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Several dicotyledonous species were infected with an Agrobacterium rhizogenes binary vector harbouring the plasmid 121.Sn which contains the maize gene Sn under the constitutive promoter CaMV35S. In maize, Sn transactivates the anthocyanin pathway in different tissues. The aim of this work was to test the efficiency of this gene to regulate the anthocyanin pathway in heterologous systems and verify its suitability as a reporter gene. The pigmentation of the hairy roots was compared with hairy roots stained for β-glucuronidase activity, which were used as a control. In two polymorphic genotypes of Lotus angustissimus, DNA integration and expression were assayed. The maize gene is competent to induce anthocyanin pigmentation in different species, but the complexity of the regulatory mechanisms of anthocyanin synthesis restricts the use of Sn as a reporter gene.

Cultivar identification is a primary concern for olive growers, breeders, and scientists. This study was aimed at examining the SSR markers retrieved from the literature and currently used in olive study, in order to select those most effective in characterizing the olive accessions and to make possible the comparison of data obtained by different laboratories. Olive microsatellite profiles were assessed by four independent laboratories, which analyzed 37 pre-selected SSR loci on a set of 21 cultivars. These SSR markers were initially tested for their reproducibility, power of discrimination and number of amplified loci/alleles. Independent segregation was tested for each pair of SSRs in a controlled cross and the allelic error rate was quantified. Some of them were finally selected as the most informative and reliable. Most of the alleles were sequenced and their sizes were determined. Profiles of the reference cultivars and a list of alleles with their sizes obtained by sequencing are reported. Several genetic parameters have been analysed on a larger set of cultivars allowing for a deeper characterization of the selected loci. Results of this study provide a list of recommended markers and protocols for olive genotyping as well as the allelic profile of a set of reference cultivars that would be useful for the establishment of a universal database of olive accessions.

Saponins, a group of glycosidic compounds present in several plant species, have aglycone moieties that are formed using triterpenoid or steroidal skeletons. In spite of their importance as antimicrobial compounds and their possible benefits for human health, knowledge of the genetic control of saponin biosynthesis is still poorly understood. In the Medicago genus, the hemolytic activity of saponins is related to the nature of their aglycone moieties. We have identified a cytochrome P450 gene (CYP716A12) involved in saponin synthesis in Medicago truncatula using a combined genetic and biochemical approach. Genetic loss-of-function analysis and complementation studies showed that CYP716A12 is responsible for an early step in the saponin biosynthetic pathway. Mutants in CYP716A12 were unable to produce hemolytic saponins and only synthetized soyasaponins, and were thus named lacking hemolytic activity (lha). In vitro enzymatic activity assays indicate that CYP716A12 catalyzes the oxidation of β-amyrin and erythrodiol at the C-28 position, yielding oleanolic acid. Transcriptome changes in the lha mutant showed a modulation in the main steps of triterpenic saponin biosynthetic pathway: squalene cyclization, β-amyrin oxidation, and glycosylation. The analysis of CYP716A12 expression in planta is reported together with the sapogenin content in different tissues and stages. This article provides evidence for CYP716A12 being a key gene in hemolytic saponin biosynthesis.

International audience; P>In light of the recent finding that Tuber melanosporum, the ectomycorrhizal ascomycete that produces the most highly prized black truffles, is a heterothallic species, we monitored the spatial distribution of strains with opposite mating types (MAT) in a natural truffle ground and followed strain dynamics in artificially inoculated host plants grown under controlled conditions. In a natural truffle ground, ectomycorrhizas (ECMs), soil samples and fruit bodies were sampled and genotyped to determine mating types. Simple sequence repeat (SSR) markers were also used to fingerprint ECMs and fruit bodies. The ECMs from nursery-inoculated host plants were analysed for mating type at 6 months and 19 months post-inoculation. In open-field conditions, all ECMs from the same sampling site showed an identical mating type and an identical haploid genotype, based on SSR analysis. Interestingly, the gleba of fruit bodies always demonstrated the same genotype as the surrounding ECMs. Although root tips from nursery-grown plants initially developed ECMs of both mating types, a dominance of ECMs of the same MAT were found after several months. The present study deepens our understanding of the vegetative and sexual propagation modes of T. melanosporum. These results are highly relevant for truffle cultivation.

