Effect of heterologous expression of FT gene from Medicago truncatula in growth and flowering behavior of olive plants.

Olive (Olea europaea L. subsp. europaea) is one of the most important crops of the Mediterranean Basin and temperate areas worldwide. Obtaining new olive varieties adapted to climatic changing conditions and to modern agricultural practices, as well as other traits such as biotic and abiotic stress resistance and increased oil quality, is currently required; however, the long juvenile phase, as in most woody plants, is the bottleneck in olive breeding programs. Overexpression of genes encoding the 'florigen' Flowering Locus T (FT), can cause the loss of the juvenile phase in many perennials including olives. In this investigation, further characterization of three transgenic olive lines containing an FT encoding gene from Medicago truncatula, MtFTa1, under the 35S CaMV promoter, was carried out. While all three lines flowered under in vitro conditions, one of the lines stopped flowering after acclimatisation. In soil, all three lines exhibited a modified plant architecture; e.g., a continuous branching behaviour and a dwarfing growth habit. Gene expression and hormone content in shoot tips, containing the meristems from which this phenotype emerged, were examined. Higher levels of OeTFL1, a gene encoding the flowering repressor TERMINAL FLOWER 1, correlated with lack of flowering. The branching phenotype correlated with higher content of salicylic acid, indole-3-acetic acid and isopentenyl adenosine, and lower content of abscisic acid. The results obtained confirm that heterologous expression of MtFTa1 in olive induced continuous flowering independently of environmental factors, but also modified plant architecture. These phenotypical changes could be related to the altered hormonal content in transgenic plants.

Modification of 13-hydroperoxide lyase expression in olive affects plant growth and results in altered volatile profile.

The C6 aldehydes, alcohols, and the corresponding esters are the most important compounds of virgin olive oil aroma. These C6 volatile compounds are synthesized via the 13-hydroperoxide lyase (13-HPL) branch of the lipoxygenase pathway. In this investigation, a functional analysis of the olive (Olea europaea L.) 13-HPL gene by its overexpression and silencing in olive transgenic lines was carried out. With this aim, sense and RNAi constructs of the olive 13-HPL gene were generated and used for the transformation of embryogenic olive cultures. Leaves from overexpressing lines showed a slight increase in 13-HPL gene expression, whereas RNAi lines exhibited a strong decrease in their transcript levels. Quantification of 13-HPL activity in two overexpressing and two RNAi lines showed a positive correlation with levels of transcripts. Interestingly, RNAi lines showed a high decrease in the content of C6 volatiles linked to a strong increase of C5 volatile compounds, altering the volatile profile in the leaves. In addition, the silencing of the 13-HPL gene severely affected plant growth and development. This investigation demonstrates the role of the 13-HPL gene in the biogenesis of olive volatile compounds and constitutes a functional genomics study in olive related to virgin olive oil quality.

Reinvigoration/Rejuvenation Induced through Micrografting of Tree Species: Signaling through Graft Union.

Trees have a distinctive and generally long juvenile period during which vegetative growth rate is rapid and floral organs do not differentiate. Among trees, the juvenile period can range from 1 year to 15-20 years, although with some forest tree species, it can be longer. Vegetative propagation of trees is usually much easier during the juvenile phase than with mature phase materials. Therefore, reversal of maturity is often necessary in order to obtain materials in which rooting ability has been restored. Micrografting has been developed for trees to address reinvigoration/rejuvenation of elite selections to facilitate vegetative propagation. Generally, shoots obtained after serial grafting have increased rooting competence and develop juvenile traits; in some cases, graft-derived shoots show enhanced in vitro proliferation. Recent advances in graft signaling have shown that several factors, e.g., plant hormones, proteins, and different types of RNA, could be responsible for changes in the scion. The focus of this review includes (1) a discussion of the differences between the juvenile and mature growth phases in trees, (2) successful restoration of juvenile traits through micrografting, and (3) the nature of the different signals passing through the graft union.

Olive (Olea europaea L.) Genetic Transformation: Current Status and Future Prospects.

