Tomato genetic resources publicly accessible via web
Tomato is one of the most important vegetable crop worldwide with a total production of around 141 million tons on a cultivated area of around 5 million hectares (FAOSTAT, 2009, http://faostat.fao.org). Among the most representative countries, Italy contributes with more than 6 million tons to the world production, on a cultivated area of around 117.000 hectares, both in open fields and greenhouses (FAOSTAT, 2009). This crop represents also one of the major products of the food industry worldwide and Italy ranks first for processing tomato production among Countries of the Mediterranean Region (World Processing Tomato Council, 2009, http://www.wptc.to). Indeed, the high variability of tomato fruits, ranging from the cherry type to the big round or elongated berry, supplies both fresh market and processing products, such as paste, juice, sauce, powder or whole. In the last years, tomato consumption has further increased since it was demonstrated that tomato fruit could protect against diseases, such as cancer and cardiovascular disorders, due to its antioxidant properties (Rein et al., 2006). Tomato fruits are particularly rich of nutritional compounds such as lycopene and alfa-carotene, vitamin C, flavonoids and hydroxycinnamic acid derivatives whose intake would account for health benefits.
The cultivated tomato (
The huge amount of researches focused on tomato allowed the development of new tools and platforms for genetics and genomics analyses (Barone et al., 2008). Since tomato is considered the model species among the
The recent release of the tomato genome sequence (Mueller et al., 2009), together with the powerful genetic and genomic resources available today for this species, allowed plant biotechnologists to implement novel methods to obtain new genotypes that could answer to new consumer, producer and processor requirements. These resources, in fact, could help the transfer of useful genes among species and/or improved genotypes through assisted breeding programs as well as through genetic transformation technologies.
In the present review, after providing some information on tomato genetic and genomic resources, we will give an overview of genetic transformation techniques and biotechnology applications investigated in this species. Several recent review reported new studies on tomato genetic transformation as a tool for the improvement of resistance to pests and pathogens (Balaji & Smart, 2011; Khan et al., 2011; Panthee & Chen, 2010; Wu et al., 2011; Zhang et al., 2010). Therefore, after a short description of main transformation techniques to which tomato is well adapted, herein we will focus on the use of genetic transformation for fruit quality engineering and pharmaceutical production.
2. Genetic and genomic resources
Among cultivated species, tomato is one of the richest in genetic and genomic resources (Table 1 and Table 2), including information now available from the complete genome sequencing that was released in the last year in a preliminary version. All these tools, used together or separately, are having a great impact on tomato breeding and genetics (Barone et al., 2009; Foolad, 2007).
This cultivated species could count on a number of wild and related species, on a wide collection of naturally or induced mutants and on many well-characterized genetic stocks, such as cultivars and landraces, cytogenetic stocks and pre-bred lines. Today this germplasm is publicly available (Table 1). In the miscellaneous group, the Backcross Recombinant Inbreds and Introgression Lines are particularly useful for the identification of genes and/or QTLs, since they constitute "immortal" population to be used for quantitative analyses (Grandillo et al., 2008). In addition, they also represent exotic libraries that allow to better exploit biodiversity exhibited by wild species. Indeed, the IL population is composed by many lines, each carrying a single homozygous genomic region from the wild species, altogether covering the whole wild genome (Eshed & Zamir, 1995; Fridman et al., 2004).
|Wild species||More than 1.100 accessions||TGRC (http://tgrc.ucdavis.edu),|
|Monogenic mutants||More than 600 mutants||TGRC|
|Miscellaneous stock||More than 1.500 accessions||TGRC, NPGS|
|Introgression lines (IL)||from ||TGRC|
|Backcross Recombinant Inbreds (RIL)||TGRC|
|Induced-mutant stocks||More than 3.400 induced-mutants||SGN (http://zamir.sgn.cornell.edu/mutants)|
|Around 1000 induced- mutants from Micro-Tom||TOMATOMA (http://tomatoma.nbrp.jp)|
|More than 5.000 mutants from ||LycoTill (www.agrobios.it/tilling/index.html)|
Currently, IL populations that derive from various wild species are available, even though others are being generated (Barone et al., 2009). The first population (from
In addition to a collection of natural mutants available at TGRC (Tomato Genetic Resource Centre), wide collections of induced mutants were generated in different genetic backgrounds, by chemical or physical mutagenesis (Emmanuel & Levy, 2002; Menda et al., 2004; Watanabe et al., 2007). These mutants were widely phenotyped for many traits and contributed to better understand some developmental processes, such as growth habit, flowering and fruit ripening (Giovannoni, 2007; Pineda et al., 2010; Saito et al., 2011). In addition, induced mutagenesis has often been implemented with gene-specific detection of single-nucleotide mutations to generate TILLING platforms. So far, TILLING was developed for the cv. M82 (Piron et al., 2010), Red Setter (Minoia et al., 2010), Tpaadasu (Gady et al., 2009) and Micro-Tom (Saito et al., 2011) and its use has allowed the pinpointing of mutations in genes of interest.
The variability displayed by the different sources of germplasm available for tomato could be explored to search for new genes or favourable alleles to be transferred by conventional breeding and/or genetic transformation in selected genotypes to obtain new varieties.
In recent years, genetic resources combined with tomato specific genomic tools (Barone et al., 2009) allowed to successfully achieve various objectives, including the development of new varieties resistant to biotic and abiotic stresses and with improved fruit quality traits and yield. Most of these resources are also publicly available for the scientific community and are accessible
|Molecular markers||Thousands markers|
(i.e RFLP, AFLP, SSR, COS, CAPS, SNP)
|Molecular maps||10 genetic maps involving crosses among different species and varieties||SGN|
|Physical map||from ||SGN|
|Complete genome sequence||released version SL2.40 January 2011||SGN|
|EST collections||Around 300.000 from various tissues and developmental stages||SOLESTdb (http://biosrv.cab.unina/solestdb)|
Tomato Gene Index
(http://compbio.dfci.harvard.edu/tgi), plantGDB (http://www.plantgdb.org), MiBASE (http://www.kazusa.or.jp/jsol/microtom)
|transcriptomic array||TOM1 (approx. 8000 unigenes)||Tomato Functional Genomics database (http://ted.bti.cornell.edu)|
|TOM2 (approx. 11.000 independent genes)||TFGD|
|Affimetrix (approx. 10.000 genes)||(http://www.affymetrix.com)|
|Combimatrix TomatoArray1.0 (more than 20.000 probes)||Functional Genomic Center (http://ddlab.sci.univr.it)|
|Metabolomic platforms||Metabolites from ||TFGD, MoToDB (http://appliedbioinformatics.wur.nl)|
|TILLING platforms||From ||LycoTill, UTill (http://urgv.evry.inra.fr/UTILLdb), (http://tilling.ucdavis.edu/index.php/TomatoTilling)|
|SNP array||SolCAP approx. 8000 SNPs from 6 genotypes||SolCAP (http://solcap.msu.edu)|
|Bioinformatic platforms||Data mining and integration, genome annotation||SGN, TFGD|
Since the beginning of 1990s, the contribution of molecular markers and maps to tomato breeding and gene identification has been widely documented (Foolad, 2007; Frary et al., 2005; Gupta et al., 2009), and more than 15.000 different markers are collected in the SGN database, where markers can be searched by name, chromosome position and mapping population. Moreover, cytological and cytogenetic maps are also available, as well as a detailed physical map, which was the foundation for the tomato genome sequencing project (Mueller et al., 2005). Contemporarily, gene expression analyses performed on different tissues and developmental stages, as well as on genotypes that differ in their answer to environmental
3. Techniques for tomato genetic transformation
Since the 1980s several
Recently, as the information provided by the tomato genome sequencing become available, the demand for efficient functional genomics tools are increasing. Functional genomics studies of the tomato plant require the use of high-throughput methods for functional analysis of many genes including simple and easily reproducible plant transformation systems. The miniature tomato cultivar MicroTom is a rapid-cycling cherry tomato variety that differs from standard tomato cultivars primarily by two recessive genes that confer the dwarf genotype (Dan et al., 2006). MicroTom shares some traits with the model plant
Another breakthrough in the field of tomato genetic transformation was the development of a system for stable genetic transformation of tomato plastids (Ruf et al., 2001). In comparison with conventional nuclear transformation, the integration of transgenes in the plastid genome presents several advantages: 1) high expression levels of recombinant proteins attainable owing to the high ploidy level of the plastid genome (up to 10,000 plastid genomes per cell); 2) efficient transgene integration since integration into the plastid genome relies on homologous recombination between the targeting regions of the transformation vector and the wild-type plastid DNA; 3) absence of epigenetic effects (gene silencing); 4) increased biosafety due to the biological containment of transgenes and recombinant products owing to maternal inheritance of plastid and plastid transgenes and absence of dispersal in the environment through the pollen; 5) possibility to express multiple transgenes from prokaryotic-like operons, thus simplifying engineering metabolic pathways (Bock & Warzecha; Cardi et al., 2010; Ruf et al., 2001; Wurbs et al., 2007).