Apomixis is a form of asexual reproduction through seed and has the potential to be applied, to great benefit, to agriculture. Understanding the genetic control of apomixis has proven to be a challenging task because the trait is mainly present in wild species and genetic mapping is often impaired by a block of recombination. A physical mapping approach has therefore been undertaken to unlock the genetic control of apomixis in Paspalum simplex Morong, a species with a relatively small genome and which exhibits a degree of genetic synteny with rice. In this paper, we report on the construction of a bacterial artificial chromosome library for Paspalum simplex with a coverage of approximately three genome equivalents and an average insert size of 94 kb. The BAC library was screened with 19 sequence characterized amplified region markers which were 100% linked to apomixis and a recombinant SCAR marker, all developed through a bulked segregant analysis strategy. A mini-sequencing procedure reported in the literature greatly aided the direct development of SCAR markers from amplified fragment length polymorphism bands. Several BAC clones linked to apomixis were identified and assembled into seven contigs and 18 singletons. Two of the BAC clones identified contained independently isolated markers. This is the first such report in an apomictic model that lacks recombination at the locus. We believe that extension of the contigs coupled to high-throughput sequencing will help the understanding of the genomic structure of the apomixis locus in P. simplex.

Creeping-rootedness (CR) is a form of vegetative propagation in lucerne (Medicago sativa L. complex) consisting of the development of new plants from adventitious shoots arising from roots with horizontal growth habit. Introgression of the CR character into cultivated lucerne may contribute to better stand persistence and greater grazing tolerance. Phenotypic selection of this trait is difficult owing to intrinsic features of the species such as autotetraploidy, allogamy and propagation by seed in addition to the polygenic control, low espressivity and variable penetrance of the trait. With the aim of developing a molecular marker-based system for the genotypic selection of the CR phenotype in lucerne, two segregating populations for this trait were screened with amplified fragment length polymorphisms (AFLPs), in order to identify CR-linked markers. Two AFLP amplicons were selected as segregating in a non-independent manner from the CR phenotype in both populations. The use of AFLP closer to the CR allows the conditional probability for CR selection to be nearly doubled. A PCR-based marker was developed for this AFLP and used to screen a collection of genotypes displaying either the CR or non-CR phenotype. The related primers were able to amplify the diagnostic amplicon in most of the CR genotypes and in approximately half of the non-CR individuals. Results are discussed in relation to the use of this marker in marker-assisted selection of the CR phenotype in lucerne.

Tuber melanosporum is an ectomycorrhizal ascomycete producing edible ascocarps. The prevalent view is that this species strictly selfs, since genetic analyses have never detected heterozygotic profiles in its putatively diploid/dikaryotic gleba. The selfing model has also forged the experimental approaches to assess the population genetic variability. Here, the hypothesis that T. melanosporum outcrosses was tested. To this end, SSR (simple sequence repeats) and ITS (internal transcribed spacer) markers were employed to fingerprint asci and the surrounding gleba within single ascocarps. The distribution of genetic variability was also investigated at different geographical levels using single (SSR and ITS) and multilocus (AFLP, amplified fragment length polymorphism) markers. It is shown that T. melanosporum outcrosses since asci display additional alleles besides those present in the surrounding, uniparental, gleba. Furthermore, SSR and AFLP data reveal a high rate of intrapopulation diversity within samples from the same ground and root apparatus and the highest rate of genetic variability within the southernmost populations of the distributional range. These data call for a profound re-examination of T. melanosporum mating system, life cycle and strategies for managing man-made plantations. They also strongly support the idea that the last glaciation restricted the species distribution to the Italian and Spanish peninsulas.

Proanthocyanidins (PAs) are plant secondary metabolites and are composed primarily of catechin and epicatechin units in higher plant species. Due to the ability of PAs to bind reversibly with plant proteins to improve digestion and reduce bloat, engineering this pathway in leaves is a major goal for forage breeders. Here, we report the cloning and expression analysis of anthocyanidin reductase (ANR) and leucoanthocyanidin 4-reductase (LAR), two genes encoding enzymes committed to epicatechin and catechin biosynthesis, respectively, in Lotus corniculatus. We show the presence of two LAR gene families (LAR1 and LAR2) and that the steady-state levels of ANR and LAR1 genes correlate with the levels of PAs in leaves of wild-type and transgenic plants. Interestingly, ANR and LAR1, but not LAR2, genes produced active proteins following heterologous expression in Escherichia coli and are affected by the same basic helix-loop-helix transcription factor that promotes PA accumulation in cells of palisade and spongy mesophyll. This study provides direct evidence that the same subclass of transcription factors can mediate the expression of the structural genes of both branches of PA biosynthesis.

