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In the present work, Agrobacterium tumefaciens-mediated genetic transformation of the model legume Medicago truncatula Gaertn. (barrel medic) was carried out using the pSIM843 vector that contains a Medicago-derived transfer DNA, delineated by a 25-bp sequence homologous to bacterial T-DNA borders. The transfer DNA contains an expression cassette for the nptII (neomycin phosphotransferase) gene and is flanked by an expression cassette for the backbone integration marker gene ipt (isopentenyl transferase). Our results demonstrate that the Medicago-derived RB-like elements efficiently support DNA mobilization from A. tumefaciens to M. truncatula. Kanamycin-resistant shoots with normal phenotype and ipt-shooty lines were recovered at a frequency of 11.7 and 7.8%, respectively. Polymerase chain reaction (PCR) analyses demonstrated that 44.4% of the independent transgenic lines were backbone-free and evidenced the occurrence of backbone-transfer events.

Cells of Saccharum officinarum submitted to hydrolyzated chitin for 1 to 8h produced phenolic compounds. These alterations were observed through cytochemical methods using Toluidine Blue and Phloroglucinol/HCl. After 4 h, besides cell wall change, there was a change in nuclear pattern of chitin treated cells. There was a 96% increase in nuclear area in 6 h chitin treated material, as observed by Feulgen reaction. The treated cells showed chromatin compacted regions and a degeneration process of nucleoli. In the outer areas of cell wall, there was a polysaccharide desagregation, confirming results obtained for different plants with the use of other elicitors. Peroxidase activity was maximal after 4 h and decreased progressively. PAL activity started to increase at 4 h of incubation. These results showed that chitin hydrolyzate stimulated a defense response in sugarcane cells.Células de Saccharum officinarum quando submetidas a quitina hidrolisada por 1 a 8h produziram material fenólico. Essas alterações foram observadas por meio de métodos citoquímicos como o Azul de Toluidina e Floroglucinol/HCl. Após 4 h, além das mudanças nas paredes celulares houve uma mudança no padrão nuclear das células tratadas com quitina. Por observação da reação de Feulgen, houve um aumento de 96% na área nuclear no material em 6h. Para as células tratadas foram observadas regiões de cromatina compactada e um processo de degeneração do nucléolo. Nas áreas externas da parede celular existia uma desagregação dos polisacarídios confirmando os resultados obtidos para diferentes plantas com o uso de outros elicitores. A atividade da peroxidase foi maxima após 4 h e então decresceu progressivamente. A atividade da PAL aumentou a partir de 4 h de incubação. Estes resultados mostram que o hidrolisado de quitina estimula as respostas de defesa em células de cana.

Triterpene saponins are a group of bioactive compounds abundant in the genus Medicago, and have been studied extensively for their biological and pharmacological properties. In this article, we evaluated the effects of the ectopic expression of AsOXA1 cDNA from Aster sedifolius on the production of triterpene saponins in barrel medic (Medicago truncatula Gaertn.). AsOXA1 cDNA encodes beta-amyrin synthase, a key enzyme involved in triterpene saponin biosynthesis. One of the four transgenic lines expressing AsOXA1 accumulated significantly larger amounts of some triterpenic compounds in leaf and root than did control plants. In particular, the leaf exhibited significantly higher levels of bayogenin, medicagenic acid and zanhic acid. The amounts of medicagenic acid and zanhic acid, which represent the core of the M. truncatula leaf saponins, were 1.7 and 2.1 times higher, respectively, than the amounts extracted from the control line. In root, the production of bayogenin, hederagenin, soyasapogenol E and 2beta-hydroxyoleanolic acid was increased significantly. The increase in the total amounts of triterpenic compounds observed in the leaves of transgenic lines correlated with the AsOXA1 expression level. Interestingly, the plants expressing AsOXA1 showed, under different growth conditions, improved nodulation when compared with the control line. Nodulation enhancement was also accompanied by a significant change in the soyasapogenol B content. Our results indicate that the ectopic expression of AsOXA1 in barrel medic leads to a greater accumulation of triterpene saponins and enhanced root nodulation.

