Dulce M. Santos

High-throughput sequencing of Medicago truncatula short RNAs identifies eight new miRNA families

BackgroundHigh-throughput sequencing technology is capable to identify novel short RNAs in plant species. We used Solexa sequencing to find new microRNAs in one of the model legume species, barrel medic (Medicago truncatula).Results3,948,871 reads were obtained from two separate short RNA libraries generated from total RNA extracted from M. truncatula leaves, representing 1,563,959 distinct sequences. 2,168,937 reads were mapped to the available M. truncatula genome corresponding to 619,175 distinct sequences. 174,504 reads representing 25 conserved miRNA families showed perfect matches to known miRNAs. We also identified 26 novel miRNA candidates that were potentially generated from 32 loci. Nine of these loci produced eight distinct sequences, for which the miRNA* sequences were also sequenced. These sequences were not described in other plant species and accumulation of these eight novel miRNAs was confirmed by Northern blot analysis. Potential target genes were predicted for most conserved and novel miRNAs.ConclusionDeep sequencing of short RNAs from M. truncatula leaves identified eight new miRNAs indicating that specific miRNAs exist in legume species.

An Efficient Transformation Method to Regenerate a High Number of Transgenic Plants Using a New Embryogenic Line of Medicago truncatula cv. Jemalong

A simple and efficient regeneration–transformation method was established to obtain transgenic plants of the model legume Medicago truncatula cv. Jemalong. This method takes advantage of a new highly embryogenic line (M9-10a) isolated in our laboratory. Leaflets of in vitro grown M9-10a plants were co-cultured with Agrobacterium tumefaciens EHA105. Plasmid constructs containing the oat arginine decarboxylase gene, Adc and the GUS reporter gene (p35SAdc–Gus) or ELIP-like drought stress protein 22 (DSP22) encoding gene from Craterostigma plantagineum (p35SDsp22) were used. Both constructs include the nptII gene as selection marker. Embryogenic calli (100–97%) were obtained on embryo induction medium containing 100 mg l −1 kanamycin and 500 mg l−1 carbenicillin. Using a two-fold increase in kanamycin concentration, instead of 50 mg l−1 usually used, we reduced the number of emerging false kanamycin-resistant (KanR) embryos, which is an important improvement to the method, making it less laborious and very efficient. Isolation of late torpedo/cotyledonary-stage embryos to lower carbenicillin/agar media reduced secondary embryogenesis and prevents hyperhydricity, improving embryo conversion. Primary transformants (T0) were regenerated within 3–4 months and those that were able to root in a 50 mg l−1 kanamycin medium were transferred to the greenhouse to produce seeds. Southern blot hybridisation analysis confirmed the integration of either the Adc or Dsp22 transgenes in the genome of the T0 transformants. Detection of β-glucuronidase (GUS) activity in Adc–Gus T0 plants demonstrated the expression of the inserted transgene. In average, 1–2 independent transgenic lines are obtained per KanR embryogenic callus, independently of the plasmid construct used for transformation. Inheritance of the transgenes is shown to be stable in the T1 generation.

Loss of DNA methylation affects somatic embryogenesis in Medicago truncatula

To investigate the involvement of methylation of DNA in somatic embryogenesis we initiated a comparative study using Medicago truncatula lines that have different capacities to produce somatic embryos. Treatment with the demethylating drug 5-azacytidine caused a loss of regeneration capacity in the embryogenic line by arresting the production of somatic embryos. Analysis with methylation-sensitive enzymes showed disruption of somatic embryogenesis competence to be correlated with rDNA demethylation. Our data suggest production of somatic embryos depends on a certain level of DNA methylation.

Trehalose and its applications in plant biotechnology

Trehalose, a nonreducing disaccharide of glucose, is one of the most effective osmoprotectants. Several strategies leading to its accumulation have been envisaged in both model and crop plants using genes of bacterial, yeast and, more recently, plant origin. Significant levels of trehalose accumulation have been shown to cause abiotic stress tolerance in transgenic plants. In this review, we describe the most biologically relevant features of trehalose: chemical and biological properties; occurrence and metabolism in organisms with special reference to plants; protective role in stabilizing molecules; physiological role in plants with special reference to carbohydrate metabolism. The emphasis of this review, however, will be on manipulation of trehalose metabolism to improve abiotic stress tolerance in plants.