Tuber spp. are ectomycorrhizal ascomycetes that produce ascocarps known as truffles. Basic aspects of Tuber biology have yet to be fully elucidated. In particular, there are conflicting hypotheses concerning the mating system and the ploidy level of the mycorrhizal and truffle hyphae. We used polymorphic microsatellites to compare the allelic configurations of asci with those from the network of the surrounding hyphae in single Tuber magnatum truffles. We then used these truffles to inoculate host plants and evaluated the microsatellite configurations of the resulting mycorrhizal root tips. These analyses provide direct evidence that T. magnatum outcrosses and that its life cycle is predominantly haploid. In addition to its scientific significance, this basic understanding of the T. magnatum life cycle may have practical importance in developing strategies to obtain and select nursery-produced mycorrhizal plants as well as in the management of artificial plantations of this and other Tuber spp.

Apomixis in plants is a form of clonal reproduction through seeds. A BAC clone linked to apomictic reproduction in Paspalum simplex was used to locate the apomixis locus on meiotic chromosome preparations. Fluorescent in situ hybridisation revealed the existence of a single locus embedded in a heterochromatin-poor region not adjacent to the centromere. We report here for the first time information regarding the sequencing of a large DNA clone from the apomixis locus. The presence of two genes whose rice homologs were mapped on the telomeric part of the long arm of rice chromosome 12 confirmed the strong synteny between the apomixis locus of P. simplex with the related area of the rice genome at the map level. Comparative analysis of this region with rice as representative of a sexual species revealed large-scale rearrangements due to transposable elements and small-scale rearrangements due to deletions and single point mutations. Both types of rearrangements induced the loss of coding capacity of large portions of the “apomictic” genes compared to their rice homologs. Our results are discussed in relation to the use of rice genome data for positional cloning of apomixis genes and to the possible role of rearranged supernumerary genes in the apomictic process of P. simplex.

Morphologically similar species such as Chinese black truffles and Tuber melanosporum were typed by restriction length polymorphism of internal transcribed spacers. This analysis together with sequence comparison revealed the presence of high genetic variability among fruit bodies collected in China. Selection of primer pairs allowed the internal transcribed spacer region of both Chinese truffles and T. melanosporum to be specifically amplified.

Anthocyanins and condensed tannins are major flavonoid end-products in higher plants. While the transactivation of anthocyanins by basic helix-loop-helix (bHLH) transcription factors is well documented, very little is known about the transregulation of the pathway to condensed tannins. The present study analyses the effect of over-expressing an Sn transgene in Lotus corniculatus, a model legume, with the aim of studying the regulation of anthocyanin and tannin end-products. Contrary to expectation, effects on anthocyanin accumulation were subtle and restricted to the leaf midrib, leaf base and petiole tissues. However, the accumulation of condensed tannin polymers was dramatically enhanced in the leaf blade and this increase was accompanied by a 50-fold increase in the number of tannin-containing cells in this tissue. A detailed analysis of selected lines indicated that this transactivational phenotype correlated with high steady-state transcript levels of the introduced transgene and the introduction of a single copy of the CaMV35S-Sn construct into these clonal genotypes. While the levels of condensed tannins in leaves were increased by up to 1% of the dry weight, other major secondary end-products (flavonols, lignins and inducible phytoalexins) were unaltered in transactivated lines. These results give an initial insight into the developmental and higher-order regulation of polyphenolic metabolism in Lotus and other higher plant species.

We have established mutant collections of the model species Medicago truncatula according to current protocols. In particular, we used a transposon (Tnt1) tagging method and an ethyl methanesulfonate (EMS) mutagenesis approach (TILLING). The collections were subjected to both forward and reverse genetics screenings, and several mutants were isolated that affect plant traits (e.g. shoot, root developments, flower morphology, etc.) and also biosynthetic pathways of secondary compounds (saponins and tannins). Genes responsible for some of the mutations were cloned and further characterized.

Segregating progenies of crosses between sexual and apomictic genotypes of Paspalum simplex were analysed for the formation of meiotic versus aposporous embryo sacs, zygotic versus parthenogenetic embryos, and autonomous versus pseudogamous endosperms by using cytoembryological and flow cytometric analyses. Reduced and unreduced 8-nucleated embryo sacs were the final product of female gametophyte development in sexual and aposporous genotypes, respectively. An incomplete penetrance of parthenogenesis was detected in aposporous genotypes. The relative DNA content of endosperm nuclei revealed the normal 2:1 maternal to paternal ratio in sexuals and a 4:1 ratio in apomicts, indicating insensitivity of the apomictic genotypes to endosperm imprinting. Apospory, parthenogenesis and pseudogamy are located on a relatively large linkage group and are inherited together with previously developed molecular markers as a single genetic unit in segregating progenies.