The availability of a technology for transgene expression from the tomato plastid genome opened up new possibilities for metabolic engineering and the use of plants as bioreactors for the production of pharmaceuticals (Ruf et al., 2001, Wurbs et al., 2007). The group of Ralph Bock investigated the possibility to elevate the pro-vitamin A content of tomatoes using the chloroplast transformation technology (Apel & Bock, 2009; Wurbs et al., 2007). Apel & Bock (2009) introduced the lycopene β-cyclase genes from the eubacterium
Today, the technology of stable plant transformation is successful in tomato; however, the lack of an efficient, simple and reliable protocol and the length of time required to produce transgenic lines complicate the analysis of gene function. In alternative, transient assays could provide a rapid tool for the functional analysis of transgenes and have been often used as an alternative to the analysis of stably transformed lines (Wroblewsky et al., 2005). A powerful tool for fast reverse genetics is the virus-induced gene silencing (VIGS) technology (Orzaez & Granell, 2009). Using this method, recombinant virus vectors carrying host-derived sequences are used to infect the plant; systemic spreading of this recombinant virus causes specific degradation of the endogenous gene transcripts by PTGS (post-transcriptional gene silencing) (Dinesh-Kumar et al., 2003; Liu et al., 2002). In 2002, Liu and colleagues demonstrated that a tobacco rattle virus (TRV)-based VIGS vector could be used in tomato to silence genes efficiently. To shorten the time and simplify the functional analysis in fruits, Orzaez et al. (2006) developed a methodology that allowed transient expression of transgenes directly in fruit tissues. However, the identification and quantification of non-visual phenotypes could be hampered by the irregular distribution of fruit VIGS. In a recent paper Orzaez et al. (2009) developed an anthocyanin-guided VIGS in order to overcome the limitations of this technique such as its irregular distribution and efficiency. To develop a visually traceable system the authors developed a method comprising: 1) a tomato line expressing
4. Biotechnology applications
4.1. Fruit quality engineering
Tomato fruit quality includes several aspects that may be grouped into two categories: organoleptic properties and nutritious contents. Organoleptic quality involves color and texture of the fruit, but also taste and aroma, whereas nutritional quality refers to the content of metabolites contributing to the intake of nutritious such as sugars, carotenoids, flavonoids, ascorbic acid and folate.
Most of the quality traits show a continuous variation, are attributed to the joint action of many genes and are strongly induced by environmental conditions. Beside their complex inheritance, fruit quality traits have often been engineered in tomato through approaches of reverse genetics, such as genetic transformation and mutagenesis, pointing at controlling the expression of single major genes involved in the regulation of a desirable phenotype. In addition, genetic transformation has often been successful in enhancing fruit quality-related traits in tomato investigating simultaneously the role of candidate genes in specific biological processes in the fruit.
In general, there are three main goals of engineering strategies in plants (Verpoorte et al., 2000): the enhancement of a desired trait, the decrease in the expression of a specific unwanted trait, and the development of a novel trait (i.e. a molecule that is produced in nature but not usually in the host plant, or a completely novel compound). Strategies aimed at inducing changes in the expression of a trait changing the synthesis of a specific metabolite are referred to as metabolic engineering. Approaches for achieving the redirection of metabolic fluxes include the engineering of single steps in a pathway to increase or decrease metabolic ﬂux to target compounds, to block competitive pathways or to introduce short cuts that divert metabolic ﬂux in a particular way. However, this strategy has only limited value because the effects of modulating single enzymatic steps are often absorbed by the system in an attempt to restore homeostasis. Recently, strategies aimed at targeting multiple steps in the same pathway are gaining increasing interest because they help to control metabolic ﬂux in a more predictable manner. This might involve up-regulating several consecutive enzymes in a pathway; up-regulating enzymes in one pathway while suppressing those in another competing pathway; or using regulatory genes such as transcription factors (TF) to establish multipoint control over one or more pathways in the cell. Since technical hurdles limits the number of genes that can be transferred to plants and pyramiding of transgenes by crossing transformants for single targets is a highly time-consuming approach, researchers developed new transformation methods to introduce multiple transgenes into plants and express them in a coordinated manner (Navqvi et al., 2009). In addition, controlling the expression of a single TF or a combination of TFs provides attractive tools for overcoming flux bottlenecks involving multiple enzymatic steps, or for deploying pathway genes in specific organs, cell types or even plants where they normally do not express.
