Transgenic Strawberry Plants Research Articles

Suppressing the rhamnogalacturonan lyase gene FaRGLyase1 preserves RGI pectin degradation and enhances strawberry fruit firmness

Plant rhamnogalacturonan lyases (RGLyases) cleave the backbone of rhamnogalacturonan I (RGI), the “hairy” pectin and polymer of the disaccharide rhamnose (Rha)-galacturonic acid (GalA) with arabinan, galactan or arabinogalactan side chains. It has been suggested that RGLyases could participate in remodeling cell walls during fruit softening, but clear evidence has not been reported. To investigate the role of RGLyases in strawberry softening, a genome-wide analysis of RGLyase genes in the genus Fragaria was performed. Seventeen genes encoding RGLyases with functional domains were identified in Fragaria × ananassa. FaRGLyase1 was the most expressed in the ripe receptacle of cv. Chandler. Transgenic strawberry plants expressing an RNAi sequence of FaRGLyase1 were obtained. Three transgenic lines yielded ripe fruits firmer than controls without other fruit quality parameters being significantly affected. The highest increase in firmness achieved was close to 32%. Cell walls were isolated from ripe fruits of two selected lines. The amount of water-soluble and chelated pectins was higher in transgenic lines than in the control. A carbohydrate microarray study showed a higher abundance of RGI epitopes in pectin fractions and in the cellulose-enriched fraction obtained from transgenic lines. Sixty-seven genes were differentially expressed in transgenic ripe fruits when compared with controls. These genes were involved in various physiological processes, including cell wall remodeling, ion homeostasis, lipid metabolism, protein degradation, stress response, and defense. The transcriptomic changes observed in FaRGLyase1 plants suggest that senescence was delayed in transgenic fruits.

The blue light signal transduction module FaCRY1-FaCOP1-FaHY5 regulates anthocyanin accumulation in cultivated strawberry.

Anthocyanins have important physiological functions and are beneficial to the improvement of fruit quality in strawberry. Light is important for anthocyanin biosynthesis, and specific light quality was identified to promote anthocyanin accumulation in many fruits. However, research on the molecular mechanisms of anthocyanin accumulation regulated by light quality in strawberry remains limited. Here we described the effects of red- and blue-light irradiation on anthocyanin accumulation in strawberry. The results showed that blue light, rather than red light, could lead to the rapid accumulation of anthocyanins after exposure to light for 48 hours. The transcriptional levels of anthocyanin structural and regulatory genes displayed similar trend to the anthocyanin content. To investigate the mechanism of blue light-induced anthocyanin accumulation, the homologs of Arabidopsis blue light signal transduction components, including the blue light photoreceptor FaCRY1, an E3 ubiquitin ligase FaCOP1 and light-responsive factor FaHY5, were cloned from the strawberry cultivar 'Benihoppe'. The protein-protein interaction of FaCRY1-FaCOP1-FaHY5 was revealed by yeast two-hybrid and fluorescence signal assays. Functional complementation analysis showed that overexpression of either FaCOP1 or FaHY5 restored the anthocyanin content and hypocotyl length in corresponding Arabidopsis mutants under blue light. Moreover, dual-luciferase assays showed that FaHY5 could increase the activity of FaRAP (anthocyanin transport gene) promoter and that this function relied on other, likely B-box protein FaBBX22, factors. The overexpression of FaHY5-VP16 (chimeric activator form of FaHY5) and FaBBX22 promoted the accumulation of anthocyanins in transgenic strawberry plants. Further, transcriptomic profiling indicated that the genes involved in the phenylpropanoid biosynthesis pathway were enriched in both FaHY5-VP16-OX and FaBBX22-OX strawberry plants. In summary, our findings provide insights into a mechanism involving the regulation of blue light-induced anthocyanin accumulation via a FaCRY1-FaCOP1-FaHY5 signal transduction module in strawberry.