Three somatic hybrid plants produced by protoplast fusion between Medicago sativa and each of the three species Medicago coerulea, Medicago falcata and Medicago arborea have been analysed for the composition of their mitochondrial DNA. Restriction fragment length polymorphism (RFLP) analysis of mitochondrial genes in somatic hybrids and their parental lines showed various degrees of rearrangement. The M. sativa+M. coerulea hybrid retained all of the M. coerulea-specific bands but lost all the major M. sativa- specific bands. The M. sativa+M. falcata hybrid showed only M. sativa-specific bands together with non-parental bands, and the M. sativa+M. arborea hybrid showed a partial incorporation of bands from both parents together with non-parental bands. The three different outcomes were attributed mainly to differences in the genetic distance between the parents of each hybrid. Analysis of the sexual progeny of the M. sativa+M. coerulea hybrid showed that a residual mitochondrial DNA subunit of M. sativa was retained in the hybrid cytoplasm. This subunit was amplified and inherited in a mutually exclusive, allelic-like fashion with its M. coerulea homologous counterpart in the sexual progeny of the hybrid. Possible mechanisms for the partitioning of mitochondrial DNA in the generative lineage of the somatic hybrids are discussed in relation to the creation of new nucleus-cytoplasm assortments otherwise impossible to obtain by a sexual cross in Medicago.

Recent reports of discrepancies between the morphological and molecular-based characterization of Tuber magnatum ectomycorrhizae suggest misleading morphological typing of these structures. This study reports a new morphological and anatomical description of T. magnatum ectomycorrhizae at different development stages using nursery-inoculated host plants. Both ascocarps, used as spore donors in the inoculation process, and the resulting ectomycorrhizae were first checked by single-step PCR analysis using a T. magnatum species-specific ITS primer pair. Direct sequence analysis of the entire ITS1–5.8S-ITS2 fragment of DNA from mycorrhizal pools demonstrated the purity of the samples processed. Thus, the validity of the criteria reported here for the morphological identification of T. magnatum symbiotic structures is supported by molecular analysis. When diagnostic morphological traits appear only occasionally, as a result of ageing or of particular edaphic conditions, only molecular markers such as the species-specific ITS primers designed here ensure reliable typing of T. magnatum mycorrhizae for the purpose of controlling and/or certifying mycorrhizal plants present on the market.

Apomixis is a form of asexual reproduction that in plants leads to the production of seed progeny that are exact copies of the mother individual. A mapping population generated by backcrossing a sexual with an apomictic genotype of Paspalum simplex, both at the tetraploid level, was used to find markers co-segregating with apomixis. Genetic analysis showed that apomixis is under the control of a single dominant allele assuming a random chromatid assortment. Five rice markers, mapped in the telomeric region of the long arm of rice chromosome 12, showed tight linkage with apomixis. Genetic and molecular data strongly indicate that the potentiality to express apomixis in P. simplex is given by a relatively large chromosome segment that is inherited as a single genetic unit.

Three seed lots, obtained from different 'foundation farms', for each of the alfalfa, Medicdgo saliva L., ecotypes 'Vogherese' and 'Maremmana', together with the variety 'Iside' as a control, were studied to test the possibility of distinguishing them through restriction fragment length polymorphism analysis. Twenty plants per seed lot were analysed with 25 DNA probes that yielded 155 informative polymorphic fragments. Variance partitioning showed that within-population variability accounted for nearly 98% of the total variance, while a small but significant contribution to the total variance was due to among-lot variability. 'Vogherese' was more homogeneous than 'Maremmana', as a consequence of the greater environmental homogeneity of the adaptation area of the former ecotype compared with the latter. Bulk analysis yielded eight variety-specific bands, 12 bands present in both ecotypes and absent in the variety and one band specific of 'Maremmana'. Results are discussed in relation to the practical use of molecular markers for alfalfa ecotype identification.

