transformation increased significantly the firmness of ripe strawberry fruit without affecting other fruit quality parameters. By contrast, the down-regulation of the expression of genes involved in the processing of hemicellulosic polymers, such as endo-b-1,4-glucanase or expansin genes, did not modify fruit firmness. Other cell wall genes, e.g. b-xylosidase, b-galactosidase, α-arabinofuranosidase, have been cloned in strawberry fruit, and their expression analyzed along fruit development, but their function on fruit softening has not been assessed yet by transgenic approaches. Overall, these results indicate that pectin processing during ripening is a critical factor in the softening of strawberry fruit, and the manipulation of this process by down-regulating pectinase genes a successful approach to improve this trait. This review summarizes recent progress on strawberry fruit softening, focusing in the role of cell wall disassembly and genes involved in that process.

Karina Alleva, Mercedes Marquez, Jorge Bellati, Natalia Villarreal, Claudia Bustamante, Gustavo Martínez, Marcos Civello, Gabriela Amodeo (Argentina) Could an Understanding of the Strawberry Softening Process Benefit from Aquaporins? (pp 49-55)

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Invited Mini-Review: The Major Intrinsic Protein (MIP) family includes a main group of key channels known as aquaporins (AQPs), described for sharing a very conservative structure with a pore that facilitates water and/or solute permeation. Since its first member was functionally reported in 1992, AQPs were found to be abundantly expressed in all kingdoms. Although many roles have been attributed to these small integral proteins, it is becoming evident that the number and type of AQPs within a membrane are major determinants of its water transport capacity. Thus, their presence is opening new perspectives to understand the role of plant cell membrane water transport in physiological and developmental processes. Strawberry is a fleshy fruit characterized by a rapid loss of firmness during ripening, limiting the shelf-life of these fruit. Even though fruit texture is influenced by various factors like structural integrity of primary cell wall, sugar accumulation, and the turgor pressure generated within cells by osmosis, main attention has been focused on degradation of cell wall polysaccharides. Turgor pressure, in spite of long being mentioned as a possible player in softening during fruit development, has not received proper consideration. In the light of AQP outbreak, it is worth questioning how these channels could contribute to strawberry fruit softening. In an attempt to answer this question, this review summarizes the current available information on plant AQPs, extending the knowledge to those specifically expressed in fruits to finally discuss the recent reported findings in strawberry, particularly those associated with ripening and softening processes.

Timo Hytönen, Paula Elomaa (Finland) Genetic and Environmental Regulation of Flowering and Runnering in Strawberry (pp 56-64)

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Invited Review: Cultivated strawberry (Fragaria × ananassa Duch.) is one of the most important berry crops worldwide. Its wild relative, woodland strawberry (Fragaria vesca L.) is also scientifically important, since recent development of molecular tools including genetic transformation methods, genetic maps and impending genome sequence is making it as one of the leading perennial model plants. Environmental regulation of strawberry reproductive development including flowering and vegetative reproduction through runners has been studied for almost hundred years and is known quite in detail. Most strawberries require short photoperiod and/or low temperature for the induction of flowering, whereas runnering is activated by opposite environmental signals. On the contrary, everbearing genotypes flower continuously in long day conditions. Some groundwork on characterization of molecular pathways controlling runnering and the induction of flowering has been done. In these studies, dozens of candidate flowering genes have been identified, the expression patterns for selected genes have been analyzed, and a marker gene for the induction of flowering has been identified. However, reports on detailed functions of the candidate genes are yet to come. Moreover, the role of gibberellin as a major signal regulating runnering, awaits further characterization. Two gene loci in F. vesca may provide keys to understand underlying regulatory pathways and are therefore major targets of further research. Runnering locus (RL) makes difference between runnering/non-runnering phenotypes and different alleles of Seasonal flowering locus (SFL) cause seasonal and everbearing flowering habits. This review aims at summarizing the recent progress on molecular control of flowering and runnering in strawberry.