A schematic description of successful metabolic engineering for enhancement of fruit quality in tomato is provided in Table 3 and Table 4. Genetic transformation targeting a single TF has been used to successfully engineer tomato for inducing development of parthenocarpic and seedless fruit. Parthenocarpy enables fruit set and growth to be independent from pollination, fertilization and seed development circumventing the environmental constraints on fruit production and ensuring yield stability. Seedless fruits enhance consumer appeal and could also be a valuable trait for industrial tomatoes because parthenocarpy increases the content of soluble solids, improves yield and flavour of paste and reduces processing costs. Reported applications involved the overexpression of an auxin response factor 8 (
Modifications of fruit softening and of the overall firmness have been achieved mostly by engineering genes controlling single enzymatic steps in cell wall-associated pathways. In particular, polygalacturonase (Kramer et al., 1992; Langley et al. 1994; Smith et al., 1990), pectin methylesterase (Tieman & Handa, 1994), expansin (Brummell et al., 1999) and β-galactosidase (Smith et al., 2002) genes showed effectiveness in controlling fruit firmness and softening in transgenic tomato plants. A dosage series of the gene
Two examples of successful metabolic engineering modifying tomato fruit flavour relayed on heterologous single-gene expression to introduce in tomato untypical traits. In the first example, a biologically active thaumatin, a sweet-tasting, flavour-enhancing protein from the African plant
In another study, the overexpression of either
In addition to organoleptic fruit quality, nutritional attributes of tomato fruit have recently received increasing attention by molecular biologists. For instance, the fruit soluble solid content was engineered by using an RNAi approach to generate transgenic plants that were exclusively altered in the expression of a speciﬁc isoform of the cell wall invertase
Several attempts have been made also to engineer higher carotenoid contents in tomato fruit and a number of tomato lines have been generated with enhanced levels of lycopene, β-carotene and xanthophylls (mainly zeaxanthin and lutein) and low levels of non-endogenous carotenoids such as ketocarotenoids (Fraser et al., 2009). One of the most interesting achievements is the HighCaro (HC) tomato plant (D’Ambrosio et al. 2004), a transgenic line carrying the tomato lycopene β-cyclase (
Due to their presumed health benefits, there is growing interest in the development of food crops with tailor-made levels and composition of flavonoids. The repertoire of case studies aimed at increasing the levels of flavonoids in tomato fruit also offers the wider range of examples of successful engineering strategies ever realized. Herein we will list some of the results recently obtained.
The first strategy is related to engineering single structural genes controlling key steps in the pathway, such as a chalcone isomerase (CHI) (Muir et al., 2001) and a chalcone synthase (CHS) (Colliver et al., 2002). More encouraging results were obtained targeting multiple constitutive genes within the flavonoid pathway. For instance, the concomitant ectopic expression of
In contrast with flavonoid metabolism, so far a reduced number of efforts have been placed into genetic transformation-mediated metabolic engineering of tomato fruit for enhanced ascorbic acid levels. Only few of them succeeded in effectively affect ascorbic acid content and only for a limited number of structural genes within the ascorbic acid pathway. In a fruit systems biological approach, transgenic tomato lines silenced for a mitochondrial ascorbic acid synthesizing enzyme L-galactono-1,4-lactone dehydrogenase performed an increased fruit ascorbic acid level (Garcia et al., 2009) whereas the silencing of an GDP-D-mannose-3’,5’-epimerase resulted in a reduced fruit ascorbic acid accumulation (Gilbert et al., 2009). On the other hand, overexpression of GDP-D-mannose-3’,5’-epimerase genes resulted in enhances ascorbic acid accumulation in tomato fruit (Zhang et al., 2011).
Similarly to ascorbic acid, the opportunity of engineering folate accumulation in tomato fruit has been mostly overlooked and only a few attempts gave rise to successful outcomes. In order to increase pteridines, which act as folate precursors and are synthesized from
Comprehensively, within genetic engineering strategies for crop improvements, the most striking advances so far have involved plants engineered to produce missing nutrients or increase the level of nutrients that are already synthesized. An important trend is to move away from plants engineered to produce single nutritional compounds towards those simultaneously engineered to produce multiple nutrients, a development made possible by the increasing use of multigene engineering or regulative genetic element with pleiotropic effects.
|induced parthenocarpy||Goetz et al., 2007; Wang et al., 2005|
|Firmness||single biosynthetic key gene||reduced softening||Langley et al|
|reduced shelf-life||Tieman and Handa, 1994|
|reduced firmness||Brummell et al., 1999|
|β-galactosidase||increased firmness||Smith et al.,|
|Size||dosage series of a single gene||increased size||Frary et al., 2000|
|Flavour||heterologous single gene||thaumatin||enhanced flavour||Bartoszewski et al., 2003|
|Flavour and aroma||heterologous single gene for diverting biosynthetic flux||changes in flavor & aroma||Davidovich-Rikanati et al., 2007|
|single biosynthetic key gene||Increased/|
decreased 2-phenylacetaldehyd, 2-phenylethanol, and 1-nitro-2-phenylethane
|Tieman et al., 2006|
|Soluble solids content||single biosynthetic|
|reduced sugars accumulation||Zanor et al., 2009|
|Carotenoid content||single biosynthetic|
|increased phytoene & carotenoids||Enfissi et al., 2005|
|increased carotenoids||Fraser et al., 2002, 2007; Wurbs et al., 2007|
|increased carotenoids||Römer et al., 2000|
lycopene & β-carotene
|Rosati et al., 2000|
D’Ambrosio et al., 2004; Ronen et al., 2000
|genes targeting biosynthetic steps||β-cryptoxanthin|
|Dharmapuri et al., 2002|
|increased carotenoid||Giliberto et al., 2005|
|increased carotenoid and flavonoid||Liu et al., 2004; Wang et al., 2008; Davuluri et al., 2005|
|increased carotenoids and volatiles||Smikin et al., 2007|
|Flavonoid content||single biosynthetic|
|increased fruit peel flavonol||Muir et al., 2001|
|genes targeting biosynthetic steps||increased flavonols||Colliver et al., 2002|
gene/genes for diverting flux
|accumulation of resveratrol, deoxychalcones,|
|Schijlen et al., 2006|
|single TF||accumulation of flavonols||Adato et al., 2009|
|Multiple TFs||high levels of anthocyanins||Butelli et al., 2008|
|Ascorbic acid content||single biosynthetic|
decreased fruit ascorbic acid
|Garcia et al., 2009; Gilbert et al., 2011; Zhang et al., 2011|
|genes targeting consecutive biosynthetic steps||increased fruit folate||Diaz de la Garza et al., 2004; 2007|
4.2. Production of pharmaceutical proteins
Genetically modified plants are currently being evaluated as promising alternative for the production of recombinant proteins and antigens. Major advantages of plant-made pharmaceuticals include low cost of production, higher scale-up capacity and lack of risk of contamination with mammalian pathogens. Several antigenic proteins have been produced in plant, examples are plant-made vaccines against smallpox, HIV and HPV (Human Papilloma Virus) (Lenzi et al., 2008; Rigano et al., 2009; Scotti et al., 2009). In addition, transgenic plants can represent a suitable vehicle for oral delivery of pharmaceuticals since the plant cell wall protects the recombinant antigen in the harsh condition of stomach and intestine (Sharma et al., 2008a). The delivery of vaccines to mucosal surface makes immunization practise safe and acceptable and is capable of inducing both humoral and cell-mediated immune responses (Salyaev et al., 2010). The production of plant-made mucosal vaccines eliminates needle-associated risks and downstream processing of traditional vaccines such as purification, sterilization and refrigeration. Recently, in addition to other systems, tomato plants have been used as vehicles for the expression and oral delivery of vaccines since tomato is edible, generates abundant biomass at low cost, has flexible growth conditions and contains the natural adjuvant α-tomatine (Salyaev et al., 2010; Soria-Guerra et al., 2011). In this regards, it is noteworthy the work from Zhang and colleagues (2006) that expressed the recombinant Norwalk virus capsid protein in tomato and potato and demonstrated that, although in mice oral immunization with both dried tomato fruit and potato tuber elicited systemic and mucosal antibody responses, the recombinant vaccine in transgenic tomato fruit, especially in air-dried material, was a more potent oral immunogen than potato. The authors speculated that the robust immunogenicity of tomato-derived vaccines was due to natural bioencapsulation by the plant cell matrix and membrane systems, larger amount of smaller 23 nm Virus-like particles and the presence of the natural adjuvant α-tomatine. In this paragraph, we will describe several examples of pharmaceuticals produced in tomato plants focusing on the most recently reported studies.