La capacite des marqueurs RFLP de distinguer entre deux ecotypes heterogenes de luzerne, « Vogherese » et «Maremmana», a ete comparee avec le nombre de plantes composant un echantillon utilise aussi bien pour l'analyse sur des plantes individuelles que sur des plantes en melange («bulk»). La partition des composantes de la variance a ete comparee dans des sous-echantillons de populations independantes de 20 et 50 plantes, et les frequences de bandes ont ete aussi comparees dans des sous-populations de Maremmana constituees par 20, 50 et 100 plantes. Le degre d'homogeneite revele a l'interieur de 3 «bulks» independants, de tailles identiques a celles qui viennent d'etre mentionnees, a ete pris comme mesure de la taille optimale du «bulk». Un minimum de 50 plantes est necessaire aussi bien pour l'analyse sur plantes individuelles qu'en «bulk», alors que pour la grande majorite des bandes analysee, aucune difference significative de leur frequence n'a ete relevee entre les populations de 20, 50 et 100 plantes, a l'exception de 6 bandes sur 48. Un marqueur specifique d'un ecotype a ete trouve au moyen de l'analyse «bulk». Les resultats sont discutes dans l'optique d'une amelioration de la methodologie RFLP servant a l'identification des cultivars chez la luzerne.

The potential use of RFLP molecular markers for the identification of four creeping bentgrass (Agrostis stolonifera L.) cultivars and one variety of A. tenuis Sibth. used as control, was investigated. Seven probes out of the total 44 screened were able to differentiate all five cultivars at once. On the basis of their genetic similarity the varieties bred at the Pennsylvania State University were grouped closely together, whereas the variety Prominent and the A. tenuis control were more distantly related.

Accumulation of zeins, the endosperm storage proteins of maize, in a heterologous plant expression system was attempted. Plants of Nicotiana tabacum and Lotus corniculatus were transformed by Agrobacterium with binary vectors harbouring genes that code for γ-zein and β-zein, two zeins rich in sulphur amino acids. Adding the ER retention signal KDEL to the C-terminal domain modified the zein polypeptides. Significant levels of γ-zein:KDEL and β-zein:KDEL were detected in primary transformants of tobacco. Moreover, the two zeins colocalized in leaf protein bodies of γ-/β-zein:KDEL plants derived from a cross between two primary transformants. Coexpression of γ-zein:KDEL and β-zein:KDEL could be a useful strategy to obtain genotypes of forage legumes which are able to accumulate sulphur amino acids to high levels. As a first step, L. corniculatus plants expressing γ-zein:KDEL in the leaves were obtained.

The chromosomes of sexual diploid plants of Paspalum simplex were colchicine-doubled and the plant obtained were crossed with their aposporous natural tetraploid counterparts to generate a F1 population segregating for apomixis. Analysis of the DNA content during megagametogenesis indicated that although the nuclei of nucellus and developing embryo sacs were in both the G1 and G2 phases, polar nuclei and egg cells of mature embryo sacs tended to remain in the G1 phase. Because both meiotic and aposporous mature embryo sacs are of the 8-nucleated-type in P. simplex and are barely distinguishable, nuclear DNA content of polar nuclei was used to distinguish apomictic and sexual phenotypes and confirmation obtained by progeny testing.

Protocols for isolating and amplifying fungal DNA from ectomycorrhizae, overcoming the presence of PCR inhibitors, were established to ensure a fast and reliable truffle mycorrhizal identification. By using species-specific ITS primers in multiplex PCR, morphologically very similar Tuber melanosporum, Tuber indicum and Tuber brumale ectomycorrhizae were identified in a single step of amplification, irrespective of their physiological stage. The molecular method assures a more accurate and reliable monitoring of nursery-inoculated plants both before and after their in-field planting, than any morphological or ITS/ RFLP analyses so far performed. It should also prevent frauds, already a problem, and the potential for environmental damage due to the incorrect typing of the allochthonous species T. indicum as European truffle species, when the identity of ectomycorrhizae is based solely on morphology.

Although burr medic (Medicago polymorpha L.) is commonly considered a self-pollinating species, intrapopulational variation for morphological, biochemical and molecular markers is relatively high. To investigate whether part of this variation is the result of outcrossing, we designed RAPD analysis experiments to reveal both inter- and intra-accession crossing. No cases of inter-accession hybrids were documented, but an intra-accessional crossing rate of 0 to 4% was estimated for one of the four accessions studied. Therefore, a rare outcrossing event between local individuals and migrating genotypes may contribute to high genetic variability observed in natural M. polymorpha. A better understanding of the factors which influence outcrossing in M. polymorpha is pertinent both for medic breeding programmes and for assessing risks associated with releasing transgenic herbicide-resistant crops.