Olive (Olea europaea L.) is the most characteristic and important oil crop of the Mediterranean region. Traditional olive cultivation is based on few tens cultivars of ancient origin. To improve this crop, novel selections with higher tolerance to biotic and abiotic stress, adaptable to high-density planting systems and resilient to climate change are needed; however, breeding programs are hindered by the long juvenile period of this species and few improved genotypes have been released so far. Genetic transformation could be of great value, in the near future, to develop new varieties or rootstocks in a shorter time; in addition, it has currently become an essential tool for functional genomic studies. The recalcitrance of olive tissues to their in vitro manipulation has been the main bottleneck in the development of genetic transformation procedures in this species; however, some important traits such as fungal resistance, flowering or lipid composition have successfully been manipulated through the genetic transformation of somatic embryos of juvenile or adult origin, providing a proof of the potential role that this technology could have in olive improvement. However, the optimization of these protocols for explants of adult origin is a prerequisite to obtain useful materials for the olive industry. In this review, initially, factors affecting plant regeneration via somatic embryogenesis are discussed. Subsequently, the different transformation approaches explored in olive are reviewed. Finally, transgenic experiments with genes of interest undertaken to manipulate selected traits are discussed.

Exploring the Use of Fruit Callus Culture as a Model System to Study Color Development and Cell Wall Remodeling during Strawberry Fruit Ripening.

Cell cultures derived from strawberry fruit at different developmental stages have been obtained to evaluate their potential use to study different aspects of strawberry ripening. Callus from leaf and cortical tissue of unripe-green, white, and mature-red strawberry fruits were induced in a medium supplemented with 11.3 µM 2,4-dichlorophenoxyacetic acid (2,4-D) under darkness. The transfer of the established callus from darkness to light induced the production of anthocyanin. The replacement of 2,4-D by abscisic acid (ABA) noticeably increased anthocyanin accumulation in green-fruit callus. Cell walls were isolated from the different fruit cell lines and from fruit receptacles at equivalent developmental stages and sequentially fractionated to obtain fractions enriched in soluble pectins, ester bound pectins, xyloglucans (XG), and matrix glycans tightly associated with cellulose microfibrils. These fractions were analyzed by cell wall carbohydrate microarrays. In fruit receptacle samples, pectins were abundant in all fractions, including those enriched in matrix glycans. The amount of pectin increased from green to white stage, and later these carbohydrates were solubilized in red fruit. Apparently, XG content was similar in white and red fruit, but the proportion of galactosylated XG increased in red fruit. Cell wall fractions from callus cultures were enriched in extensin and displayed a minor amount of pectins. Stronger signals of extensin Abs were detected in sodium carbonate fraction, suggesting that these proteins could be linked to pectins. Overall, the results obtained suggest that fruit cell lines could be used to analyze hormonal regulation of color development in strawberry but that the cell wall remodeling process associated with fruit softening might be masked by the high presence of extensin in callus cultures.

Heterologous Expression of the AtNPR1 Gene in Olive and Its Effects on Fungal Tolerance.

The NPR1 gene encodes a key component of systemic acquired resistance (SAR) signaling mediated by salicylic acid (SA). Overexpression of NPR1 confers resistance to biotrophic and hemibiotrophic fungi in several plant species. The NPR1 gene has also been shown to be involved in the crosstalk between SAR signaling and the jasmonic acid-ethylene (JA/Et) pathway, which is involved in the response to necrotrophic fungi. The aim of this research was to generate transgenic olive plants expressing the NPR1 gene from Arabidopsis thaliana to evaluate their differential response to the hemibiotrophic fungus Verticillium dahliae and the necrotroph Rosellinia necatrix. Three transgenic lines expressing the AtNPR1 gene under the control of the constitutive promoter CaMV35S were obtained using an embryogenic line derived from a seed of cv. Picual. After maturation and germination of the transgenic somatic embryos, the plants were micropropagated and acclimated to ex vitro conditions. The level of AtNPR1 expression in the transgenic materials varied greatly among the different lines and was higher in the NPR1-780 line. The expression of AtNPR1 did not alter the growth of transgenic plants either in vitro or in the greenhouse. Different levels of transgene expression also did not affect basal endochitinase activity in the leaves, which was similar to that of control plants. Response to the hemibiotrophic pathogen V. dahliae varied with pathotype. All plants died by 50 days after inoculation with defoliating (D) pathotype V-138, but the response to non-defoliating (ND) strains differed by race: following inoculation with the V-1242 strain (ND, race 2), symptoms appeared after 44-55 days, with line NPR1-780 showing the lowest disease severity index. This line also showed good performance when inoculated with the V-1558 strain (ND, race 1), although the differences from the control were not statistically significant. In response to the necrotroph R. necatrix, all the transgenic lines showed a slight delay in disease development, with mean area under the disease progress curve (AUDPC) values 7-15% lower than that of the control.

