Leandro Peña

Constitutive expression of Arabidopsis LEAFY or APETALA1 genes in citrus reduces their generation time

Citrus trees have a long juvenile phase that delays their reproductive development by between 6 and 20 years, depending on the species. With the aim of accelerating their flowering time, we transformed juvenile citrus seedlings to constitutively express the Arabidopsis LEAFY (LFY) or APETALA1 (AP1) genes, which promote flower initiation in Arabidopsis. Both types of transgenic citrus produced fertile flowers and fruits as early as the first year, notably through a mechanism involving an appreciable shortening of their juvenile phase. Furthermore, expression of AP1 was as efficient as LFY in the initiation of flowers, and did not produce any severe developmental abnormality. Both types of transgenic trees flowered in consecutive years, and their flowering response was under environmental control. In addition, zygotic and nucellar derived transgenic seedlings had a very short juvenile phase and flowered in their first spring, demonstrating the stability and inheritance of this trait. These results open new possibilities for domestication, genetic improvement, and experimental research in citrus and other woody species.

Agrobacterium-mediated transformation of citrange: factors affecting transformation and regeneration

Abstract The effects of cocultivation with Agrobacterium tumefaciens, regeneration and selection conditions on the transformation efficiency of citrange (Citrus sinensis L. Osbeck×Poncirus trifoliata L. Raf.) have been investigated. Factors such as cocultivation period, preculture of explants, use of acetosyringone or feeder plates during cocultivation, cocultivation on a medium rich in auxins, postcultivation in darkness, and different kanamycin concentrations for selection were assessed. A 3-day cocultivation on a medium rich in auxins improved transformation frequencies, since it increased the number of dividing cells competent for transformation, at the cut ends of the explants. Exposure of explants to darkness for 4 weeks on selection medium resulted in further callus development and increased the regeneration frequency of transgenic shoots. Furthermore, this treatment drastically reduced the number of regenerated escape shoots. A transformation efficiency of 41.3% was achieved using the optimized transformation procedure.

High efficiency Agrobacterium-mediated transformation and regeneration of citrus

Abstract A procedure for increased efficiency of production of transgenic citrus plants was developed by extending the exposure of the explants to the selection agent and by grafting in vitro the regenerated shoot apices onto seedling rootstocks. Carrizo citrange ( Citrus sinensis L. Osbeck × Poncirus trifoliata L. Raf.) stem segments from in vitro grown seedlings were cocultivated with Agrobacterium tumefaciens EHA 105 carrying the binary vector p35SGUSINT. The introncontaining β-glucuronidase (GUS) gene in the T-DNA served as reporter in the histochemical assay and the neomycin phosphotransferase II (NPT II) gene provided resistance to kanamycin and was used as selectable marker. Regenerated shoots were harvested from the stem segments within 5 to 6 months. Extended time periods of selection greatly improved recovering of transformed shoots and reduced the occurrence of escapes. However, prolonged exposure to kanamycin favoured the regeneration of chimeric shoots. Shoot basal portions were GUS-assayed for screening transformants and the remaining portions were shoot tip grafted in vitro for production of whole plants. Citrus genetic transformation was confirmed by polymerase chain reaction (PCR). Southern and Northern analysis. The transformation efficiencies obtained are the highest reported so far for citrus.

Agrobacterium-mediated transformation of sweet orange and regeneration of transgenic plants

SummaryTransgenic sweet orange (Citrus sinensis L. Osbeck) plants have been obtained by Agrobacterium tumefaciens-mediated gene transfer. An hypervirulent A. tumefaciens strain harboring a binary vector that contains the chimeric neomycin phosphotransferase II (NPT II) and ß-glucuronidase (GUS) genes was cocultivated with stem segments from in vivo grown seedlings. Shoots regenerated under kanamycin selection were harvested from the stem segments within 12 weeks. Shoot basal portions were assayed for GUS activity and the remaining portions were shoot tip grafted in vitro for production of plants. Integration of the GUS gene was confirmed by Southern analysis. This transformation procedure showed the highest transgenic plant production efficiency reported for Citrus.