Apomictic tetraploid Paspalum simplex was crossed with colchicine-doubled diploid sexual plants belonging to the same species. Homologous genomic probes were selected from a partial PstI genomic library for their capacity to detect alleles specific to the apomictic parent, and their segregation was analyzed in the F1progeny. High levels of polymorphism between apomictic and sexual genotypes were recorded. The heterozygosity was high in both tetraploid and diploid genotypes but the differences between them were not as great as expected. In the sexual parent, some markers segregated as either a monoallelic duplex or a diallelic duplex, while several allelic configurations were observed in the apomictic parent. The segregation of double-dose monoallelic fragments demonstrated the tetrasomic inheritance of apomictic P. simplex. The correlations between apomixis, ploidy level, and tetrasomic inheritance are discussed.Key words: apomixis, tetrasomic inheritance, Paspalum simplex, molecular markers.

The aim of this study was to produce somatic hybrids between tetraploid (2n=4x=32) M. sativa and diploid (2n=2x=16) M. falcata and analyse their genomic structure. Protoplasts from genotypes selected for regeneration ability from the cultivar Rangelander of M. sativa and Wisfal-1 of M. falcata were electrofused. Seven somatic hybrid calli were produced and one of them regenerated plants. The hybrid nature of these plants and their genetic composition were assessed with morphological, cytological, and molecular analyses. The resulting plants were hyper-aneuploid (2n=33) and contained one extra long chromosome, indicating that a translocation had taken place. The presence of both types of parental sequences in the RAPDs analysis confirmed the true hybrid nature of the plants. Rearrangements within the parental genomes and the presence of somaclonal variation among hybrid plants were observed through an RFLP analysis of the nucleolar organizing region (NOR). The possible causes for the gross genomic alterations, and the suitability of this method for transferring useful agronomic traits from wild species to cultivated alfalfa, are discussed.

With the aim of increasing the content of sulphur amino acids (SAAs) in the aerial part of the plants, Nicotiana tabacum and two leguminous species, Medicago sativa and Lotus corniculatus, were transformed with a maize γ-zein gene (G1L), coding for a SAAs-rich protein. Gene fusions with two different promoters were constructed; one promoter was the constitutive cauliflower mosaic virus (CaMV) 35S and the other was the light-regulated ribulose bisphosphate carboxylase small subunit (rbcS) from a tobacco gene. The constructs were introduced by Agrobacterium-mediated gene transfer. All the transformants produced the G1L RNA (two differently sized transcripts were detected) and no relevant differences were found between the two promoters. Despite the presence of G1L message the γ-zein was found only in tobacco plants (up to 0.05% of the alcohol soluble proteins). Post-transcriptional mechanisms seemed to inhibit protein accumulation. The low level of G1L RNA in the transformed plants was probably the main factor that limited the production of γ-zein protein.

Medicago truncatula is one of the model species for legume molecular genetics. In the last decade different types of mutant populations have been created in this species that can be screened by forward and reverse-genetic approaches to identify and functionally characterize genes of interest. TILLING is a reverse-genetic method combining random chemical mutagenesis and a PCR-based screen to identify point mutations in regions of interest. The different steps of the TILLING analysis are described in a mutant collection of ~2,300 M2 individuals for which genomic DNA and M3 seed were obtained. A two-dimensional DNA pooling strategy was adopted to reduce the number of PCR reactions necessary to screen the collection and to unambigously identify the individual M2 plant carrying the mutation. The genotypic and phenotypic analyses of the mutant M3 progeny provide the possibility to study the gene function. In spite of its reduced size, this mutant collection has proved valid in the study of the biosynthetic pathway of a class of secondary metabolites present in the genus Medicago, the triterpenic saponins.

A cytological examination of the nucleolus organizing regions (NORs) of three species from the Medicago sativa complex was conducted to evaluate the structural and functional evolution of the ribosomal RNA (rRNA) loci that encode the 18S, 5.8S, and 26S rRNAs. Mitotic chromosomes in root-tip preparations from tetraploid M. sativa and diploids Medicago coerulea and Medicago falcata were visualized by four methods that provide new data. Fluorescent in situ hybridization using the M. sativa 18S gene as probe localized the structural rDNA to the constricted regions of the satellited chromosomes only. Chromomycin A3 (CMA3) staining and 4′,6-diamidino-2-phenylindole (DAPI) staining identified these chromosomal segments as the most GC-rich regions in the alfalfa karyotype. Medicago falcata exhibited fewer DAPI bands and chromocenters than did M. sativa and M. coerulea. Positive silver nitrate staining showed that all four rDNA regions in M. sativa (located in two chromosome pairs) and both rDNA sites in both diploid species remain transcriptionally active. Counts of nucleoli confirmed that all rDNA regions are independently capable of nucleolus organization. Thus, the number of active NORs in M. sativa is double the number found in M. coerulea or M. falcata. Consequently, if M. sativa originated from sexual hybridization of 2n gametes involving one or both diploid species, no major reorganization or loss of structural or functional rDNA loci has occurred. Key words : alfalfa evolution, CMA3 banding, DAPI banding, fluorescent in situ hybridization, silver nitrate staining.