Kati Hanhineva, Sirpa O. Kärenlampi (Finland), Asaph Aharoni (Israel) Recent Advances in Strawberry Metabolomics (pp 65-75)

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Invited Review: The recent developments in metabolomics technologies have facilitated a comprehensive examination of the rich chemical composition of plants. Both gas and liquid chromatography based separation combined to high mass accuracy mass spectrometry as well as structural elucidation utilizing 2D NMR are currently frequently applied in metabolite profiling approaches for various plant species, including strawberry. With these technologies, the knowledge of the metabolite composition of strawberry has been expanded to include numerous different derivatives of well-known metabolites. Furthermore, metabolite classes previously unknown for this species have been identified. As in other plants, the array of natural products generated in different organs and cell layers of strawberry forms the basis for the chemical defense and interaction with the environment. The same compounds, when consumed in the diet are responsible for the bioactivity mediating beneficial health effects in humans. Strawberry produces large amounts of commonly occurring phenolic compounds such as phenolic acids, flavonols and anthocyanidins. The early developmental stages of strawberry fruit are characterized by abundant accumulation of proanthocyanidin polymers that protect the developing fruit against pests, and give an astringent taste rendering it unappealing for consumption. One of the most abundant metabolite classes of strawberry fruit is ellagitannins, group of compounds restricted to a small number of plant species. Ellagitannins are likely to contribute to the beneficial health effects claimed for strawberry, as these compounds show e.g. anticarcinogenic activity in vitro. In this review we discuss the phytochemicals produced in the vegetative and reproductive organs of strawberry, both in terms of the plant's physiology and as a constituent of the human diet. The metabolome of strawberry is described in light of recent developments and application of cutting-edge analytical chemistry-based approaches for metabolomics analysis of complex plant matrices.

Julio Bonet, Amparo Monfort (Spain) Structural Genomic Resources in Fragaria Genus (pp 76-84)

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Invited Mini-Review: Strawberry is the most widely grown of the berries, and the most economically important soft fruit. It has an interesting history. Fragaria vesca, known as the wood strawberry, a diploid species (2n=14) with hermaphrodite flowers, has small, aromatic fruit and is found in localised markets in Europe, northern Asia, northern Africa and North America. Additionally, many species of Fragaria L. (Rosaceae) occur throughout northern temperate regions with one species extending into South America. A range of ploidy levels is observed within the genus, with naturally occurring diploid, tetraploid, hexaploid and octoploid species and natural hybrids. The strawberry cultivated worldwide, F. x ananassa,is derived from the chance hybridisation between two octoploid (2n=56) native American species, F. virginiana, and F. chiloensis, both usually dioecious, in European botanic gardens. There is a well characterised collection of molecular markers in Fragaria species, used principally to develop a saturated genetic linkage map of diploid Fragaria. The map has over 600 transferable molecular loci from an interspecific cross between F. vesca and another closely-related diploid species, F. bucharica (FV×FB) and includes microsatellites (SSRs), T-DNA insertion site markers, gene-specific and STS markers, RFLPs and SCAR markers and single nucleotide polymorphism (SNP) markers. Comparative mapping studies have shown a high level of macrosynteny between the diploid and octoploid Fragaria genomes.

Guillermo Schmeda-Hirschmann, Mario Simirgiotis (Chile), José Cheel (Czech Republic) Chemistry of the Chilean Strawberry (Fragaria chiloensis spp. chiloensis) (pp 85-90)

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Invited Mini-Review: The Chilean strawberry (Fragaria chiloensis spp. chiloensis) is one of the progenitors of the cultivated strawberry, Fragaria × ananassa. Recent studies have started to disclose the chemical composition of the native fruit, the secondary metabolite occurrence and distribution in different plant parts as well as the biological activity of the main fruit constituents. This article revises the chemistry of the Chilean strawberry and compares some of the most characteristic constituents with that of the highly cultivated and worldwide known F. × ananassa. The main phenolic in the white strawberry are ellagic acid or hexahydroxydiphenolic acid-based hydrolysable tannins and procyanidins as well as flavonoid glycosides from quercetin and kaempferol. The general trend in the plant is the accumulation of condensed tannins of increasing molecular weight in the rhizomes while the leaf contains mainly hydrolysable tannins and flavonoids.