Heterologous expression of Arabidopsis thaliana rty gene in strawberry (Fragaria \xd7 ananassa Duch.) improves drought tolerance

BackgroundStrawberry (Fragaria × ananassa Duch.) is an important fruit crop worldwide. It was particularly sensitive to drought stress because of their fibrous and shallow root systems. Mutant rty of Arabidopsis thaliana ROOTY (RTY) results in increased endogenous auxin levels, more roots, and shoot growth. It is still unclear whether the rty gene improves stress tolerance in strawberry.Resultsrty gene was isolated from Arabidopsis thaliana and placed under the control of the cauliflower mosaic virus (CaMV) 35S promoter in the pBI121-rty binary vector carrying the selectable marker of neomycin phosphotransferase II (NPT II). Seven transgenic lines were confirmed by PCR and western blot analysis. Accumulations of IAA and ABA were significantly increased in the transgenic plants. The endogenous IAA contents were 46.5 ng g− 1 and 66.0 ng g− 1in control and transgenic plants respectively. The endogenous ABA contents in the control plant were 236.3 ng g− 1 and in transgenic plants were 543.8 ng g− 1. The production of adventitious roots and trichomes were enhanced in the transgenic plants. Furthermore, transcript levels of the genes including IAA and ABA biosynthetic, and stress-responsive genes, were higher in the transgenic plants than in the control plants under drought conditions. Water use efficiency and a reduced water loss rate were enhanced in the transgenic strawberry plants. Additionally, peroxidase and catalase activities were significantly higher in the transgenic plants than in the control plants. The experiment results revealed a novel function for rty related to ABA and drought responses.ConclusionsThe rty gene improved hormone-mediated drought tolerance in transgenic strawberry. The heterologous expression of rty in strawberry improved drought tolerance by promoting auxin and ABA accumulation. These phytohormones together brought about various physiological changes that improved drought tolerance via increased root production, trichome density, and stomatal closure. Our results suggested that a transgenic approach can be used to overcome the inherent trade-off between plant growth and drought tolerance by enhancing water use efficiency and reducing water loss rate under water shortage conditions.

Transcriptomic Analysis in Strawberry Fruits Reveals Active Auxin Biosynthesis and Signaling in the Ripe Receptacle.

The role of auxin in ripening strawberry (Fragaria ×ananassa) fruits has been restricted to the early stages of development where the growth of the receptacle is dependent on the delivery of auxin from the achenes. At later stages, during enlargement of the receptacle, other hormones have been demonstrated to participate to different degrees, from the general involvement of gibberellins and abscisic acid to the more specific of ethylene. Here we report the involvement of auxin at the late stages of receptacle ripening. The auxin content of the receptacle remains constant during ripening. Analysis of the transcriptome of ripening strawberry fruit revealed the changing expression pattern of the genes of auxin synthesis, perception, signaling and transport along with achene and receptacle development from the green to red stage. Specific members of the corresponding gene families show active transcription in the ripe receptacle. For the synthesis of auxin, two genes encoding tryptophan aminotransferases, FaTAA1 and FaTAR2, were expressed in the red receptacle, with FaTAR2 expression peaking at this stage. Transient silencing of this gene in ripening receptacle was accompanied by a diminished responsiveness to auxin. The auxin activity in the ripening receptacle is supported by the DR5-directed expression of a GUS reporter gene in the ripening receptacle of DR5-GUS transgenic strawberry plants. Clustering by co-expression of members of the FaAux/IAA and FaARF families identified five members whose transcriptional activity was increased with the onset of receptacle ripening. Among these, FaAux/IAA11 and FaARF6a appeared, by their expression level and fold-change, as the most likely candidates for their involvement in the auxin activity in the ripening receptacle. The association of the corresponding ARF6 gene in Arabidopsis to cell elongation constitutes a suggestive hypothesis for FaARF6a involvement in the same cellular process in the growing and ripening receptacle.