Interspecific somatic hybrid plants were obtained by symmetrical electrofusion of mesophyll protoplasts of Medicago sativa with callus protoplasts of Medicago arborea. Somatic hybrid calli were picked manually from semi-solid culture medium after they were identified by their dual color in fluorescent light. Twelve putative hybrid calli were selected and one of them regenerated plants. The morphogenesis of the somatic hybrid calli was induced by the synthetic growth regulator 1,2 benzisoxazole-3-acetic acid. Somatic hybrid plants showed intensive genome rearrangements, as evidenced by isozyme and RFLP analysis. The morphology of somatic hybrid plants was in general intermediate between the parents. The production of hybrids by protoplast fusion between sexually incompatible Medicago species is related to the in vitro respon siveness of the parental protoplasts. The possibility of using somatic hybrid plants in alfalfa breeding is discussed.

Culture conditions have been established for callus induction and growth from different explants in L. angustissimus L. Calli were obtained from hypocotyls, leaves, stems, cotyledons and roots cultured on media containing 2,4-dichlorophenoxyacetic acid or α-naphthaleneacetic acid with kinetin, N6 − Δ2 or benzyladenine in different combinations and concentrations. Only those calli induced in presence of α-naphthaleneacetic acid with benzyladenine or kinetin produced shoots. Calli induced from hypocotyl explants were the most efficient in regeneration of shoots. Transformation with an Agrobacterium rhizogenes binary vector carrying the plasmid pBI 121.1 is reported. The percentage of cotransformation was estimated by testing GUS activity in hairy roots. The integration of Ri T-DNA and the NPTII gene in transformed plants was confirmed by molecular analyses and in vitro culture of transgenic tissues in the presence of kanamycin.

Tuber spp. are fungi that establish symbiosis with several trees and shrubs. Some of these fungal species produce edible ascomata, also known as truffles, which are highly appreciated for their taste and odour. We isolated and characterized eight polymorphic microsatellite loci from Tuber magnatum, the finest white truffle species, and assessed their variability in 370 individuals collected from all over the species range of distribution. Although two to 18 alleles per locus were found, no heterozygous individuals were observed. The availability of simple sequence repeat loci provides valuable tools for assessment of the genetic structure and population dynamics in this species.

Medicago truncatula is one of the model species for legume molecular genetics. In the last decade different types of mutant populations have been created in this species that can be screened by forward and reverse-genetic approaches to identify and functionally characterize genes of interest. TILLING is a reverse-genetic method combining random chemical mutagenesis and a PCR-based screen to identify point mutations in regions of interest. The different steps of the TILLING analysis are described in a mutant collection of ~2,300 M2 individuals for which genomic DNA and M3 seed were obtained. A two-dimensional DNA pooling strategy was adopted to reduce the number of PCR reactions necessary to screen the collection and to unambigously identify the individual M2 plant carrying the mutation. The genotypic and phenotypic analyses of the mutant M3 progeny provide the possibility to study the gene function. In spite of its reduced size, this mutant collection has proved valid in the study of the biosynthetic pathway of a class of secondary metabolites present in the genus Medicago, the triterpenic saponins.

Analyses, based on the structure of the rDNA region, were developed to examine the possibility of identifying and typing some of the most important Tuber spp at both the symbiotic and non-symbiotic levels. Internal transcribed spacers (ITS) were amplified by the polymerase chain reaction using specific primer pairs on genomic DNAs, isolated from ascocarps and ectomycorrhizae of various Tuber spp and from rootlets of uninoculated host plants. The variability in the lengths of ITS amplification products of DNA isolated from fruit bodies allowed a primary discrimination of the Tuber spp considered. At the ectomycorrhizal level the same species showed a pattern which was the combination of those typical of Tuber plus the host plants. Southern analyses, performed using the ITS product of Tuber brumale as a probe, confirmed the previous results : each fungus species showed a specific pattern, as well as each Tuber-host plant combination. It is concluded that molecular markers from the rDNA region can be used to differentiate Tuber spp and that the method proposed could be used to check and certify mycorrhizally infected plants.