Wilfried Schwab, Thomas Hoffmann, Gregor Kalinowski, Anja Preuß (Germany) Functional Genomics in Strawberry Fruit through RNAi-mediated Silencing (pp 91-101)

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Invited Review: Down-regulation of gene expression by RNA interference (RNAi) has become a powerful tool to investigate gene functions in vivo. In this mini-review we examine how RNAi has been used to assess gene function in strawberry fruit, including molecular mechanisms and analysis of silenced phenotypes. The down-regulated genes include FaCHS, FaOMT, FaGT1, FaDFR, FaANS, and Fra encoding proteins functioning in the flavonoid biosynthetic pathway. A comparison with stably transformed genotypes shows how spatial silencing of gene expression in fruit may affect metabolite patterns.

Jan G. Schaart (The Netherlands), Trygve D. Kjellsen, Lisbeth Mehli, Reidun Heggem, Tor-Henning Iversen (Norway), Henk J. Schouten, Frans A. Krens (The Netherlands) Towards the Production of Genetically Modified Strawberries which are Acceptable to Consumers (pp 102-107)

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Original Research Paper: This manuscript discusses different aspects that are relevant to genetically modified strawberry plants with improved characteristics and ‘acceptable’ to consumers and growers of strawberry. It starts with a consumer acceptance survey, held in Norway, Denmark and the UK, studying public perception of genetic modification in general and specifically of genetically modified strawberries with altered properties. This study revealed that genetically modified plants are better accepted by consumers if only genes from the species itself are used for the genetic modification. Subsequently, the results of a functional analysis of the strawberry polygalacturonase inhibiting protein gene (FaPGIP) are described. This indicates that this gene is a possible candidate to induce resistance to Botrytis cinerea when upregulated in strawberry fruits. For this analysis, the FaPGIP gene was overexpressed in transgenic strawberry plants using the cauliflower mosaic virus 35S (CaMV35S) promoter. This showed that FaPGIP overexpression led to resistance to Botrytis in transgenic leaves. For the generation of intragenic (i.e. genetically modification using native genetic elements only) strawberry plants, a transformation vector was constructed in which FaPGIP was combined with a strawberry fruit-specific promoter and terminator that were isolated from a strawberry expansin gene (FaExp2). This vector also included elements that allow the elimination of (foreign) selectable marker genes after genetically modified plant lines have been established. Using this vector, genetically modified strawberry plants were produced that contained only genes from the species itself, and therefore these plants were called intragenic, rather than transgenic. Unfortunately, further evaluations of the intragenic strawberry plants could not demonstrate any enhanced level of resistance to Botrytis in fruits.

Jan G. Schaart, Elma M. J. Salentijn, Koen T. B. Pelgrom, Asaph Aharoni, Frans A. Krens (The Netherlands) Isolation and Characterisation of a Strawberry Fruit-Specific Promoter (pp 108-114)

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Original Research Paper: In order to achieve specific expression of transgenes in strawberry fruits, the availability of tissue- (receptacle) specific promoter sequences is desired. For this reason, 5′-upstream sequences of the strawberry expansin gene FaExp2, which is expressed in a fruit-specific manner, have been isolated. To characterise the promoter activity of the isolated sequences, fragments of 0.7 kb (0.7pFaExp2)and 1.6 kb (1.6pFaExp2) have been fused to the β-glucuronidase reporter gene (gus). In transgenic strawberry plants transformed with either 0.7pFaExp2-gus or1.6pFaExp2-gus, a fruit-specific expression pattern was observed for both promoter constructs. However, quantitative RT-PCR revealed that gus expression levels driven by the 1.6pFaExp2 promoter fragment were much more higher. In addition to the expression in fruits, both promoter fragments also seemed to direct gene expression in the achenes and to some extent in epidermal and subepidermal tissues of petioles and stems of flowers and fruits. It is concluded that both promoter sequences are suitable for directing transgene expression in strawberry fruits in a specific way.