Plant Regeneration via Somatic Embryogenesis in Mature Wild Olive Genotypes Resistant to the Defoliating Pathotype of Verticillium dahliae.

Regeneration capacity, via somatic embryogenesis, of four wild olive genotypes differing in their response to defoliating Verticillium dahliae (resistant genotypes StopVert, OutVert, Ac-18 and the susceptible one, Ac-15) has been evaluated. To induce somatic embryogenesis, methodologies previously used in wild or cultivated olive were used. Results revealed the importance of genotype, explant type, and hormonal balance in the induction process. Use of apical buds obtained from micropropagated shoots following a methodology used in cultivated olive (4 days induction in liquid 1/2 MS medium supplemented with 30 µM TDZ-0.54 µM NAA, followed by 8 weeks in basal 1/2 MS medium) was adequate to obtain somatic embryos in two genotypes, StopVert and Ac-18, with a 5.0 and 2.5% induction rates, respectively; however, no embryogenic response was observed in the other two genotypes. Embryogenic cultures were transferred to basal ECO medium supplemented with 0.5 µM 2iP, 0.44 µM BA, and 0.25 µM indole-3-butyric acid (IBA) for further proliferation. Somatic embryos from StopVert were maturated and germinated achieving a 35.4% conversion rate. An analysis of genetic stability on StopVert, using Simple Sequence Repeats (SSRs) and Random Amplified Polymorphic DNA (RAPDs) markers, was carried out in embryogenic callus, plants regenerated from this callus and two controls, micropropagated shoots used as explant source, and the original mother plant. Polymorphism was only observed in the banding pattern generated by RAPDs in 1 of the 10 callus samples evaluated, resulting in a variation rate of 0.07%. This is the first time in which plants have been regenerated via somatic embryogenesis in wild olive.

The Strawberry FaWRKY1 Transcription Factor Negatively Regulates Resistance to Colletotrichum acutatum in Fruit Upon Infection.

Strawberry (Fragaria ×ananassa) is a major food crop worldwide, due to the flavor, aroma and health benefits of the fruit, but its productivity and quality are seriously limited by a large variety of phytopathogens, including Colletotrichum spp. So far, key factors regulating strawberry immune response remain unknown. The FaWRKY1 gene has been previously proposed as an important element mediating defense responses in strawberry to Colletotrichum acutatum. To get further insight into the functional role that FaWRKY1 plays in the defense mechanism, Agrobacterium-mediated transient transformation was used both to silence and overexpress the FaWRKY1 gene in strawberry fruits (Fragaria ×ananassa cv. Primoris), which were later analyzed upon C. acutatum inoculation. Susceptibility tests were performed after pathogen infection comparing the severity of disease between the two agroinfiltrated opposite halves of the same fruit, one half bearing a construct either for FaWRKY1 overexpression or RNAi-mediated silencing and the other half bearing the empty vector, as control. The severity of tissue damage was monitored and found to be visibly reduced at five days after pathogen inoculation in the fruit half where FaWRKY1 was transiently silenced compared to that of the opposite control half and statistical analysis corroborated a significant reduction in disease susceptibility. Contrarily, a similar level of susceptibility was found when FaWRKY1 overexpression and control fruit samples, was compared. These results unravel a negative regulatory role of FaWRKY1 in resistance to the phytopathogenic fungus C. acutatum in strawberry fruit and contrast with the previous role described for this gene in Arabidopsis as positive regulator of resistance against the bacteria Pseudomonas syringae. Based on previous results, a tentative working model for WRKY75 like genes after pathogen infection is proposed and the expression pattern of potential downstream FaWRKY1 target genes was also analyzed in strawberry fruit upon C. acutatum infection. Our results highlight that FaWRKY1 might display different function according to species, plant tissue and/or type of pathogen and underline the intricate FaWRKY1 responsive defense regulatory mechanism taking place in strawberry against this important crop pathogen.

Rosellinia necatrix infection induces differential gene expression between tolerant and susceptible avocado rootstocks.

