Eugénia Nunes

Cotton (Gossypium hirsutum L.).

Cotton continues to be a crop of great economic importance in many developing and some developed countries. Cotton plants expressing the Bt gene to deter some of the major pests have been enthusiastically and widely accepted by the farmers in three of the major producing countries, i.e., China, India, and the USA. Considering the constraints related to its production and the wide variety of products derived from the cotton plant, it offers several target traits that can be improved through genetic engineering. Thus, there is a great need to accelerate the application of biotechnological tools for cotton improvement. This requires a simple, yet robust gene delivery/transformant recovery system. Recently, a protocol, involving large-scale, mechanical isolation of embryonic axes from germinating cottonseeds followed by direct transformation of the meristematic cells has been developed by an industrial laboratory. However, complexity of the mechanical device and the patent restrictions are likely to keep this method out of reach of most academic laboratories. In this chapter, we describe the method developed in our laboratory that has undergone further refinements and involves Agrobacterium-mediated transformation of cotton cells, selection of stable transgenic callus lines, and recovery of plants via somatic embryogenesis.

Optimization of matrix solid-phase dispersion extraction method for the analysis of isoflavones in Trifolium pratense.

A method based on matrix solid-phase dispersion (MSPD) has been developed for the determination of 12 isoflavones in Trifolium pratense L. Dried leaf samples were blended with C(18), placed in small columns and isoflavones extracted with dichloromethane-methanol. Analyses were performed by high performance liquid chromatography with diode array detection (HPLC-DAD) with 2-methoxyflavone as internal standard. Several dispersants, eluents and clean-up steps were tested during the optimization of the process in order to obtain the best selectivity and yields. Mean recoveries ranged from 70% to 119%, with relative standard deviations <18%. The limits of detection were between 0.006 mg/l for biochanin A and 0.108 mg/l for daidzin. The performance of the optimized method in real samples was compared with a conventional method based in solid-liquid extraction (SLE).

Advances in Isoflavone Profile Characterisation using Matrix Solid‐phase Dispersion Coupled to HPLC/DAD in Medicago Species

INTRODUCTION Analytical methods used in phytochemistry analysis are limited by the sample preparation step, which should ideally be fast, accurate, ecofriendly and achievable using low quantities of the sample. Matrix solid-phase dispersion (MSPD) may be a good alternative for combining extraction and purification procedures, thereby reducing the indicated limitations. OBJECTIVE Applying an MSPD extraction procedure coupled to high-performance liquid chromatography diode-array detection (HPLC/DAD) as an alternative methodology to evaluate isoflavone profiles. METHODS Isoflavone profiles were determined for the leaves of nine species of Medicago in the late flower phenological stage (one or more nodes with 50% open flowers, no seed pods). Extraction was performed following MSPD, and isoflavone profiles were characterised using HPLC/DAD. The quantified amounts were compared with previous results in different species commonly recognised as good sources of isoflavones. RESULTS Formononetin was the major isoflavone in most species, except M. polymorpha and M. truncatula. The isoflavone amounts were significantly different among the assayed species, with M. orbicularis and M. arabica as the major isoflavone sources, while M. rigidula presented the lowest contents. Furthermore, the detected differences allow electing the best species as a primary source of a specific isoflavone. CONCLUSION The MSPD allowed good extraction efficiency, reproducibility and recovery. Some of the species showed relevant isoflavone contents, even when compared with acknowledged plant sources such as soy or red clover. To the best of our knowledge the results presented are reported for the first time in these species.

Medicago spp. as potential sources of bioactive isoflavones: Characterization according to phylogenetic and phenologic factors.

A high variety of plant species are often proposed as potential natural sources of specific bioactive components, with emphasis in phenolic compounds. However, the ability to produce a determined phytochemical might be variable, even among species with close phylogeny. Furthermore, the metabolic dynamics vary greatly according to phenologic factors. Herein, it was verified whether isoflavone production in Medicago spp. is more associated with phylogenetic or phenologic determinants, to define the optimal productive conditions. Isoflavone profiles were characterized in field-grown Medicago species in three phenologic stages. Isoflavones were extracted by matrix solid-phase dispersion method and analyzed using high-performance liquid chromatography coupled with a diode-array detector. The obtained data were evaluated by a generalized linear model (GLM) and linear discriminant analysis (LDA). Formononetin, genistein and irilone were the most abundant isoflavones, reaching values higher than those present in acknowledged plant sources like soy or red clover. Outputs from GLM and LDA indicate that the phylogenetic factors are the most defining criteria. This study promotes Medicago spp. as potential isoflavone sources, particularly because the effects of these compounds are highly dependent on their type and concentration, with potential application as foodstuff, feedstuff, or in the nutraceutical and pharmaceutical industry.

Isoflavone determination in spontaneous legumes identified by DNA barcodes.