In Medicago truncatula Gaertn. cv. Jemalong plants some mechanisms involved in drought resistance were analysed in response to a progressive water deficit imposed by suppression of soil irrigation. Withholding water supply until the soil had reached one-half of its maximum water content had no significant effect on leaf RWC, gas exchanges or chlorophyll fluorescence parameters. Under severe drought conditions, the plants resistance to water shortage involved mainly drought avoidance mechanisms through a decrease in stomatal conductance. The consequent decrease in the internal CO2 concentration (Ci) should have limited the net CO2 fixation (A). Since A decreased slightly more than Ci under severe water deficit, non-stomatal limitations of photosynthesis may have also occurred. Analysis of A/Ci curves showed reduced carboxylation efficiency due to limitations in RuBP regeneration and Rubisco activity, confirming the presence of non-stomatal limitations of photosynthesis. Drought tolerance mechanisms involving osmotic adjustment and an increase in cell membrane integrity were also present. Altogether, these mechanisms allowed M. truncatula cv. Jemalong plants to still maintain a quite elevated level of net CO2 fixation rate under severe water deficit conditions. These results may contribute to identify useful physiological traits for breeding programs concerning drought adaptation in legumes.

International audience; Agriculture faces many challenges to maximize yields while it is required to operate in anenvironmentally sustainable manner. In the present study, we analyze the major agriculturalchallenges identified by European farmers (primarily related to biotic stresses) in 13 countries,namely Belgium, Bulgaria, the Czech Republic, France, Germany, Hungary, Italy, Portugal,Romania, Spain, Sweden, UK and Turkey, for nine major crops (barley, beet, grapevine, maize,oilseed rape, olive, potato, sunflower and wheat). Most biotic stresses (BSs) are related to fungior insects, but viral diseases, bacterial diseases and even parasitic plants have an importantimpact on yield and harvest quality. We examine how these challenges have been addressed bypublic and private research sectors, using either conventional breeding, marker-assistedselection, transgenesis, cisgenesis, RNAi technology or mutagenesis. Both national surveys andscientific literature analysis followed by text mining were employed to evaluate geneticengineering (GE) and non-GE approaches. This is the first report of text mining of the scientificliterature on plant breeding and agricultural biotechnology research. For the nine major cropsin Europe, 128 BS challenges were identified with 40% of these addressed neither in thescientific literature nor in recent European public research programs. We found evidence thatthe private sector was addressing only a few of these ‘‘neglected’’ challenges. Consequently,there are considerable gaps between farmer’s needs and current breeding and biotechnologyresearch. We also provide evidence that the current political situation in certain Europeancountries is an impediment to GE research in order to address these agricultural challengesin the future. This study should also contribute to the decision-making process on futurepertinent international consortia to fill the identified research gaps.

We developed an alternative methodology for in vitro selection of transgenic Medicago truncatula cv. Jemalong plants using a bifunctional construct in which the coding sequences for the green fluorescent protein (GFP) and the β-glucuronidase protein (GUS) are fused. An Agrobacterium-mediated transformation protocol was used followed by regeneration via somatic embryogenesis in the dark, to avoid the synthesis and the consequent autofluorescence of chlorophyll. This method is a clear advantage over antibiotic and herbicide selection in which survival of non-transformed tissue is commonly reported, with the reassurance that all the somatic embryos selected as GFP positive are transformed. This was subsequently corroborated by the detection of GUS activity in leaves, stems and roots of the regenerated plants. Without antibiotic selection, and performing the embryo induction in the dark, it was possible to attest the advantage of using GFP as an in vivo detectable reporter for early embryo selection. The fusion with the GUS coding sequence provided additional evidence for the transformation of the previously selected embryos.

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.

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.

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.

Plant microRNAs have been implicated in various abiotic stress responses. We identified several conserved microRNAs that showed differential expression in Medicago truncatula plants subjected to water deficit: miR169 is down-regulated only in the roots and miR398a/b and miR408 are strongly up-regulated in both shoots and roots. Down-regulation of miR169 in the roots did not correlate with accumulation of its target MtHAP2-1 transcripts, suggesting that its regulation may not occur at the mRNA level or may depend on other regulatory mechanisms, which do not involve this miRNA, in water-deficit conditions. The up-regulation of miR398a/b and miR408 and the clear down-regulation of their respective target genes, which encode the copper proteins COX5b (subunit 5b of mitochondrial cytochrome c oxidase) and plantacyanin, highlight the involvement of these miRNAs in response to water deprivation in M. truncatula. Also, miR398 up-regulation is inversely correlated with the down-regulation of copper superoxide dismutase, CSD1, during water deficit. The regulation of genes encoding copper proteins by miR398a/b and miR408 suggests a link between copper homeostasis and M. truncatula adaptation to progressive water deficit.

Background High-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). Results 3,948,871 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. Conclusion Deep sequencing of short RNAs from M. truncatula leaves identified eight new miRNAs indicating that specific miRNAs exist in legume species.