Rosellinia necatrix is the causal agent of avocado white root rot (WRR). Control of this soil-borne disease is difficult, and the use of tolerant rootstocks may present an effective method to lessen its impact. To date, no studies on the molecular mechanisms regulating the avocado plant response towards this pathogen have been undertaken. To shed light on the mechanisms underpinning disease susceptibility and tolerance, molecular analysis of the gene's response in two avocado rootstocks with a contrasting disease reaction was assessed. Gene expression profiles against R. necatrix were carried out in the susceptible 'Dusa' and the tolerant selection BG83 avocado genotypes by micro-array analysis. In 'Dusa', the early response was mainly related to redox processes and cell-wall degradation activities, all becoming enhanced after disease progression affected photosynthetic capacity, whereas tolerance to R. necatrix in BG83 relied on the induction of protease inhibitors and their negative regulators, as well as genes related to tolerance to salt and osmotic stress such as aspartic peptidase domain-containing proteins and gdsl esterase lipase proteins. In addition, three protease inhibitors were identified, glu protease, trypsin and endopeptidase inhibitors, which were highly overexpressed in the tolerant genotype when compared to susceptible 'Dusa', after infection with R. necatrix, reaching fold change values of 52, 19 and 38, respectively. The contrasting results between 'Dusa' and BG83 provide new insights into the different mechanisms involved in avocado tolerance to Phytophthora cinnamomi and R. necatrix, which are consistent with their biotrophic and necrotrophic lifestyles, respectively. The differential induction of genes involved in salt and osmotic stress in BG83 could indicate that R. necatrix penetration into the roots is associated with osmotic effects, suggesting that BG83's tolerance to R. necatrix is related to the ability to withstand osmotic imbalance. In addition, the high expression of protease inhibitors in tolerant BG83 compared to susceptible 'Dusa' after infection with the pathogen suggests the important role that these proteins may play in the defence of avocado rootstocks against R. necatrix.

Usage of the Heterologous Expression of the Antimicrobial Gene afp From Aspergillus giganteus for Increasing Fungal Resistance in Olive.

The antifungal protein (AFP) produced by Aspergillus giganteus, encoded by the afp gene, has been used to confer resistance against a broad range of fungal pathogens in several crops. In this research, transgenic olive plants expressing the afp gene under the control of the constitutive promoter CaMV35S were generated and their disease response against two root infecting fungal pathogens, Verticillium dahliae and Rosellinia necatrix, was evaluated. Embryogenic cultures derived from a mature zygotic embryo of cv. 'Picual' were used for A. tumefaciens transformation. Five independent transgenic lines were obtained, showing a variable level of afp expression in leaves and roots. None of these transgenic lines showed enhanced resistance to Verticillium wilt. However, some of the lines displayed a degree of incomplete resistance to white root rot caused by R. necatrix compared with disease reaction of non-transformed plants or transgenic plants expressing only the GUS gene. The level of resistance to this pathogen correlated with that of the afp expression in root and leaves. Our results indicate that the afp gene can be useful for enhanced partial resistance to R. necatrix in olive, but this gene does not protect against V. dahliae.

A possible role for flowering locus T-encoding genes in interpreting environmental and internal cues affecting olive (Olea europaea L.) flower induction.

Olive (Olea europaea L.) inflorescences, formed in lateral buds, flower in spring. However, there is some debate regarding time of flower induction and inflorescence initiation. Olive juvenility and seasonality of flowering were altered by overexpressing genes encoding flowering locus T (FT). OeFT1 and OeFT2 caused early flowering under short days when expressed in Arabidopsis. Expression of OeFT1/2 in olive leaves and OeFT2 in buds increased in winter, while initiation of inflorescences occurred i n late winter. Trees exposed to an artificial warm winter expressed low levels of OeFT1/2 in leaves and did not flower. Olive flower induction thus seems to be mediated by an increase in FT levels in response to cold winters. Olive flowering is dependent on additional internal factors. It was severely reduced in trees that carried a heavy fruit load the previous season (harvested in November) and in trees without fruit to which cold temperatures were artificially applied in summer. Expression analysis suggested that these internal factors work either by reducing the increase in OeFT1/2 expression or through putative flowering repressors such as TFL1. With expected warmer winters, future consumption of olive oil, as part of a healthy Mediterranean diet, should benefit from better understanding these factors.

Generation and Selection of Transgenic Olive Plants.

