Classical breeding programmes applied to produce new or improved varieties of floricultural species resulted in a range of cultivars with excellent traits, such as colour, shape, fragrance, vase life in cut-flower species, rooting potential or overall plant morphology. However, some of these aims have not yet been achieved in many ornamental species. Gene transfer by means of Agrobacterium tumefaciens enables the introduction of new genes/traits from unrelated species and would be a helpful tool in pelargonium breeding.
Efficient regeneration protocols that could be applicable to a broad spectrum of different genotypes are a prerequisite for developing a transformation system for a plant species or genotype . We have developed a simple and reliable in vitro regeneration protocol for the genetic transformation of Pelargonium spp. The two Pelargonium genotypes used in the present study showed different regeneration ability. Using the same culture medium, regeneration was carried out via somatic embryogenesis in P. peltatum and via organogenesis in P. zonale. Interestingly, the percentage of regenerating explants obtained from both genotypes was similar (90% in P. peltatum and 80% in P. zonale) and also the transformation efficiency (2-3%).
We have also evaluated the use of the gfp gene as an in vivo selectable marker in Pelargonium. It has been reported that gfp expression in transformed cells is useful to select transformation events in early stages, so that antibiotic selection is not needed [47, 54–61]. In the case of Pelargonium spp., the use of gfp as a selectable in vivo marker gene is restricted to identify transformation events, because at late developmental stages it becomes undetectable due to the presence of chlorophyll in the green tissues (leaves). In adult plants, gfp expression is only detectable in non green tissues like roots, petals or anther filaments.
Using this transformation protocol, we introduced two new traits in P. zonale cv. 370, one to produce long-life plants by inducing the ipt gene during plant senescence and the other to produce male sterile plants without pollen. During the initial transformation assays we used the gfp reporter gene to in vivo identify the transformations events and to evaluate transformation efficiency in the regenerating plantlets. The gfp gene has been used to successfully transform sugar cane, tobacco, maize, lettuce , walnut , citrus , peach , potato , pear , carrot  and other species . In P. zonale, we have observed gfp expression along the regeneration process, but, at times longer than three weeks after inoculation, we could not correlate explants that regenerated transgenic plants with green fluorescence. Low levels of gfp fluorescence coincided in time with increased content of chlorophyll and the red autofluorescence of chlorophyll interferes with the gfp green fluorescence . This interference could also be caused by pigments that are opaque to exciting UV or blue light . Some authors state that transformation efficiencies based on resistance to a selective marker are probably underestimating the actual rate of regenerated transgenic plants . Therefore PCR analysis has been proposed to identify transgenic plants in addition to the use of selectable or visual screening markers . In the present work, the gfp gene was useful to confirm genetic transformation of P. zonale in vivo. All transgenic plants showed green fluorescence in nearly all tissues analyzed but there were large differences in green fluorescence between organs and tissues, depending on the chlorophyll content of each one. PCR analysis corroborated the presence of transgenes in the regenerated plantlets.
In genetic transformation experiments, the analyses generally focus on the molecular characterization of the transgenic plants but the ploidy level of the transgenic material is checked in relatively few cases. The confirmation of the ploidy level in transgenic material is particularly important during the selection of transgenic lines. Our results indicate that leaf tissues of the Pelargonium cultivars used here have a diploid number of chromosomes and regeneration from these explants leads to plants with the same ploidy level as adult material of the original cultivar.
Transgenic pSAG12::ipt plants showed delayed leaf senescence, which was more evident at the basal leaves. They also showed increased branching and reduced internodal length when compared with non transformed plants. In addition, the transgenic pSAG12::ipt plants displayed a more compact architecture than the WT plants. Other interesting phenotypic difference among the WT and the transgenic pSAG12::ipt plants was the reduction of transgenic leaf size. The plant architecture was compact in the transgenic plants, including tight inflorescences and flowers. In some pSAG12::ipt inflorescences, flowers coexisted with new vegetative structures which are produced at the same time as flowers or new inflorescences. This occasional phenomenon might be due to a change in the determination of the floral meristem leading to inflorescence reversion and new vegetative organs instead of flowers in the inflorescences.
Inflorescence or flower reversion may occur when the level of the floral signal is insufficient for the completion of flower development and the suppression of indeterminacy . Flower and inflorescence reversion involve a switch from floral development back to vegetative development, thus rendering flowering a phase in an ongoing growth pattern rather than a terminal act of the meristem. Although it can be considered an unusual event, it is linked to environmental conditions and is most often a response to conditions opposite to those that induce flowering. A clear-cut reversion to leaf production has been described in Impatiens balsamina. In I. balsamina, a leaf-derived signal is critical for completion of flowering and can be considered to be the basis of a plant-wide floral commitment that is achieved without accompanying meristem autonomy. It has been proposed that cytokinins can be involved in floral induction as the leaf-generated signal that produces completion of the flowering process making it irreversible [66, 67]. These cytokinin fluxes during floral induction in LD plants could be altered in the pSAG12::ipt transgenic plants during senescence due to the continued production of cytokinin and this fact could be influencing the reversion process in some inflorescences. To elucidate if there was a correlation between the inflorescence reversion phenotype and the expression level of the exogenous gene in these plants, we carried out real-time RT-PCR experiments. Our results indicated that the expression level of the transgene is higher in those transgenic lines showing inflorescence reversion. However, this phenotype could be considered as an undesirable collateral effect from a commercial point of view and transgenic lines showing occasional inflorescence reversion were discarded.