Several studies reported the production of transgenic tomato plants for the expression of viral antigens. In 2008, Perea Arango and colleagues reported high-level expression of the entire coding region of the nucleoprotein (N) gene of rabies virus in transgenic tomato plants. When mice were immunized both intraperitoneally (i.p.) and orally with the tomato-made N protein, the antibody titer of mice immunized i.p. was at least four times higher than that of mice immunized orally. In addition, only mice immunized i.p. were partially protected against a peripheral virus challenge. In the same year, Pan et al. (2008) described the production of genetically modified tomato plants that expressed the structural polyprotein, P1-2A, and protease, 3C, from foot-and-mouth disease virus (FMDV). Guinea pigs vaccinated intramuscularly with foliar extracts from the transgenic material developed a virus-specific antibody response and were protected against a challenge infection. Recently, in order to develop a vaccine against HPV Paz De la Rosa et al. (2009) expressed in tomato plants chimeric particles containing the HPV 16 L1 sequence fused to a string of T-cell epitopes from HPV 16 E6 and E7 proteins. L1 fused to the string of epitopes was able to assemble into chimeric VLPs (Virus-like particles); in addition, intraperitoneal administration in mice of the transgenic material was able to induce both neutralizing antibodies against the viral particle and a cytotoxic T-lymphocytes activity against the epitopes. Up to date, several groups investigated the production of a mucosal vaccine against HIV and HBV (Hepatitis B virus) in genetically modified tomato plants (Lou et al., 2007; Salyaev et al., 2010; Zhou et al., 2008). For instance, Shchelkunov et al. (2006) investigated the production of transgenic plants expressing a synthetic chimeric gene,
Bacterial antigens have also been expressed in transgenic tomato plants. Alvarez and colleagues (2006) expressed in transgenic tomato plants the FI-V antigen fusion protein for the production of a vaccine against pneumonic and bubonic plague. The authors tested the immunogenicity of the tomato-made vaccine in mice which were primed subcutaneously with bacterially produced F1-V and boosted orally with freeze-dried, powdered transgenic tomato fruit and demonstrated that the vaccine elicited IgG1 in serum and mucosal IgA in fecal pellets. In 2007, Soria-Guerra and collegues expressed in tomato a plant-optimized synthetic gene encoding the recombinant polypeptide sDTP (diphtheria-pertussis-tetanus), containing six DTP immunoprotective exotoxin epitopes and two adjuvants in order to develop an edible multicomponent DPT vaccine. Recently, the same group examined whether immunization of mice fed with freeze-dried tomato material elicited specific antibody responses. Sera of immunized mice tested for IgG antibody response to pertussis, tetanus and diphtheria toxin showed responses to the foreign antigens; in addition, high response of IgA against tetanus toxin was evident in gut (Soria-Guerra et al., 2011). In addition, several studies investigated the feasibility of production of a safe, inexpensive plant-based mucosal vaccine against cholera. For instance, Jang et al. (2007) expressed the Cholera toxin B subunit (CTB) in transgenic tomato fruits and demonstrated the immunogenicity of the tomato-made vaccine in mice. In alternative, Sharma and colleagues (2008b) produced the toxin co-regulated pilus subunit A (TCPA) of
Another advantage of using transgenic plants for the production of recombinant protein of biopharmaceutical and industrial importance is that plant cells are able to perform complex post-translational modification, including glycosylation (Agarwal et al., 2008). In this regard, the feasibility of expression of glycosylated and biologically active recombinant human (-1-antitrypsin (AAT) protein in transgenic tomato plants was demonstrated. In this study, in order to achieve high-level expression of recombinant protein in transgenic plant cells, the gene encoding human AAT protein was optimized by codon adjustment and elimination of mRNA destabilizing sequences. In addition, the synthetic gene was expressed with different signal sequences, translation initiation context sequence, Alfalfa mosaic virus UTR (untranslated region) at 5’ end and ER (endoplasmic reticulum) retention signal sequence (KDEL) at 3’ end. The modified gene driven by CaMV35S duplicated enhancer promoter resulted in high-level expression (up to 1.55% of TSP) of recombinant protein in transgenic tomato plants. Elias-Lopez et al. (2008) described the production of transgenic tomato plants expressing interleukin-12. BALB/c mice were infected with either
Another alternative for the production of recombinant antigens in plant cells is transgene expression from the plastid genome. Chloroplast transformation offers a number of advantages, including the potential to accumulate enormous amounts of recombinant protein, uniform transgene expression rates, no gene silencing and transgene containment. Recently, Zhou et al. (2008) expressed HIV antigens p24 and Nef from tomato`s plastid genome. In tomato, antigen accumulation reached values of approximately 40% of total leaf protein. When the authors determined p24-Nef accumulation in fruits they found that although green tomatoes accumulated the HIV antigens to approximately 2.5% of the TSP, there was no expression in ripe fruits. The authors speculated that this was due to the presence in red-fruited tomatoes of chromoplasts that, compared to chloroplasts, are usually less active in plastid gene expression.
Up to date, several studies demonstrated the feasibility of using tomato plants as vehicles for the production of pharmaceuticals. One drawback of a tomato-made vaccine could be the short shelf-life of fresh fruits. To provide antigen stability during storage, food-processing techniques, such as freeze-drying, could be applied to transgenic tomato fruits expressing recombinant proteins. Freeze-dried plant material could be stored for long time and consumed without cooking; in addition, this technique could allow to standardize and concentrate the plant-made vaccine. Several studies applied this technique to vaccine produced in transgenic tomato and demonstrated that freeze-dried produced stable formulations for oral delivery (Alvarez et al., 2006; Salyaev et al., 2010; Shchelkunov et al., 2006; Soria-Guerra et al., 2011; Zhang et al., 2006).
In the present review, we underlined the role of genetic transformation as method to improve fruit quality and pharmaceutical production. In addition, we highlighted the double role of genetic transformation as tool for biotechnology applications and functional analyses of genes of interest (Figure 1). For tomato, these approaches are feasible following strategies of gene/QTL identification based on the use of genetic and genomic resources today available for this species.
Nowadays, European politicians often debate about perceived risks of genetically modified crops, while ignoring potential benefits; therefore, it is highly unlikely that engineered crops will be adopted in the short-to-medium term.
Considering these constraints, mutants could be envisaged as valid alternative to engineer tomato plants for enhanced fruit quality (Figure 1). Mutants could be selected from natural variation or generated using different approaches. In addition, if the mutant exhibits superior alleles, it could be used as improved genotypes or as donor parent in backcrossing breeding schemes to deliver the desirable trait. The isogenic mutant resources available today for tomato are useful for dissecting the mechanisms underlying mutant phenotypes, and such mutagenized populations are also being used to develop targeting induced local lesions in genomes (TILLING) platforms, which represent a high-throughput genetic strategy to screen for point mutations in specific regions of targeted genes, and to validate gene function (McCallum et al., 2000).
