José Juárez

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.

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.

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.

Cytological and molecular characterization of three gametoclones of Citrus clementina

BackgroundThree gametoclonal plants of Citrus clementina Hort. ex Tan., cv. Nules, designated ESP, FRA, and ITA (derived from three labs in Spain, France, and Italy, respectively), were selected for cytological and molecular characterization in order to elucidate genomic rearrangements provoked by haploidization. The study included comparisons of their ploidy, homozygosity, genome integrity, and gene dosage, using chromosome counting, flow cytometry, SSR marker genotyping, and array-Comparative Genomic Hybridization (array-CGH).ResultsChromosome counting and flow cytometry revealed that ESP and FRA were haploid, but ITA was tri-haploid. Homozygous patterns, represented by a single peak (allele), were observed among the three plants at almost all SSR loci distributed across the entire diploid donor genome. Those few loci with extra peaks visualized as output from automated sequencing runs, generally low or ambiguous, might result from amplicons of paralogous members at the locus, non-specific sites, or unexpected recombinant alleles. No new alleles were found, suggesting the genomes remained stable and intact during gametogenesis and regeneration. The integrity of the haploid genome also was supported by array-CGH studies, in which genomic profiles were comparable to the diploid control.ConclusionsThe presence of few gene hybridization abnormalities, corroborated by gene dosage measurements, were hypothetically due to the segregation of hemizygous alleles and minor genomic rearrangements occurring during the haploidization procedure. In conclusion, these plants that are valuable genetic and breeding materials contain completely homozygous and essentially intact genomes.