Dina B. Shokaeva, Natalya V. Solovykh, Dmitry N. Skovorodnikov (Russia) In Vitro Selection and Strawberry Plant Regeneration for Developing Resistance to Botrytis cinerea Pers., Phytophthora cactorum Leb. et Cohn (Schroet) and Salinity Stress (pp 115-125)

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Original Research Paper: In vitro screening techniques were employed to obtain strawberry (Fragaria × ananassa Duch.) lines with resistance/tolerance to environmental stressors. The aim was to select genotypes resistant to Phytophthora cactorum and Botrytis cinerea and tolerant to chloride salinity. Callus tissues were screened for tolerance on MS medium, supplemented with 2 mg l^-1 indole-3-butyric acid (IBA), which contained selective agents: either monthly culture filtrate of one of the two pathogens at 10, 20 and 30% or 0.05, 0.10 and 0.15 M sodium chloride. Tolerant tissues were used for adventitious shoot regeneration on MS medium with 2 mg l^-1 6-benzylaminopurine (BAP), 0.5 mg l^-1 IBA and 0.2 mg l^-1 gibberellic acid (GA₃), which were then micropropagated on MS medium supplemented with 1 mg l^-1 BAP and 0.1 mg l^-1 IBA. Rooting was achieved on MS medium supplemented with 0.5–1 mg l^-1 IBA. The obtained plants were acclimatized to in vivo conditions in a plastic greenhouse and subsequently planted in the field. Tests for disease resistance were conducted using artificial leaf inoculation with spore-mycelium suspensions of the phytopathogens. Three methods were employed to assess tolerance to salinity: (1) evaluation of growth parameters on selective media containing 0.05 and 0.1 М NaCl, and determination of propagation coefficient and leaf necrosis scores, (2) laser analysis of tissue microstructure (LAM) and (3) leaf diagnostics. Lines exceeding the level of resistance to P. cactorum and B. cinerea (‘Lvovskaya Rannyaya’, ‘Urozhainaya CGL’ and ‘Zolushka’), and those with superior tolerance to salt toxicity (‘Feierverk’, ‘Lvovskaya Rannyaya’, ‘Urozhainaya CGL’ and ‘Zolushka’) were selected. In addition to either trait, the most promising lines produced high yield and large fruit.

Yoh-ichi Matsubara (Japan) Tolerance to Fusarium Wilt and Changes in Antioxidative Ability and Free Amino Acid Content in Mycorrhizal Strawberry Plants (pp 126-131)

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Original Research Paper: The influence of arbuscular mycorrhizal fungi (AMF) colonization on tolerance to fusarium wilt and the changes in antioxidative ability and free amino acid content in strawberry (Fragaria×ananassa Duch. cv. ‘Nohime’) plants was investigated. Strawberry runner plants were treated with split root system and inoculated with Glomus mosseae. Mycorrhizal plants showed higher dry weight of shoots and roots than did non-mycorrhizal plants among most of the plots. Five weeks after Fusarium oxysporum f. sp. fragariae (Fof) inoculation, severity of disease symptoms was eased in shoots and roots of mycorrhizal plants. Plant mycorrhization did not modified the antioxidative ability before Fof inoculation. However, 5 weeks after Fof inoculation, mycorrhizal plants showed higher SOD activity, DPPH radical scavenging activity, polyphenol content, ascorbic acid content in some plant portions. Free amino acid content GABA, aspartic acid, threonine, serine, glycine, citrulline and arginine increased in mycorrhizal plants with different entity depending on plant portions. In addition, some among the amino acids increased in mycorrhizal plants showed suppressed by 20 to 70% the propagation of Fof cultured by Czapec-Dox media in vitro depending their concentration. From these findings, plant growth enhancement and tolerance to fusarium wilt including induced tolerance occurred in mycorrhizal strawberry plants. In this case, the disease tolerance might be associated with the increase in antioxidative ability and symbiosis-specific amino acids.