Olive (Olea europaea L.) is one of the most important oil crops in the Mediterranean basin. Biotechnological improvement of this species is hampered by the recalcitrant nature of olive tissue to regenerate in vitro. In previous investigations, our group has developed a reliable Agrobacterium-mediated transformation protocol using olive somatic embryos as explants ( Torreblanca et al., 2010 ). Embryogenic cultures derived from radicles of matured zygotic embryos are infected with Agrobacterium tumefaciens, AGL1 strain, containing a binary plasmid with the gene of interest and the nptII selection gene. After a meticulous selection procedure, carried out using solid and liquid media supplemented with paromomycin, the putative transformed lines are established. A preliminary confirmation of their transgenic nature is carried out through PCR amplification. Afterwards, plants can be obtained through an efficient regeneration protocol, whose main characteristics are the use of a low-ionic-strength mineral formulation, a phase in liquid medium for synchronization of cultures and the use of semi-permeable cellulose acetate membranes for embryo maturation ( Cerezo et al., 2011 ). Final confirmation of transgene insertion is carried out through Southern or Northern analysis using leaf samples of regenerated plants.

Partial Activation of SA- and JA-Defensive Pathways in Strawberry upon Colletotrichum acutatum Interaction.

Understanding the nature of pathogen host interaction may help improve strawberry (Fragaria × ananassa) cultivars. Plant resistance to pathogenic agents usually operates through a complex network of defense mechanisms mediated by a diverse array of signaling molecules. In strawberry, resistance to a variety of pathogens has been reported to be mostly polygenic and quantitatively inherited, making it difficult to associate molecular markers with disease resistance genes. Colletotrichum acutatum spp. is a major strawberry pathogen, and completely resistant cultivars have not been reported. Moreover, strawberry defense network components and mechanisms remain largely unknown and poorly understood. Assessment of the strawberry response to C. acutatum included a global transcript analysis, and acidic hormones SA and JA measurements were analyzed after challenge with the pathogen. Induction of transcripts corresponding to the SA and JA signaling pathways and key genes controlling major steps within these defense pathways was detected. Accordingly, SA and JA accumulated in strawberry after infection. Contrastingly, induction of several important SA, JA, and oxidative stress-responsive defense genes, including FaPR1-1, FaLOX2, FaJAR1, FaPDF1, and FaGST1, was not detected, which suggests that specific branches in these defense pathways (those leading to FaPR1-2, FaPR2-1, FaPR2-2, FaAOS, FaPR5, and FaPR10) were activated. Our results reveal that specific aspects in SA and JA dependent signaling pathways are activated in strawberry upon interaction with C. acutatum. Certain described defense-associated transcripts related to these two known signaling pathways do not increase in abundance following infection. This finding suggests new insight into a specific putative molecular strategy for defense against this pathogen.

Expression of the ß-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 ß-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.

Water relations in culture media influence maturation of avocado somatic embryos.

Application of transformation and other biotechnological tools in avocado (Persea americana Mill.) is hampered by difficulties in obtaining mature somatic embryos capable of germination at an acceptable rate. In this work, we evaluated the effect of different compounds affecting medium water relations on maturation of avocado somatic embryos. Culture media were characterized with respect to gel strength, water potential and osmotic potential. Improved production of mature somatic embryos was achieved with gelling agent concentrations higher than those considered standard. The osmotic agents such as sorbitol and PEG did not have positive effects on embryo maturation. The number of w-o mature somatic embryos per culture was positively correlated with medium gel strength. Gel strength was significantly affected by gelling agent type as well as by gelling agent and PEG concentration. Medium water potential was influenced by sorbitol concentration; incorporation of PEG to a culture medium did not affect medium water potential. The highest maturation results were achieved on a medium gelled with 10 gl(-1) agar. Moreover, these somatic embryos had improved germination rates. These results corroborate the role of water restriction as a key factor controlling maturation of somatic embryos.

Evidence for a positive regulatory role of strawberry (Fragaria x ananassa) Fa WRKY1 and Arabidopsis At WRKY75 proteins in resistance.