All the pSAG12::ipt P. zonale plants cultivated in the greenhouse exhibited delayed senescence when compared with WT control plants, especially in the basal leaves. A high number of adult leaves of control plants exhibited an evident senescence phenotype while similar leaves at the same positions in the transgenic plants remained green and fully expanded. To better characterize and determine the delay of senescence in the transgenic plants, young and healthy leaves of similar age from both transgenic and control plants were detached and their petioles were placed in glass tubes with water at 28°C in darkness. The analysis of these leaves over time showed that leaves from the pSAG12::ipt transgenic plants remained green longer than the controls. While the WT leaves exhibited evident symptoms of chlorophyll degradation after 6 days of incubation in the darkness, the transgenic leaves exhibited similar symptoms after 22 days of incubation, indicating a delay in the senescence process. Likewise, necrotic symptoms appeared early in the WT leaves than in the transgenic ones. Quantification of chlorophyll content of detached WT and pSAG12::ipt leaves indicated that the decline of chlorophyll was higher in the WT leaves when compared with the transgenic ones. Moreover, the loss of water in leaves from transgenic plants was minor over the time course analyzed. These data reinforce the idea that the chimaeric pSAG12::ipt construct could be useful in Pelargonium spp. to delay the senescence process and to produce long-lived plants.
We have obtained engineered male sterile plants of P. zonale to prevent undesirable lateral gene flow of the introduced transgenes to related species. The PsEND1 promoter specifically directed expression of the barnase gene to different anther tissues involved in anther architecture (epidermis, endothecium, middle layer, connective tissue). Expression of the barnase gene under control of this promoter caused specific ablation of these tissues at early stages of anther development in the transgenic plants. We readily observed small structures that developed instead of normal anthers in the third floral whorl of transgenic flowers, which displayed premature senescence and collapse of the pollen sacs, microspores and tapetum and a lack of pollen at anthesis. Ablation of the structural anther tissues also produces the improper formation of the tapetum tissue and the subsequent degeneration of the microspores is accompanied by a change in anther wall thickness, by a size reduction and by a change in the epidermal cell types . Since this phenotype is unlikely to be due to expression of barnase in structural tissues, it is likely to be an indirect effect of the loss of the tapetum and microspore cells.
No pollen grains were observed in any section of the ablated anthers from the male sterile plants, indicating that barnase effectively ablates specific cell lines that will form the structural tissues of the anther, preventing pollen development. Transgenic anthers appeared to show effects of barnase expression at every stage examined. This is likely due to the developmentally earlier expression of the PsEND1 promoter. The anther filaments of the transgenic plants were shorter than WT filaments. The formation of short filaments is commonly associated with male sterility or reduced fertility [43, 68–70]. These observations reinforce our previous results in other crop species using the same chimaeric construct .
Due to the extremely toxic nature of barnase, other researchers have reported a general lack of vigour and a decline in plant growth in transgenic plants carrying the barnase gene [71–73]. The lack of significant effects on growth characteristics is important to know when considering the use of barnase for male sterility in landscaping plants. To prevent the possible effects of ectopic barnase expression, Gardner et al.  proposed the introduction of the male and/or female sterility genes in combination with a gene protecting against inappropriate barnase expression (enhanced 35S::barstar). In all the lines of transgenic P. zonale plants expressing barnase under control of the PsEND1 promoter, we did not observe differences with respect to wild type plants in vegetative growth, flowering time or inflorescence number. Morphological analysis of the transgenic plants showed that, under greenhouse conditions, the expression of the PsEND1::barnase construct does not significantly affect the vegetative and floral development, thus confirming the anther specificity of the PsEND1 promoter region previously observed by means of the GUS expression studies in different dicots and monocots [38, 39, 41, 42]. The potential biotechnological applications of the PsEND1 promoter largely depend on both its spatial and temporal expression pattern, since the ectopic expression of the cytotoxic agent would damage other plant tissues and organs, decreasing the agronomic value of hybrid plants.
Expression of the barnase gene in ornamental plants under control of the anther-specific PsEND1 promoter may be used to create efficient male sterile versions of existing popular cultivars without adversely affecting their respective phenotypes. Therefore, this technology would be especially useful to produce environmentally friendly transgenic plants carrying new traits by preventing gene flow between the genetically modified ornamentals.