Another alternative approach to obtain tomato with desirable traits is to discover gene markers that discriminate contrasting alleles in genes or QTLs that control the trait(s) of interest (Figure 1). Following their identiﬁcation, useful genes or QTLs can be introgressed into desirable genetic backgrounds via Marker Assisted Selection (MAS), where the selection for a trait is based on the genotype rather that the trait itself (Foolad, 2007). The knowledge of the tomato genome sequence dramatically enhances identification of novel molecular markers. Indeed we can envisage that, notwithstanding the implementation of recently developed Next Generation Sequencing technologies, the routine application of markers in tomato breeding will increase (Varshney et al., 2009).
In conclusion, the use of different approaches, such as tomato genetic transformation, exploiting of mutants and identification of allele-specific markers, could not only speed up the process of gene transfer, but it could also allow pyramiding of desirable genes and QTLs from different genetic backgrounds. The rapid integration of new alleles in elite tomato lines will allow new cultivars with desirable traits to enter the market in a shorter time compared to cultivar obtained through traditional breeding.
Contribution no.Book 007 from the DISSPAPA
Adato A. Mandel T. Mintz-Oron S. Venger I. Levy D. Yativ M. Dominguez E. Wang Z. De Vos R. C. Jetter R. Schreiber L. Heredia A. Rogachev I. Aharoni A. 2009Fruit-surface flavonoid accumulation in tomato is controlled by a SlMYB12-regulated transcriptional network.
Agarwal S. Singh R. Sanyal I. Amla D. V. 2008Expression of modified gene encoding functional human α-1-antitrypsin protein in transgenic tomato plants.
Alvarez M. L. Pinyeard H. L. Crisantes J. D. Rigano M. M. Pinkhasov J. Walsmley A. M. Mason H. S. Cardineau G. A. 2006Plant-made subunit vaccine against pneumonic and bubonic plague is orally immunogenic in mice.
Apel W. Bock R. 2009Enhancement of carotenoid biosynthesis in transplastomic tomatoes by induced lycopene-to-provitamin A conversion.
Bai Y. Lindhout P. 2007Domestication and Breeding of Tomatoes: What have We Gained and What Can We Gain in the Future?
Balaji V. Smart C. D. 2011Over-expression of snakin-2 and extensin-like protein genes restricts pathogen invasiveness and enhances tolerance to Clavibacter michiganensis subsp. michiganensis in transgenic tomato (
Barone A. Chiusano M. L. Ercolano M. R. Giuliano G. Grandillo S. Frusciante L. 2008Structural and functional genomics of tomato.
Barone A. Di Matteo A. Carputo D. Frusciante L. 2009High-throughput genomics enhances tomato breeding efficiency.
Bartoszewski G. Niedziela A. Szwacka M. Niemirowicz-Szczytt K. 2003Modification of tomato taste in transgenic plants carrying a thaumatin gene from Thaumatococcus daniellii Benth. Plant Breeding, 122 4 347 351
Baxter C. J. Carrari F. Bauke A. Overy S. Hill S. A. Quick P. W. Fernie A. R. Sweetlove L. J. 2005aFruit carbohydrate metabolism in an introgression line of tomato with increased fruit soluble solids.
Baxter C. J. Sabar M. Quick W. P. Sweetlove L. J. 2005bComparison of changes in fruit gene expression in tomato introgression lines provides evidence of genome-wide transcriptional changes and reveals links to mapped QTLs and described traits.
Bock R. Warzecha H. 2010Solar-powered factories for new vaccines and antibiotics.
Bovy A. de Vos R. Kemper M. Schijlen E. Pertejo M. A. Muir S. Collins G. Robinson S. Verhoeyen M. Hughes S. Santos-Buelga C. van Tunen A. 2002High flavonol tomatoes resulting from the heterologous expression of the maize transcription factor genes Lc and C1.
Brummell D. A. Harpster M. H. Civello P. M. Palys J. M. Bennett A. B. Dunsmuir P. 1999Modification of Expansin Protein Abundance in Tomato Fruit Alters Softening and Cell Wall Polymer Metabolism during Ripening.
Butelli E. Titta L. Giorgio M. Mock H. P. Matros A. Peterek S. Schijlen E. G. Hall R. D. Bovy A. G. Luo J. Martin C. 2008Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors.
Cardi T. Lenzi P. Maliga P. 2010Chloroplasts as expression platforms for plant-produced vaccines.
Chen H. Chang M. Chiang B. Jeng S. 2006Oral immunization of mice using transgenic tomato fruit expressing VP1 protein from enterovirus 71.
Colliver S. Bovy A. Collins G. Muir S. Robinson S. de Vos C. H. R. Verhoeyen M. E. 2002Improving the nutritional content of tomatoes through reprogramming their flavonoid biosynthetic pathway.
Cueno M. E. Hibi Y. Karamatsu K. Yasutomi Y. Imai K. Laurena A. C. Okamoto T. 2010Preferential expression and immunogenicity of HIV-1 Tat fusion protein expressed in tomato plant.
D’Ambrosio C. Giorio G. Marino I. Merendino A. Petrozza A. Salﬁ L. Stigliani A. L. Cellini F. 2004Virtually complete conversion of lycopene into β-carotene in fruits of tomato plants transformed with the tomato lycopene β-cyclase (tlcy-b) cDNA.
Dan Y. Yan H. Munyikw T. Dong J. Zhang Y. Armstrong C. L. 2006MicroTom- a high-throughput model transformation system for functional genomics.
Davidovich-Rikanati R. Sitrit Y. Tadmor Y. Iijima Y. Bilenko N. Bar E. Carmona B. Fallik E. Dudai N. Simon J. E. Pichersky E. Lewinsohn E. 2007Enrichment of tomato flavor by diversion of the early plastidial terpenoid pathway.
Davuluri G. R. van Tuinen A. Fraser P. D. Manfredonia A. Newman R. Burgess D. Brummell D. A. King S. R. Palys J. Uhlig J. Bramley P. M. Pennings H. M. Bowler C. 2005Fruit-specific RNAi-mediated suppression of DET1 enhances carotenoid and flavonoid content in tomatoes.
de la Garza R. I. D. Gregory J. F. Hanson A. D. 2007Folate biofortification of tomato fruit.
de la Garza R. I. D. Quinlivan R. Klaus E. P. Basset S. M. Gregory G. J. Hanson A. D. 2004Folate biofortification in tomatoes by engineering the pteridine branch of folate synthesis.
Dharmapuri S. Rosati C. Pallara P. Aquilani R. Bouvier F. Camara B. Giuliano G. 2002Metabolic engineering of xanthophyll content in tomato fruits.
Di Matteo A. Sacco A. Anacleria M. Pezzotti M. Delledonne M. Ferrarini A. Frusciante L. Barone A. 2010The ascorbic acid content of tomato fruits is associated with the expression of genes involved in pectin degradation.
Dinesh-Kumar S. P. Anandalakshmi R. Marathe R. Schiff M. Liu Y. 2003Virus-Induced gene silencing.
Diretto G. Al-Babili S. Tavazza R. Papacchioli V. Beyer P. Giuliano G. 2007Metabolic engineering of potato carotenoid content through tuber-speciﬁc overexpression of a bacterial mini-pathway.