Knowledge of the molecular basis of plant resistance to pathogens in species other than Arabidopsis is limited. The function of Fa WRKY1, the first WRKY gene isolated from strawberry (Fragaria x ananassa), an important agronomical fruit crop, has been investigated here. Fa WRKY1 encodes a IIc WRKY transcription factor and is up-regulated in strawberry following Colletotrichum acutatum infection, treatments with elicitors, and wounding. Its Arabidopsis sequence homologue, At WRKY75, has been described as playing a role in regulating phosphate starvation responses. However, using T-DNA insertion mutants, a role for the At WRKY75 and Fa WRKY1 in the activation of basal and R-mediated resistance in Arabidopsis is demonstrated. At wrky75 mutants are more susceptible to virulent and avirulent isolates of Pseudomonas syringae. Overexpression of Fa WRKY1 in At wrky75 mutant and wild type reverts the enhanced susceptible phenotype of the mutant, and even increases resistance to avirulent strains of P. syringae. The resistance phenotype is uncoupled to PATHOGENESIS-RELATED (PR) gene expression, but it is associated with a strong oxidative burst and glutathione-S-transferase (GST) induction. Taken together, these results indicate that At WRKY75 and Fa WRKY1 act as positive regulators of defence during compatible and incompatible interactions in Arabidopsis and, very likely, Fa WRKY1 is an important element mediating defence responses to C. acutatum in strawberry. Moreover, these results provide evidence that Arabidopsis can be a useful model for functional studies in Rosacea species like strawberry.

Antisense down-regulation of the FaPG1 gene reveals an unexpected central role for polygalacturonase in strawberry fruit softening.

The strawberry (Fragaria x ananassa 'Chandler') fruit undergoes a fast softening during ripening. Polygalacturonase (PG) activity is low during this process, but two ripening-related PG genes, FaPG1 and FaPG2, have been cloned. Both genes were up-regulated during fruit ripening and were also negatively regulated by auxin. To further assess the role of FaPG1 on strawberry softening, transgenic plants containing an antisense sequence of this gene under the control of the 35S promoter (APG lines) were obtained. Sixteen out of 30 independent transgenic lines showed fruit yields similar to those of the control. Several quality parameters were measured in ripe fruits from these 16 lines. Fruit weight was slightly reduced in four lines, and most of them showed an increase in soluble solid content. Half of these lines yielded fruits significantly firmer than did the control. Four APG lines were selected, their ripened fruits being on average 163% firmer than the control. The postharvest softening of APG fruits was also diminished. Ripened fruits from the four selected lines showed a 90% to 95% decrease in FaPG1 transcript abundance, whereas the level of FaPG2 was not significantly altered. Total PG activity was reduced in three of these lines when compared with control fruits. Cell wall extracts from APG fruits showed a reduction in pectin solubilization and an increase in pectins covalently bound to the cell wall. A comparative transcriptomic analysis of gene expression between the ripened receptacle of the control and those of the APG fruits (comprising 1,250 receptacle expressed sequence tags) did not show any statistically significant change. These results indicate that FaPG1 plays a central role in strawberry softening.

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.

Manipulation of strawberry fruit softening by antisense expression of a pectate lyase gene.

Strawberry (Fragaria x ananassa, Duch., cv Chandler) is a soft fruit with a short postharvest life, mainly due to a rapid lost of firm texture. To control the strawberry fruit softening, we obtained transgenic plants that incorporate an antisense sequence of a strawberry pectate lyase gene under the control of the 35S promoter. Forty-one independent transgenic lines (Apel lines) were obtained, propagated in the greenhouse for agronomical analysis, and compared with control plants, non-transformed plants, and transgenic lines transformed with the pGUSINT plasmid. Total yield was significantly reduced in 33 of the 41 Apel lines. At the stage of full ripen, no differences in color, size, shape, and weight were observed between Apel and control fruit. However, in most of the Apel lines, ripened fruits were significantly firmer than controls. Six Apel lines were selected for further analysis. In all these lines, the pectate lyase gene expression in ripened fruit was 30% lower than in control, being totally suppressed in three of them. Cell wall material isolated from ripened Apel fruit showed a lower degree of in vitro swelling and a lower amount of ionically bound pectins than control fruit. An analysis of firmness at three different stages of fruit development (green, white, and red) showed that the highest reduction of softening in Apel fruit occurred during the transition from the white to the red stage. The postharvest softening of Apel fruit was also diminished. Our results indicate that pectate lyase gene is an excellent candidate for biotechnological improvement of fruit softening in strawberry.