Evaluation of a MdMYB10/GFP43 fusion gene for its suitability to act as reporter gene in promoter studies in Fragaria vesca L. \u2018R\xfcgen\u2019

A Malus domestica MdMYB10 transcription factor gene was previously used as visible marker for successful plant transformation. We combined the MdMYB10 transcription factor gene with a GFP gene to test its viability as a non-destructive, visual, double reporter system for functional promoter studies in transgenic strawberry plants. The GFP gene was fused to MdMYB10 to provide evidence for promoter activity in red colored cells of transformed plant tissue and to exclude artefacts resulting from stress response or due to other environmental cues. To test this system in a first approach, we evaluated the MdMYB10-GFP43 construct in transgenic strawberries in combination with two constitutive promoters of varying strength, the strong CaMV 35S promoter and a weak flavonoid 3′-hydroxylase (F3′H) promoter isolated from the ornamental plant Cosmos sulphureus. Agrobacterium tumefaciens mediated transformation of Fragaria vesca with the MdMYB10-GFP43 construct combined with the CaMV 35S or F3′H promoter sequences resulted in the regeneration of 6 and 4 transgenic lines, respectively. A complete red coloration of all plant organs was found in four out of six transgenic lines harboring the 35S-MdMYB10-GFP43 construct. Less red coloration of plant organs was found for lines transformed with the F3′H-MdMYB10-GFP43 construct. The MdMYB10 gene shows only limited suitability as a reporter gene for promoter studies in strawberries because weak promoter activity is difficult to distinguish, particularly in tissues showing a strongly colored background such as green leaves. GFP specific fluorescence signals were detectable neither in tissue strongly expressing MdMYB10 nor in green tissue of any transgenic line. The reason for this remained unclear but it can be excluded that it was due to incorrect splicing.

Development of transgenic strawberry plants expressing monellin, a sweet protein

Leaf discs from ‘Yeobong’ and ‘Maehyang’ strawberry plants were used as explants for transformation. The Agrobacterium tumefaciens strain EHA105 harboring the monellin gene under the control of the CaMV 35S promoter was used in co-cultivation experiments. The frequencies of callus formation and plant regeneration from leaf explants after co-cultivation in ‘Yeobong’ were higher than those of ‘Maehyang’. These transgenic plants showed normal growth patterns and flowering. PCR and Southern hybridization confirmed that 1 to 2 copies of the monellin gene were integrated into genome of the transgenic strawberry plants. Northern blot analysis confirm that the transcripts were expressed in transgenic strawberry plants. Although long-term subcultured transgenic strawberry plants showed a phenomenon to escape the transgene, the transformation system established in this study provides new opportunities for genetic improvement of strawberry plants.

Expression of the β-1,3-glucanase gene bgn13.1 from Trichoderma harzianum in strawberry increases tolerance to crown rot diseases but interferes with plant growth.

The expression of antifungal genes from Trichoderma harzianum, mainly chitinases, has been used to confer plant resistance to fungal diseases. However, the biotechnological potential of glucanase genes from Trichoderma has been scarcely assessed. In this research, transgenic strawberry plants expressing the β-1,3-glucanase gene bgn13.1 from T. harzianum, under the control of the CaMV35S promoter, have been generated. After acclimatization, five out of 12 independent lines analysed showed a stunted phenotype when growing in the greenhouse. Moreover, most of the lines displayed a reduced yield due to both a reduction in the number of fruit per plant and a lower fruit size. Several transgenic lines showing higher glucanase activity in leaves than control plants were selected for pathogenicity tests. When inoculated with Colletotrichum acutatum, one of the most important strawberry pathogens, transgenic lines showed lower anthracnose symptoms in leaf and crown than control. In the three lines selected, the percentage of plants showing anthracnose symptoms in crown decreased from 61 % to a mean value of 16.5 %, in control and transgenic lines, respectively. Some transgenic lines also showed an enhanced resistance to Rosellinia necatrix, a soil-borne pathogen causing root and crown rot in strawberry. These results indicate that bgn13.1 from T. harzianum can be used to increase strawberry tolerance to crown rot diseases, although its constitutive expression affects plant growth and fruit yield. Alternative strategies such as the use of tissue specific promoters might avoid the negative effects of bgn13.1 expression in plant performance.