Elías-López A. L. Marquina B. Gutiérrez-Ortega A. Aguilar D. Gomez-Lim M. Hernández-Pando R. 2008Transgenic tomato expressing interleukin-12 has a therapeutic effect in a murine model of progressive pulmonary tuberculosis.
Emmanuel E. Levy A. A. 2002Tomato mutants as tools for functional genomics.
Enfissi E. M. A. Fraser P. D. Lois L. M. Boronat A. Schuch W. Bramley P. M. 2005Metabolic engineering of the mevalonate and non-mevalonate isopentenyl diphosphate-forming pathways for the production of health-promoting isoprenoids.
Eshed Y. Zamir D. 1995An introgression line population of Lycopersicon pennellii in the cultivated tomato enables the identification and fine mapping of yield-associated QTL.
Estornell L. H. Orzaez D. Lopez-Pena L. Pineda B. Anton M. T. Moreno V. Granell A. 2009A multisite gateway-based toolkit for targeted gene expression and hairpin RNA silencing in tomato fruits.
Fernandez A. I. Viron N. Alhagdow M. Karimi M. Jones M. Amsellem Z. Sicard A. Czerednik A. Angenent G. Grierson D. May S. Seymour G. Eshed Y. Lemaire-Chamkey M. Rothan C. Hilson P. 2009Flexible tools for gene expression and silencing in tomato.
Foolad M. R. 2007Genome Mapping and Molecular Breeding of Tomato.
Frary A. Nesbitt T. C. Grandillo S. van der Knaap E. Cong B. Liu J. Meller J. Elber R. Alpert K. B. Tanksley S. D. 2000fw2.2: A Quantitative Trait Locus Key to the Evolution of Tomato Fruit Size.
Frary A. Xu Y. Liu J. et al. 2005Development of a set of PCR-based anchor markers encompassing the tomato genome and evaluation of their usefulness for genetics and breeding experiments.
Fraser P. D. Enfissi E. M. Bramley P. M. 2009Genetic engineering of carotenoid formation in tomato fruit and the potential application of systems and synthetic biology approaches. Archives of Biochemistry and Biophysics, 483 196 204
Fraser P. D. Enfissi E. M. Halket J. M. Truesdale M. R. Yu D. Gerrish C. Bramley P. M. 2007Manipulation of phytoene levels in tomato fruit: effects on isoprenoids, plastids, and intermediary metabolism.
Fraser P. D. Roemer S. Shipton C. A. Mills P. B. Kiano J. W. Misawa N. Drake R. G. Schuch W. Bramley P. M. 2002Evaluation of transgenic tomato plants expressing an additional phytoene synthase in a fruit-specific manner.
Fray R. G. Wallace A. Fraser P. D. Valero D. Hedden P. Bramley P. M. Grierson D. 1995Constitutive expression of a fruit phytoene synthase gene in transgenic tomatoes causes dwarfism by redirecting metabolites from the gibberellin pathway.
Fridman E. Carrari F. Liu Y. S. Fernie A. R. Zamir D. 2004Zooming in on a quantitative trait for tomato yield using interspecific introgressions.
Fridman E. Pleban T. Zamir D. 2000A recombination hotspot delimits a wild-species quantitative trait locus for tomato sugar content to 484 bp within an invertase gene.
Fuentes A. D. Ramos P. L. Sanchez Y. Callard D. Ferreira A. Tiel K. Cobas K. Rodriguez R. Borroto C. Doreste V. Pujol M. 2008A transformation procedure for recalcitrant tomato by addressing transgenic plant-recovery limiting factors. Biotechnology Journal, 3 1088 1093
H. B. J.; van Loo, E. N.; Visser, R. G. F. & Bachem, C. W. B. ( Gady A. L. F. Hermans F. W. K. Van de Wal M. 2009Implementation of two high through-put tech- niques in a novel application: detecting point mutations in large EMS mutated plant populations.
Garcia V. Stevens R. Gil L. Gilbert L. Gest N. Petit J. Faurobert M. Maucourt M. Deborde C. Moing A. Poessel J. L. Jacob D. Bouchet J. P. Giraudel J. L. Gouble B. Page D. Alhagdow M. Massot C. Gautier H. Lemaire-Chamley M. Rolin D. Usadel B. Lahaye M. Causse M. Baldet P. Rothan C. 2009An integrative genomics approach for deciphering the complex interactions between ascorbate metabolism and fruit growth and composition in tomato.
Gilbert L. Alhagdow M. Nunes-Nesi A. Quemener B. Guillon F. Bouchet B. Faurobert M. Gouble B. Page D. Garcia V. Petit J. Stevens R. Causse M. Fernie A. R. Lahaye M. Rothan C. Baldet P. 2009GDP-D-mannose 3,5-epimerase (GME) plays a key role at the intersection of ascorbate and non-cellulosic cell-wall biosynthesis in tomato.
Giliberto L. Perrotta G. Pallara P. Weller J. L. Fraser P. D. Bramley P. M. Fiore A. Tavazza M. Giuliano G. 2005Manipulation of the Blue Light Photoreceptor Cryptochrome 2 in Tomato Affects Vegetative Development, Flowering Time, and Fruit Antioxidant Content.
Giovannoni J. J. 2007Fruit ripening mutants yield insights into ripening control.
Goetz M. Hooper L. C. Johnson S. D. Rodrigues J. C. Vivian-Smith A. Koltunow A. M. 2007Expression of aberrant forms of AUXIN RESPONSE FACTOR8 stimulates parthenocarpy in Arabidopsis and tomato.
Grandillo S. Tanksley S. D. Zamir D. 2008Exploitation of natural biodiversity through genomics,
Gupta V. Mathur S. Solanke A. U. Sharma M. K. Kumar R. Vyas S. Khurana P. Khurana J. P. Tyagi A. K. Sharma A. K. 2009Genome analysis and genetic enhancement of tomato.
Gur A. Zamir D. 2004Unused natural variation can lift yield barriers in plant breeding.
Jang X. He Z. Peng Z. Qi Y. Chen Q. Yu S. 2007Cholera toxin B protein in transgenic tomato fruit induces systemic immune response in mice.
Khan R. S. Nakamura I. Mii M. 2011Development of disease-resistant marker-free tomato by R/RS site-specific recombination.
Kramer M. Sanders R. Bolkan H. Waters C. Sheeny R. E. Hiatt W. R. 1992Postharvest evaluation of transgenic tomatoes with reduced levels of polygalacturonase: processing, firmness and disease resistance.
Lai L. Huang T. Wang Y. Liu Y. Zhang J. Song Y. 2009The expression of analgesic-antitumor peptide (AGAP) from Chinese
Langley K. R. Martin A. Stenning R. Murray A. J. Hobson G. E. Schuch W. W. Bird C. R. 1994Mechanical and optical assessment of the ripening of tomato fruit with reduced polygalacturonase activity. Journal of the Science of Food and Agriculture, 66 4 547 554
Lenzi P. Scotti N. Alagna F. Tornesello M. L. Pompa A. Vitale A. De Stradis A. Monti L. Grillo S. Buonaguro F. M. Maliga P. Cardi T. 2008Translational fusion of chloroplast-expressed human papillomavirus type 16 L1 capsid protein enhances antigen accumulation in transplastomic tobacco.