Isoflavones have been associated with several health protective effects. In this work spontaneous legume plants were screened as putative sources of dietary isoflavones. A molecular identification of the collected species was performed throughout DNA barcoding using ITS, rbcL, rpoC1 and matK sequences. The use of a multi-locus barcoding system complemented with basic morphological information allowed the unequivocal identification at the species level of 90% of the samples. The determination of isoflavone content was performed by high-performance liquid chromatography with diode-array detection. Total average contents in the studied species were significantly different, Ononis natrix and Cytisus scoparius possessing the highest total isoflavones content (396 and 273 mg kg(-1), respectively) and Lotus creticus, the lowest (20 mg kg(-1)). The correlation of total isoflavone content with the phylogeny of this set of plants as determined by the rpoC1 sequences was evaluated for the first time.

ptxD gene in combination with phosphite serves as a highly effective selection system to generate transgenic cotton ( Gossypium hirsutum L.)

Key messageThis report demonstrates the usefulness of ptxD/phosphite as a selection system that not only provides a highly efficient and simple means to generate transgenic cotton plants, but also helps address many of the concerns related to the use of antibiotic and herbicide resistance genes in the production of transgenic crops.AbstractTwo of the most popular dominant selectable marker systems for plant transformation are based on either antibiotic or herbicide resistance genes. Due to concerns regarding their safety and in order to stack multiple traits in a single plant, there is a need for alternative selectable marker genes. The ptxD gene, derived from Pseudomonas stutzeri WM88, that confers to cells the ability to convert phosphite (Phi) into orthophosphate (Pi) offers an alternative selectable marker gene as demonstrated for tobacco and maize. Here, we show that the ptxD gene in combination with a protocol based on selection medium containing Phi, as the sole source of phosphorus (P), can serve as an effective and efficient system to select for transformed cells and generate transgenic cotton plants. Fluorescence microscopy examination of the cultures under selection and molecular analyses on the regenerated plants demonstrate the efficacy of the system in recovering cotton transformants following Agrobacterium-mediated transformation. Under the ptxD/Phi selection, an average of 3.43 transgenic events per 100 infected explants were recovered as opposed to only 0.41% recovery when bar/phosphinothricin (PPT) selection was used. The event recovery rates for nptII/kanamycin and hpt/hygromycin systems were 2.88 and 2.47%, respectively. Molecular analysis on regenerated events showed a selection efficiency of ~ 97% under the ptxD/Phi system. Thus, ptxD/Phi has proven to be a very efficient, positive selection system for the generation of transgenic cotton plants with equal or higher transformation efficiencies compared to the commonly used, negative selection systems.

Phytochemical profiling of underexploited Fabaceae species: Insights on the ontogenic and phylogenetic effects over isoflavone levels

There is an increasing trend towards finding alternative sources of valued phytochemicals due to their diverse potentialities in food industry and pharmaceutical applications. Phenolic compounds, in particular, have been the focus of several profiling reports, but isoflavones characterization has been studied in fewer cases and in a very limited group of plant species. Despite their acknowledged bioactivity, theres actually a strict number of plants validated for their isoflavones contents. In a previous report, we have identified nine Leguminosae species (from genera Biserrula, Lotus, Ornithopus and Scorpiurus) as potential alternative sources of these phenolic compounds. However, the isoflavone profiles are highly modulated by the ontogenic stage. Therefore, the present study was conducted in the same Leguminosae species, but harvested at three sequential vegetative development stages: vegetative elongation, late bud and late flowering, with the main purpose of assessing the evolution of isoflavones content throughout the plant development. In general, the plant species from Biserrula and Lotus genera showed the highest potential as new natural sources of isoflavones, especially owing their high levels of biochanin A. Independently of the plant species, it was possible to identify the phenologic stages where each of the quantified isoflavones is maximized. These findings are useful to predict isoflavone yields according to harvesting time, validating the potential use of the studied plants in innovative food formulations.

The correct phylogenetic position of Lotus conimbricensis Brot. (Leguminosae, Loteae) based on nuclear ribosomal ITS sequences

The correct phylogenetic position of Lotus conimbricensis Brot. (Leguminosae, Loteae) based on nuclear ribosomal ITS sequences The nrDNA ITS sequence determined in Lotus conimbricensis in a previous phylogenetic study was unusual, in that it was almost identical to those retrieved from the morphologically distinct species L. subbiflorus. In the present study we sequenced new specimens of both species to reassess the phylogenetic position of L. conimbricensis. We conclude that the ITS sequence of L. conimbricensis used in the earlier analyses was most likely erroneous, and in fact L. conimbricensis is not closely related to L. subbiflorus. Critical reexamination of previously published data indicates that several other similar errors may exist for other Lotus species, and these should be checked before taxonomic conclusions are made.