Transcription factors and anthocyanin genes related to low-temperature tolerance in rd29A:RdreB1BI transgenic strawberry

Dehydration-responsive element-binding (DREB) transcription factors play critical roles in plant stress responses and signal transduction. To further understand how DREB regulates genes expression to promote cold-hardiness, Illumina/Solexa sequencing technology was used to compare the transcriptomes of non-transgenic and rd29A:RdreB1BI transgenic strawberry plants exposed to low temperatures. Approximately 3.5 million sequence tags were obtained from non-transgenic (NT) and transgenic (T) strawberry untreated (C) or low-temperature treated (LT) leaf samples. Over 1000 genes were differentially expressed between the NT-C and T-C plants, and also the NT-C and NT-LT, as well as the T-C and T-LT plants. Analysis of the genes up-regulated following low-temperature treatment revealed that the majority are linked to metabolism, biosynthesis, transcription and signal transduction. Uniquely up-regulated transcription factors as well as anthocyanin biosynthetic pathway genes are discussed. Accumulation of anthocyanin in the stolon and the base of the petiole differed between non-treated NT and T plants, and this correlated with gene expression patterns. The differentially expressed genes that encode transcription factors and anthocyanin enzymes may contribute to the cold hardiness of RdreB1BI transgenic strawberry. The transcriptome data provide a valuable resource for further studies of strawberry growth and development and DREB-mediated gene regulation under low-temperature stress.

Ethylene is involved in strawberry fruit ripening in an organ-specific manner.

The fruit of the strawberry Fragaria×ananassa has traditionally been classified as non-climacteric because its ripening process is not governed by ethylene. However, previous studies have reported the timely endogenous production of minor amounts of ethylene by the fruit as well as the differential expression of genes of the ethylene synthesis, reception, and signalling pathways during fruit development. Mining of the Fragaria vesca genome allowed for the identification of the two main ethylene biosynthetic genes, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase. Their expression pattern during fruit ripening was found to be stage and organ (achene or receptacle) specific. Strawberry plants with altered sensitivity to ethylene could be employed to unravel the role of ethylene in the ripening process of the strawberry fruit. To this end, independent lines of transgenic strawberry plants were generated that overexpress the Arabidopsis etr1-1 mutant ethylene receptor, which is a dominant negative allele, causing diminished sensitivity to ethylene. Genes involved in ethylene perception as well as in its related downstream processes, such as flavonoid biosynthesis, pectin metabolism, and volatile biosynthesis, were differently expressed in two transgenic tissues, the achene and the receptacle. The different transcriptional responsiveness of the achene and the receptacle to ethylene was also revealed by the metabolic profiling of the primary metabolites in these two organs. The free amino acid content was higher in the transgenic lines compared with the control in the mature achene, while glucose and fructose, and citric and malic acids were at lower levels. In the receptacle, the most conspicuous change in the transgenic lines was the depletion of the tricarboxylic acid cycle intermediates at the white stage of development, most probably as a consequence of diminished respiration. The results are discussed in the context of the importance of ethylene during strawberry fruit ripening.

Antisense expression of peach mildew resistance locus O (PpMlo1) gene confers cross-species resistance to powdery mildew in Fragaria x ananassa

Powdery mildew (PM) is one of the major plant pathogens. The conventional method of PM control includes frequent use of sulfur-based fungicides adding to production costs and potential harm to the environment. PM remains a major scourge for Rosaceae crops where breeding approaches mainly resort to gene-for-gene resistance. We have tested an alternate source of PM resistance in Rosaceae. Mildew resistance locus O (MLO) has been well studied in barley due to its role in imparting broad spectrum resistance to PM. We identified PpMlo1 (Prunus persica Mlo) in peach and characterized it further to test if a similar mechanism of resistance is conserved in Rosaceae. Due to its recalcitrance in tissue culture, reverse genetic studies involving PpMloI were not feasible in peach. Therefore, Fragaria x ananassa LF9 line, a taxonomic surrogate, was used for functional analysis of PpMlo1. Agrobacterium-mediated transformation yielded transgenic strawberry plants expressing PpMlo1 in sense and antisense orientation. Antisense expression of PpMlo1 in transgenic strawberry plants conferred resistance to Fragaria-specific powdery mildew, Podosphaera macularis. Phylogenetic analysis of 208 putative Mlo gene copies from 35 plant species suggests a large number of duplications of this gene family prior to the divergence of monocots and eudicots, early in eudicot diversification. Our results indicate that the Mlo-based resistance mechanism is functional in Rosaceae, and that Fragaria can be used as a host to test mechanistic function of genes derived from related tree species. To the best of our knowledge, this work is one of the first attempts at testing the potential of using a Mlo-based resistance strategy to combat powdery mildew in Rosaceae.