Lippman Z. B. Semel Y. Zamir D. 2007An integrated view of quantitative trait variation using tomato interspecific introgression lines.
Liu J. Cong B. Tanksley S. D. 2003Generation and analysis of an artificial gene dosage series in tomato to study the mechanisms by which the cloned quantitative trait locus fw2.2 controls fruit size.
Liu Y. Roof S. Ye Z. Barry C. van Tuinen A. Vrebalov J. Bowler C. Giovannoni J. 2004Manipulation of light signal transduction as a means of modifying fruit nutritional quality in tomato.
Liu Y. Schiff M. Dinesh-Kumar S. P. 2002Virus-Induced gene silencing in tomato.
Lou X. Yao Q. Zhang Z. Peng R. Xiong A. Wang H. 2007Expression of the human hepatitis B virus large surface antigen gene in transgenic tomato plants.
Mc Callum C. M. Comai L. Greene E. A. Henikoff S. 2000Targeting induced local lesions in genomes (TILLING) for plant functional genomics.
Menda N. Semel Y. Peled D. Eshed Y. Zamir D. 2004In silico screening of a saturated mutation library of tomato.
Minoia S. Petrozza A. D’Onofrio O. Piron F. Mosca G. Sozio G. Cellini F. Bendahmane A. Carriero F. 2010A new mutant genetic resource for tomato crop improvement by TILLING technology.
Mueller L. A. Lankhorst R. K. Tanksley S. D. et al. 2009A Snapshot of the Emerging Tomato Genome Sequence.
Mueller L. A. Tanksley S. D. Giovannoni J. J. et al. 2005The Tomato Sequencing Project, the first cornerstone of the International Solanaceae Project (SOL).
Muir S. R. Collins G. J. Robinson S. Hughes S. G. Bovy A. G. de Vos C. H. van Tunen A. J. Verhoeyen M. E. 2001Overexpression of petunia chalcone isomerase in tomato results in fruit containing dramatically increased levels of flavonols.
Naqvi S. Farré G. Sanahuja G. Capell T. Zhu C. Christou P. 2009When more is better: multigene engineering in plants.
Neily M. H. Matsukura C. Maucourt M. Bernillon S. Deborde C. Moing A. Yin Y. G. Saito T. Mori K. Asamizu E. Rolin D. Moriguchi T. Ezura H. 2011Enhanced polyamine accumulation alters carotenoid metabolism at the transcriptional level in tomato fruit over-expressing spermidine synthase.
Orzaez D. Granell A. 2009Reverse genetics and transient gene expression in fleshy fruits.
Orzaez D. Medina A. Torre S. Fernandez-Moreno J. P. Rambla J. L. Fernandez-del-Carmen A. Butelli E. Martin C. Granell A. 2009A visual-reporter system for virus-induced gene silencing in tomato fruit based on anthocyanin accumulation.
Orzaez D. Mirabel S. Wieland W. H. Granell A. 2006Agroinjection of tomato fruits. A tool for rapid functional analysis of transgenes directly in fruit.
Pan L. Zhang Y. Wang Y. Wang B. Wang W. Fang Y. Jiang S. Lv J. Wang W. Sun Y. Xie Q. 2008Foliar extracts from transgenic tomato plants expressing the structural polyprotein, P1-2A, and protease, 3C, from foot-and-mouth disease virus elicit a protective response in guinea pigs.
Panthee D. R. Chen F. 2010Genomics of Fungal Disease Resistance in Tomato.
Paz la Rosa. G. Monroy-Garcia A. de Lourdes-Garcia Mora. M. Reynaga Peña. C. G. Hernández-Montes J. Weiss-Steider B. Gómez Lim. A. M. 2009An HPV 16 L1-based chimeric human papilloma virus-like particles containing a string of epitopes produced in plants is able to elicit humoral and cytotoxic T-cell activity in mice.
Peña Ramírez. Y. J. Tasciotti E. Gutierrez-Ortega A. Donayre Torres. A. J. Olivera Flores. M. T. Giacca M. Gómez Lim. M. A. 2007Fruit-specific expression of the human immunodeficiency virus type 1 tat gene in tomato plants and its immunogenic potential in mice.
Gomez Lim, M.A. ( Perea Arango. I. Loza Rubio. E. Rojas Anaya. E. Olivera Flores. T. Gonzalez la Vara. 2008Expression of the rabies virus nucleoprotein in plants at high-levels and evaluation of immune responses in mice.
Pineda B. Gimenez-Caminero E. Garcia-Sogo B. Anton M. T. Atares A. Capel J. et al. 2010Genetic and physiological charac- terization of the arlequin insertional mutant reveals a key regulator of reproductive development in tomato.
Pino L. E. Lombardi-Crestana S. Azevedo M. S. Scotton D. C. Borgo L. Quecini V. Figueira A. Peres L. E. P. 2010The
Piron F. Nicolai M. Minoia S. Piednoir E. Moretti A. Salgues A. Zamir D. Caranta C. Bendahmane A. 2010An induced mutation in tomato eIF4E leads to immunity to two potyviruses.
Qiu D. Diretto G. Tavarza R. Giuliano G. 2007Improved protocol for Agrobacterium mediated transformation of tomato and production of transgenic plants containing carotenoid biosynthetic gene
Ralley L. Enfissi E. M. Misawa N. Schuch W. Bramley P. M. Fraser P. D. 2004Metabolic engineering of ketocarotenoid formation in higher plants.
Rein D. Schijlen E. Kooistra T. Herbers K. Verschuren L. Hall R. Sonnewald U. Bovy A. Kleemann R. 2006Transgenic flavonoid tomato intake reduces C- reactive protein in human C-reactive protein transgenic mice more than wild-type tomato.
Rigano M. M. Manna C. Giulini A. Pedrazzini E. Capobianchi M. Castilletti C. Di Caro A. Ippolito G. Beggio P. De Giuli Morghen. C. Monti L. Vitale A. Cardi T. (2009 2009Transgenic chloroplasts are efficient sites for high-yield production of the vaccinia virus envelope protein A27L in plant cells.
Römer S. Fraser P. D. Kiano J. W. Shipton C. A. Mills P. B. Drake R. Schuch W. Bramley P. M. 2000Elevation of the provitamin A content of transgenic tomato plants.
Ronen G. Carmel-Goren L. Zamir D. Hirschberg J. 2000An alternative pathway to β-carotene formation in plant chromoplasts discovered by map-based cloning of beta and old-gold color mutations in tomato.
Rosati C. Aquilani R. Dharmapuri S. Pallara P. Marusic C. Tavazza R. Bouvier F. Camara B. Giuliano G. 2000Metabolic engineering of β-carotene and lycopene content in tomato fruit.
Ruf S. Hermann M. Berger I. J. Carrer H. Bock R. 2001Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit.
Saito T. Ariizumi T. Okabe Y. Asamizu E. Hiwasa-Tanase K. Fukuda N. Mizoguchi T. Yamazaki Y. Aoki K. Ezura H. 2011TOMATOMA: A Novel Tomato Mutant Database Distributing Micro-Tom Mutant Collections.