Water deficit and recovery response of Medicago truncatula plants expressing the ELIP-like DSP22

In this article, we present the response of Medicago truncatula Gaert. cv. Jemalong plants expressing constitutively the Dsp22 gene from Craterostigma plantagineum to water stress and rehydration. The Dsp22 gene encodes an ELIP-like protein thought to protect the chloroplast against photooxidative damage during the dehydration and rehydration. The Dsp22 transgenic homozygous M. truncatula plants showed higher amount of chlorophyll (Chl), lower Chl a/Chl b ratio and higher actual efficiency of energy conversion in photosystem 2 (ΦPSII) after rehydration, when compared to the wild type. The combined data from the Chl a fluorescence analysis, pigment quantification and biomass accumulation showed that transgenic M. truncatula plants are able to recover from water deprivation better than wild type plants.

Optimisation of a Selection Scheme using Kanamycin to Improve Transformation of Medicago truncatula cv. Jemalong

We developed an efficient method for in vitro selection of Medicago truncatula cv. Jemalong lines transformed with the nptII gene and subsequent confirmation of phenotype inheritance in these lines. For in vitro selection, the concentration of kanamycin inhibitory to embryogenic callus development and somatic embryo differentiation was identified by placing wounded leaves of non-transformed M. truncatula cv. Jemalong on Embryo Inducing Medium supplemented with 0, 85.8, 128.7, 171.6, 214.6, 257.5 and 343.3 µM of kanamycin. Differentiation of somatic embryos was inhibited with 171.6 µM of kanamycin but callus development was not altered. To confirm transgene inheritance, the kanamycin concentration to distinguish between resistant and non-resistant seedlings was identified by germinating non-transformed seeds of M. truncatula cv. Jemalong on 0.8% (w/v) water-agar plates containing 0, 171.6, 343.3, 514.9 and 686.6 µM of kanamycin. These concentrations did not impair seed germination since all the seedlings exhibited green cotyledons. The effect of kanamycin was only observed at 514.9 and 686.6 µM and on the first pair of leaves, which became white. Due to the high level of resistance to kanamycin of the seedlings the highest concentration is thought to be use to assure the selection efficiency. This optimised antibiotic selection scheme eliminates the regeneration of non-transformed escapes and discriminates between resistant and non-resistant seedlings, confirming the inheritance of the phenotype in transformed M. truncatula cv. Jemalong lines.

Reverse transcription-PCR assay to verify gene integrity within plasmid constructs for plant transformation

We have developed a rapid and simple RT-PCR based method to check the integrity of chimeric genes within plasmid constructs for plant transformation. It exploits the Agrobacterium tumefaciens-mediated transient expression of plasmid constructs in plant tissue. Total RNA was isolated from tobacco leaves co-cultivated for 3 days with Agrobacterium tumefaciens harbouring the plant transformation vectors constructed in our laboratory. First strand cDNA synthesis with oligo(dT) primers generate a pool of cDNA that was used for PCR amplification with primers specific for each of the genes present within the constructs. PCR amplification reactions were successful for all chimeric genes tested, thus confirming their intactness and suitability to be used for stable plant transformation.

Development and assessment of efficacy of an anti-cellulite formulation containing bentonite from Porto Santo Island

Clays are part of the natural resources that have become prominent in cosmetic treatments and wellness. The relevant physical, chemical and thermal properties of bentonite clay from Serra de Dentro, Porto Santo Island, allow its use in cosmetic and therapeutic applications. In this work we developed an anticellulite gel formulation containing 10% of Ivy extract, 10% of Horse Chestnut Extract and 20% of Bentonite clay (Smectitic), dee <180 m, from Porto Santo Island. After physicochemical characterization of the formulation, efficacy tests were performed on volunteers who had gynoid hydrolipodystrophy, commonly referred as cellulite.