Development of an efficient transformation method by Agrobacterium tumefaciens and high throughput spray assay to identify transgenic plants for woodland strawberry (Fragaria vesca) using NPTII selection

KEY MESSAGE : We developed an efficient Agrobacterium -mediated transformation method using an Ac/Ds transposon tagging construct for F. vesca and high throughput paromomycin spray assay to identify its transformants for strawberry functional genomics. Genomic resources for Rosaceae species are now readily available, including the Fragaria vesca genome, EST sequences, markers, linkage maps, and physical maps. The Rosaceae Genomic Executive Committee has promoted strawberry as a translational genomics model due to its unique biological features and transformability for fruit trait improvement. Our overall research goal is to use functional genomic and metabolic approaches to pursue high throughput gene discovery in the diploid woodland strawberry. F. vesca offers several advantages of a fleshy fruit typical of most fruit crops, short life cycle (seed to seed in 12-16weeks), small genome size (206Mbb/C), small plant size, self-compatibility, and many seeds per plant. We have developed an efficient Agrobacterium tumefaciens-mediated strawberry transformation method using kanamycin selection, and high throughput paromomycin spray assay to efficiently identify transgenic strawberry plants. Using our kanamycin transformation method, we were able to produce up to 98 independent kanamycin resistant insertional mutant lines using a T-DNA construct carrying an Ac/Ds transposon Launchpad system from a single transformation experiment involving inoculation of 22 leaf explants of F. vesca accession 551572 within approx. 11weeks (from inoculation to soil). Transgenic plants with 1-2 copies of a transgene were confirmed by Southern blot analysis. Using our paromomycin spray assay, transgenic F. vesca plants were rapidly identified within 10days after spraying.

Characterization of the Hormone and Stress-Induced Expression of FaRE1 Retrotransposon Promoter in Strawberry

Retrotransposons are the most abundant mobile elements in the plant genome and seem to play an important role in genome reorganization induced by environmental challenges. Their success in this function depends on the ability of their promoters to regulate plant adaptation to biotic and abiotic stresses. In this study, the promoter region of FaRE1 was amplified in the strawberry genome, and promoter::GUS fusion was constructed. We produced transgenic strawberry plants carrying FaRE1 promoter::GUS-fusion genes, and monitored GUS reporter activity. Histochemical and fluorimetric GUS analysis these plants showed the characteristics of the FaRE1 promoter were activated by either hormones treatments with ABA, NAA, and 2,4-D or cold stress. In addition, we found the GUS reporter was activated in the leaves of transgenic strawberry plants using 5-azaC. These results suggest that the promoter of FaRE1 may act as different signal transduction pathways, allowing FaRE1 retrotransposon to be activated in response to multiples challenges.

A strawberry KNOX gene regulates leaf, flower and meristem architecture.

The KNOTTED-LIKE HOMEODOMAIN (KNOX) genes play a central role in maintenance of the shoot apical meristem. They also contribute to the morphology of simple and compound leaves. In this report we characterize the FaKNOX1 gene from strawberry (Fragaria spp.) and demonstrate its function in trasgenic plants. The FaKNOX1 cDNA was isolated from a cultivated strawberry (F.×ananassa) flower EST library. The sequence is most similar to Class I KNOX genes, and was mapped to linkage group VI of the diploid strawberry genome. Unlike most KNOX genes studied, steady-state transcript levels were highest in flowers and fruits. Transcripts were also detected in emerging leaf primordia and the apical dome. Transgenic strawberry plants suppressing or overexpressing FaKNOX1 exhibited conspicuous changes in plant form. The FaKNOX1 RNAi plants presented a dwarfed phenotype with deeply serrated leaflets and exaggerated petiolules. They also exhibited a high level of cellular disorganization of the shoot apical meristem and leaves. Overexpression of FaKNOX1 caused dwarfed stature with wrinkled leaves. These gain- and loss-of-function assays in strawberry functionally demonstrate the contributions of a KNOX domain protein in a rosaceous species.