Salyaev R. K. Rigano M. M. Rekoslavskaya N. I. 2010Development of plant-based mucosal vaccines against widespread infectious diseases.
Schauer N. Semel Y. Roessner U. Gur A. Balbo I. Carrari F. Pleban T. Perez-Melis A. Bruedigam C. Kopka J. Willmitzer L. Zamir D. Fernie A. R. 2006Comprehensive metabolic profiling and phenotyping of interspecific introgression lines for tomato improvement.
Schijlen E. de Vos C. H. R. Jonker H. van den Broeck. H. Molthoff J. van Tunen A. Martens S. Bovy A. 2006Pathway engineering for healthy phytochemicals leading to the production of novel flavonoids in tomato fruit.
Scotti N. Alagna F. Ferraiolo E. Formisano G. Sannino L. Buonaguro L. De Stradis A. Vitale A. Monti L. Grillo S. Buonaguro F. M. Cardi T. 2009High-level expression of the HIV-1 Pr55gag polyprotein in transgenic tobacco chloroplasts.
Sharma M. K. Jani D. Thungapathra M. Gautam J. K. Meena L. S. Singh Y. Ghosh A. Tyagi A. K. Sharma A. K. 2008aExpression of accessory colonization factor subunit A (ACFA) of
Sharma M. K. Singh N. K. Jani D. Sisodia R. Thungapathra M. Gautam J. K. Meena L. S. Singh Y. Ghosh A. Tyagi A. K. Sharma A. K. 2008bExpression of toxin co-regulated pilus subunit A (TCPA) of
Sharma M. K. Solanke A. U. Jani D. Singh Y. Sharma A. K. 2009A simple and efficient
Shchelkunov S. N. Salyaev R. K. Posdnyakov S. G. Rekoslavskaya N. I. Nesterov A. E. Ryzhova T. S. Sumtsova V. M. Pakova N. V. Mishutina U. O. Kopytina T. V. Hammond R. W. 2006Immunogenicity of a novel, bivalent, plant-based oral vaccine against hepatitis B and human immunodeficiency viruses.
Simkin A. J. Gaffé J. Alcaraz J. P. Carde J. P. Bramley P. M. Fraser P. D. Kuntz M. 2007Fibrillin influence on plastid ultrastructure and pigment content in tomato fruit.
Smith C. J. S. Watson C. F. Bird C. R. Ray J. Schuch W. Grierson D. 1990Expression of a truncated tomato polygalacturonase gene inhibits expression of the endogenous gene in transgenic plants.
Smith D. L. Abbott J. A. Gross K. C. 2002Down-regulation of tomato beta-galactosidase 4 results in decreased fruit softening.
Soria-Guerra R. E. Rosales-Mendoza S. Márquez-Mercado C. López-Revilla R. Castilli-Collazo R. Alpuche-Solís A. G. 2007Transgenic tomatoes express an antigenic polypeptide containing epitopes of the diphtheria, pertussis and tetanus exotoxins, encoded by a synthetic gene.
Soria-Guerra R. E. Rosales-Mendoza S. Moreno-Fierros L. López-Revilla R. Alpuche-Solís A. G. 2011Oral immunogenicity of tomato derived sDPT polypeptide containing
Sun H. Uchii S. Watanabe S. Ezura H. 2006A highly efficient transformation protocol for Micro-Tom, a model cultivar for tomato functional genomics.
Tieman D. M. Handa A. K. 1994Reduction in pectin methyltransferase activity modifies tissue integrity and cation levels in ripening tomato (Lycopersicon esculentum Mill.) fruits. Plant Physiology, 106 429 436
Tieman D. M. Zeigler M. Schmelz E. A. Taylor M. G. Bliss P. Kirst M. Klee H. J. 2006Identification of loci affecting flavour volatile emissions in tomato fruits.
Van Eck J. Kirk D. D. Walsmley A. M. 2006Tomato (
Varshney R. K. Nayak S. N. May G. D. Jackson S. A. 2009Next-generation sequencing technologies and their implications for crop genetics and breeding.
Verpoorte R. van der Heijdenvan R. Memelink J. 2000Engineering the plant cell factory for secondary metabolite production.
Wang H. Jones B. Li Z. Frasse P. Delalande C. Regad F. Chaabouni S. Latche A. Pech J. C. Bouzayen M. 2005The tomato Aux/IAA transcription factor IAA9 is involved in fruit development and leaf morphogenesis. Plant Cell, 17 2676 2692
Wang S. Liu J. Feng Y. Niu X. Giovannoni J. Liu Y. 2008Altered plastid levels and potential for improved fruit nutrient content by downregulation of the tomato DDB1-interacting protein CUL4. The Plant Journal, 55 1 89 103
Watanabe S. Mizoguchi T. Aoki K. Kubo Y. Mori H. Imanishi S. Yamazaki Y. Shibata D. Ezura H. 2007Ethylmethanesulfonate (EMS) mutagenesis of Solanum lycopersicum cv. Micro-Tom for large-scale mutant screens.
Wroblewski T. Tomczak A. Michelmore R. 2005Optimization of
Wu F. Tanksley S. D. 2010Chromosomal evolution in the plant family Solanaceae.
Wu Y. He Y. Ge X. 2011Functional characterization of the recombinant antimicrobial peptide Trx-Ace-AMP1 and its application on the control of tomato early blight disease.
Wurbs D. Ruf S. Bock B. 2007Contained metabolic engineering in tomatoes by expression of carotenoid biosynthesis genes from the plastid genome.
Yasmeen A. Mirza B. Inayatullah S. Safdar N. Jamil M. Ali S. Choudhry M. F. 2009In planta transformation of tomato.
Youm J. W. Jeon J. H. Kim H. Kim Y. H. Ko K. Joung H. Kim H. 2008Transgenic tomatoes expressing human beta-amyloid for use as a vaccine against Alzheimer`s disease.
Zanor M. I. Osorio S. Nunes-Nesi A. Carrari F. Lohse M. Usadel B. Kuhn C. Bleiss W. Giavalisco P. Willmitzer L. Sulpice R. Zhou Y. H. Fernie A. R. 2009RNA interference of LIN5 in tomato confirms its role in controlling Brix content, uncovers the influence of sugars on the levels of fruit hormones, and demonstrates the importance of sucrose cleavage for normal fruit development and fertility.
Zhang C. Liu J. Zhang Y. Cai X. Gong P. Zhang J. Wang T. Li H. Ye Z. 2011Overexpression of SlGMEs leads to ascorbate accumulation with enhanced oxidative stress, cold, and salt tolerance in tomato.
Zhang X. Buehner N. A. Hutson A. M. Estes M. L. Mason H. S. 2006Tomato is a highly effective vehicle for expression and oral immunization with Norwalk virus capsid protein.
Zhang X. Li H. Zhang J. Zhang C. Gong P. Ziaf K. Xiao F. Ye Z. 2010Expression of artificial microRNAs in tomato confers efficient and stable virus resistance in a cell-autonomous manner.
Zhou F. Badillo-Corona J. A. Karcher D. Gonzalez-Rabade N. Piepenburg K. Borchers A. M. Maloney A. P. Kavanagh T. A. Gray J. C. Bock R. 2008High-level expression of human immunodeficiency virus antigens from the tobacco and tomato plastid genomes.