Efficient production of transgenic citrus plants expressing the coat protein gene of citrus tristeza virus.

Abstract The coat protein gene of citrus tristeza virus (CTV) has been introduced into Mexican lime (Citrus aurantifolia Swing.) plants by using an improved Agrobacterium-mediated genetic transformation system. Internodal stem segments from greenhouse-grown seedlings were co-cultivated with A. tumefaciens strain EHA 105 carrying the binary plasmid pBI 121/CTV-CP in a medium rich in auxins that provided the explant cells with the proper treatment to shift them to a competent state for transformation. The transformation frequency was enhanced, and this allowed us to recover 42 transgenic plants from 1200 explants. Regenerated shoots were identified as transformants by performing β-glucuronidase (GUS) assays and subsequently by PCR amplifications of the CTV-CP transgene. Southern analyses revealed that at least one copy of the CTV-CP gene was integrated in all PCR positive plants. Interestingly, 70% of them had linked T-DNAs arranged at one locus. Copy number of the CTV-CP gene varied from one to six among the transgenic lines. Half of them showed truncated T-DNAs in which the left border was lost. Expression of the CTV-CP transgene was demonstrated in 38 out of 42 plants by western analysis and DASI-ELISA. No correlation was found between coat protein expression and transgene copy number or integration pattern.

Genetic transformation and regeneration of mature tissues of woody fruit plants bypassing the juvenile stage

Regeneration and transformation systems from mature plant material of woody fruit species have to be achieved as a necessary requirement for the introduction of useful genes into specific cultivars and the rapid evaluation of resulting horticultural traits. We report here, for the first time, a procedure for genetic transformation and regeneration of mature tissues of woody plants that overcomes the long juvenile periods and high heterozygosity that are characteristic of most of these species. An improved regeneration frequency from mature explants was obtained by invigoration of the plant material through grafting of mature buds on juvenile seedlings. Co-cultivation of the explants in feederplates after inoculation with Agrobacterium tumefaciens resulted in enhanced transformation frequencies. Furthermore, in vitro shoot-tip grafting of the regenerated mature shoots on seedling rootstocks provided a rapid and efficient system for plant production. Citrus is the most extensivel y grown fruit crop worldwide and sweet orange (Citrus sinensis L. Osbeck) accounts for approximately 70% of the Citrus total production. Mature transgenic sweet orange plants have been obtained, which flowered and bore fruit in 14 months

Genetic transformation of lime (Citrus aurantifolia Swing.): factors affecting transformation and regeneration

Abstract We have previously developed procedures for the efficient production of sweet orange (Citrus sinensis L. Osbeck) and Carrizo citrange (C. sinensis L. Osbeck×Poncirus trifoliata L. Raf.) transgenic plants using an Agrobacterium tumefaciens-mediated transformation and shoot tip grafting in vitro regeneration system. We now report on the optimization of the cocultivation, regeneration and selection conditions for efficient and reliable production of transgenic lime (C. aurantifolia Swing.) plants. Improved transformation frequencies were obtained by cocultivating the explants with Agrobacterium on feeder plates. Optimum regeneration of transgenic shoots was obtained by exposing the explants to darkness for 2 weeks and by using kanamycin at 100 mg/l as selective agent. Attempts to use geneticin as selection antibiotic were not successful. Shoot tip grafting of regenerated shoots on Troyer citrange seedlings resulted in 100% successful production of transgenic plants. The presence and expression of the transferred genes in the regenerated plants was verified by β-glucuronidase histochemical and fluorimetric assays, neomycin phosphotransferase ELISA assays, PCR and Southern analyses.