Modulation of fruit softening by antisense suppression of endo-β-1,4-glucanase in strawberry

To test the effect of endo-β-1,4-glucanase (EGase) in strawberry, we produced transgenic strawberry plants that contained sense and antisense cDNA encoding strawberry EGase under the CaMV 35S promoter (CaMV35S-P) and the strawberry fruit dominant ascorbate peroxidase (APX) promoter. Independent transgenic lines were generated and the firmness of the fruit was characterized after harvest. Interestingly, transgenic lines that harbored the cDNA antisense under the CaMV 35S promoter were not generated, but transgenic lines that contained sense EGase cDNA (FraCel1) under the CaMV 35S promoter and sense and antisense EGase cDNA under the APX promoter were successfully obtained using a tissue culture system. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that the steady-state transcript levels of CaMV35S-P:FraCel1 sense transgenic lines were dramatically increased in fruit evaluated at all stages, as well as in the leaves. Moreover, real-time PCR analysis demonstrated that the steady-state transcript level of FraCel1 in independent transgenic lines that contained antisense FraCel1 under the APX promoter was reduced in fruit during the turning and red stages when compared with wild-type strawberries. These results were consistent with the firmness of strawberries that contained the antisense FraCel1 under the APX promoter, which was 19–36% greater in the turning stage and 22–25% greater in the red stage when compared to wild-type strawberries. Taken together, these findings indicate that fruit-specific down-regulation of the EGase gene using the APX promoter is an effective technique for increasing fruit firmness in strawberries.

Pre- and post-agroinfection strategies for efficient leaf disk transformation and regeneration of transgenic strawberry plants

Following previously described Agrobacterium tumefaciens-mediated transformation procedures for Fragaria x ananassa Duch. 'Chandler', we undertook several experiments to establish the importance of some parameters affecting transformation. The most important factor that increased the percent recovery of transformants was the introduction of a pre-selection phase, in-between co-cultivation and selection, in which leaf disks were cultured on pre-selection regeneration medium containing validamycin A, timentin, and cefotaxime. The average percentage of leaf disks forming shoots on selection medium containing cefotaxime (250 mg l(-1)) + timentin (250 mg l(-1)) was 5.4% and about three shoots per regenerating leaf disk. Maximum transformation percentage, based on polymerase chain reaction, was 31.25%. Transgene integration and copy number were assessed by Southern hybridization confirming single copy as well as multiple copies of transgene integration in shoots as well as roots separately. This confirmed the non-chimeric nature of these transgenic plants. The system is very promising for the regeneration of genetically transformed cells and obtaining transgenic strawberry plants at high efficiency.

Evaluation of the role of the endo-β-(1,4)-glucanase gene FaEG3 in strawberry fruit softening

In strawberry, Fragaria × ananassa Duch., fruit, two different endo-β-1,4-glucanase genes, FaEG1 and FaEG3, also named Cel1 and Cel2, are expressed during the softening process that occurs during fruit ripening. It has also been suggested that FaEG3, which contains a putative cellulose-binding domain, could play a key role in fruit development, since previous attempts to down-regulate this gene through transgenesis have been unsuccessful. In this investigation, we obtained transgenic strawberry plants containing an antisense sequence of the FaEG3 gene under the control of the 35S promoter. Ripened fruit from transgenic lines (Acel lines) showed large variation in FaEG3 silencing, but fruit firmness was similar to control fruit in all the lines. Two Acel lines showing almost 95% reductions in FaEG3 mRNA levels were selected for further study. In these lines, FaEG3 down-regulation was high, from 78 to 95%, at all fruit developmental stages, whereas FaEG1 was only slightly suppressed. In spite of the high FaEG3 silencing achieved, EGase activity was not modified in ripe fruit. At the cell wall level, walls from transgenic ripe fruit contained a significantly higher amount of the 4 M KOH fraction, which is enriched in hemicellulosic polymers. The analysis of this fraction by size exclusion chromatography showed that transgenic cell walls contained a smaller amount of higher molecular mass polymers than controls. Altogether, these results indicate that FaEG3 does not play a key role either in fruit development or fruit softening. However, its silencing affects the amount and, in a minor way, the size of hemicellulosic polymers.

The polygalacturonase

The loss of firm texture is one of the most characteristic physiological processes that occur during the ripening of fleshy fruits. It is generally accepted that the disassembly of primary cell wall and middle lamella is the main factor involved in fruit softening. In this process, polygalacturonase (PG) has been implicated in the degradation of the polyuronide network in several fruits. However, the minor effect of PG down-regulation on tomato softening, reported during the nineties, minimized the role of this enzyme in softening. Further works in other fruits are challenging this general assumption, as is occurring in strawberry. The strawberry (Fragaria × ananassa) fruit undergoes an extensive and fast softening that limit its shelf life and postharvest. Traditionally, it has also been considered that PG plays a minor role on this process, due to the low PG activity found in ripened strawberry fruits. Transgenic strawberry plants expressing an antisense sequence of the ripening-specific PG gene FaPG1 have been generated to get an insight into the role of this gene in softening. Half of the transgenic lines analyzed yielded fruits significantly firmer than control, without being affected other fruit parameters such as weight, color or soluble solids. The increase on firmness was maintained after several days of posharvest. In these firmer lines, FaPG1 was silenced to 95%, but total PG activity was only minor reduced. At the cell wall level, transgenic fruits contained a higher amount of covalently bound pectins whereas the soluble fraction was diminished. A microarray analysis of genes expressed in ripened receptacle did not show any significant change between control and transgenic fruits. Thus, contrary to the most accepted view, it is concluded that PG plays a key role on pectin metabolism and softening of strawberry fruit.

STILBENE SYNTHASE GENE TRANSFER RESULTED IN DOWN REGULATION OF ENDOGENOUS CHALCONE SYNTHASE IN STRAWBERRY (FRAGARIA × ANANASSA) AND LED TO THE IDENTIFICATION OF NOVEL PHENYLPROPANOID GLUCOSIDES

Modification of plants by introducing the stilbene synthase (STS) gene has often resulted in novel production of the phytoalexin resveratrol, and provided enhanced resistance against several pathogenic fungi. In the present study, a grapevine STS expressing strawberry (Fragaria × ananassa) was investigated for the effect of the transgene on plant metabolism by techniques including quantitative real-time PCR, UPLC-qTOF-MS profiling and NMR analysis. The introduced STS gene caused down-regulation of the strawberry endogenous chalcone synthase (CHS) gene expression. The perturbation of the gene expression was reflected at the metabolite content, as the CHS enzyme downstream products, mainly flavonols, were found at reduced levels concomitantly with the accumulation of the precursor molecules like phenolic acid derivatives. In addition, several previously unidentified metabolites were accumulating in the leaves of the transgenic strawberry plants. A detailed metabolite analysis was pertinent for the detection of unintended consequences of the gene transfer, and eventually provided deeper insight in the phenolic compound metabolism of strawberry.

Transgenic strawberry expressing the taste-modifying protein miraculin

The gene encoding the taste-modifying protein miraculin was introduced under the control of the 35S or El2 promoter into strawberry (Fragaria x ananassa) by Agrobacterium-mediated transformation to produce transgenic plants. Although miraculin was detected in the leaves and fruits of the transgenic plants, the level of accumulation among the transgenic lines, which ranged from 0.5 to 2.0 μg g−1 fresh fruit, was not significantly different and was lower than that in miracle fruits (145 μg g−1 fresh fruit). High levels of miraculin accumulation were detected in the mature fruits. The transgenic lines were subsequently propagated via the runners for three vegetative generations, and miraculin was detected at equal levels in the leaves and fruits of the plants from each generation. In conclusion, although the level of accumulation was not high, miraculin was stably expressed and accumulated in the vegetative progeny of the transgenic strawberry plants.

Evidence of frequent integration of non-T-DNA vector backbone sequences in transgenic strawberry plant

We have studied the occurrence of the integration of non-T-DNA sequences in transgenic strawberry plants obtained through Agrobacterium inoculation. DNA from these plants was subjected to PCR amplification of the sequence of the gene trfA, which is located outside the T-DNA. The percentage of trfA-positive plants varied from 40% to 90%, with a mean of 65.7%. Backbone sequences were confirmed by Southern blot analysis.