Increased tolerance to Phytophthora citrophthora in transgenic orange plants constitutively expressing a tomato pathogenesis related protein PR-5

Phytophthora citrophthora is the most widely spread oomycete plant pathogen over all the citrus growing areas and represents one of the major causes of crop losses. Constitutive over-expression of genes encoding proteins involved in plant defence mechanisms to disease is one of the strategies proposed to increase plant tolerance to oomycete and fungal pathogens. P23 (PR-5), a 23-kDa pathogenesis-related protein similar to osmotins, is induced in tomato (Lycopersicon esculentum Mill. cv. Rutgers) plants when they are infected with citrus exocortis viroid, and its antifungal activity has been demonstrated in in vitro assays. We have successfully produced transgenic orange (Citrus sinensis L. Obs. cv. Pineapple) plants bearing a chimeric gene construct consisting of the cauliflower mosaic virus 35S promoter and the coding region of the tomato pathogenesis-related PR-5. Nine regenerated transgenic lines constitutively expressed the PR protein. They were challenged with Phytophthora citrophthora using a detached bark assay. A significant reduction in lesion development was consistently observed in one transgenic line in comparison to the control plants. This same line achieved plant survival rates higher than control plants when transgenic trees were inoculated with oomycete cultures. These results provide evidence for the in vivo activity of the tomato PR-5 protein against Phytophthora citrophthora, and suggest that this may be employed as a strategy aimed at engineering Phytophthora disease resistance in citrus.

Post-Transcriptional Gene Silencing of the p23 Silencing Suppressor of Citrus tristeza Virus Confers Resistance to the Virus in Transgenic Mexican Lime

Previously, we have shown that most Mexican limes (Citrus aurantifolia (Christ.) Swing.) expressing the p23 gene of Citrus tristeza virus (CTV) exhibit aberrations resembling viral leaf symptoms. Here we report that five independent transgenic lines having normal phenotype displayed characteristics typical of post-transcriptional gene silencing (PTGS): multiple copies of the transgene, low levels of the corresponding mRNA, methylation of the silenced transgene, and accumulation of p23-specific small interfering RNAs (siRNAs). When graft- or aphid-inoculated with CTV, some propagations of these silenced lines were immune: they neither expressed symptoms nor accumulated virions and viral RNA as estimated by DAS-ELISA and Northern blot hybridization, respectively. Other propagations were moderately resistant because they became infected later and showed attenuated symptoms compared to controls. The susceptible propagations, in addition to symptom expression and elevated virus titer, accumulated p23-specific siRNAs at levels significantly higher than immune or non-inoculated propagations, and showed transgene demethylation. This variable response among clonal transformants indicates that factors other than the genetic background of the transgenic plants play a key role in PTGS-mediated resistance.

Characterisation of regenerants obtained under selective conditions after Agrobacterium-mediated transformation of citrus explants reveals production of silenced and chimeric plants at unexpected high frequencies

Genetic transformation has been achieved for several citrus genotypes. However, regeneration of escapes at high frequency is a major problem, making the available procedures rather inefficient. Attempts to improve selection by increasing the concentration of kanamycin, used as the selective agent, or substituting it by geneticin have been unsuccessful. Here, we have critically assessed the actual frequency and origin of escapes in citrus by using visual screening with β-glucuronidase (gusA) and green fluorescent protein (gfp) markers, by studying the persistence of engineered Agrobacterium in the explants, and by characterising through Southern blot analysis all the regenerants obtained under kanamycin selection. Our results show that inefficient selection could be attributed to the protection of the non-transformed cells from the selective agent by the surrounding transformed cells, and to the persistence of kanamycin-resistance Agrobacterium in explant tissues over long periods of time after co-cultivation. This also explained the high frequency (12%) of chimeric shoots that were commonly recovered. High frequency regeneration of chimeras that resulted from the fusion of different transformation events is reported for the first time. On the other hand, molecular analysis of all the regenerants reveals that transformation frequency is underestimated when based on the expression of a screenable marker gene, and that low expressors and silenced lines could account for at least 25% of those plants considered escapes based on selectable and screenable marker analysis. Consequences of these results at the practical level are also discussed.