Plant regeneration from explant and protoplast derived callus has been achieved in Medicago littoralis cv. Harbinger 1886, an annual legume resistant to the fungus Pseudopeziza medicaginis. Callus was induced from different tissue explants and the fastest growth rate was observed for hypocotyls in B5 medium with 2 mg l−1 2,4-dichlorophenoxyacetic acid and 0.5 mg l−1 N6-benzyladenine. Protoplasts were isolated from cotyledons and leaves of sterile plants and from callus; the first two kinds of protoplasts showed a plating efficiency of 5.6% and 5%, respectively, when embedded in agarose. Plant regeneration occurred on media containing % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9qq-f0-yqaqVeLsFr0-vr% 0-vr0db8meaabaqaciGacaGaaeqabaWaaeaaeaaakeaacaqGobWaaW% baaSqabeaacaqG2aaaaOGaaeOVfiaabs5adaahaaWcbeqaaiaaikda% aaGccaqG+waaaa!3F97!\[{\text{N}}^{\text{6}} {\text{\Delta }}^2 {\text{}}\]isopentenyl-adenine combined with indole-3-acetic acid or 1,2-benzisoxazole-3-acetic acid, and on media with N6-benzyladenine plus α-naphtaleneacetic acid; a cytokinin/auxin ratio higher than 1 induced embryos while a ratio around 1 stimulated shoot formation. Embryo development and rooting of shoots were performed in RL medium without growth regulators.

International audience; The genome of Tuber melanosporum has recently been sequenced. Here, we used this information to identify genes involved in the reproductive processes of this edible fungus. The sequenced strain (Mel28) possesses only one of the two master genes required for mating, that is, the gene that codes for the high mobility group (HMG) transcription factor (MAT1-2-1), whereas it lacks the gene that codes for the protein containing the alpha-box- domain (MAT1-1-1), suggesting that this fungus is heterothallic. A PCR-based approach was initially employed to screen truffles for the presence of the MAT1-2-1 gene and amplify the conserved regions flanking the mating type (MAT) locus. The MAT1-1-1 gene was finally identified using primers designed from the conserved regions of strains that lack the MAT1-2-1 gene. Mating type-specific primer pairs were developed to screen asci and gleba from truffles of different origins and to genotype single ascospores within the asci. These analyses provided definitive evidence that T. melanosporum is a heterothallic species with a MAT locus that is organized similarly to those of ancient fungal lineages. A greater understanding of the reproductive mechanisms that exist in Tuber spp. allows for optimization of truffle plantation management strategies.

Belowground ectomycorrhizal communities are often species rich. Characterization of the ectomycorrhizas (ECMs) underneath native truffle areas and/or cultivation sites is particularly relevant to identifying fungal species that might interfere with or promote truffle propagation and fruiting. Fungal identification at the genus/species level can now be achieved by combining detailed morphological and anatomical descriptions with molecular approaches. In a survey of the mycorrhizal biodiversity of Tuber melanosporum orchards and inoculated host plants in nurseries, we repeatedly sampled ECMs with morphological features resembling those of the ECMs widely known as the AD type. Despite the fact that the AD type is regarded as one of the most competitive fungal species towards Tuber spp., its taxonomical rank has yet to be resolved. By analyzing the 28S and internal transcribed spacer (ITS) rDNA regions, here, we show that AD-type ECMs result from host plant colonization by the pyronemataceous species Trichophaea woolhopeia. Further to this, the 28S and ITS phylogenetic trees built from the AD-type ECMs analyzed sustain the hypothesis that T. woolhopeia is a species complex.

The speciesLotus corniculatus andL. tenuis were transformed with anAgrobacterium rhizogenes binary vector, conferring resistance to the antibiotic hygromycin. Transgenic plants recovered from both species were tested for the ability of leaf-derived calluses to grow in a hygromycin-supplemented medium. Molecular analysis showed the integration of the Ri T-DNA and of the gene for hygromycin resistance, with a high frequency of co-transformation. Progeny analysis of the hygromycin resistance indicated this to be a single Mendelian trait in test plants. The transformed plants will be utilized in somatic hybridization experiments with lucerne for producing non-bloating genotypes with condensed tannins in leaves.

Seventeen ecotypes of the wild species Medicago polymorpha adapted to a Sardinian (Italy) environment have been evaluated for their response to tissue culture. The accession Samughero-Albi was the more respondent for callus induction and, together with Usassai, showed the highest regeneration capacity on media containing 1 mg l-1 2iP and 0.1 mg l-1 IAA. The morphogenetic response was also affected by the explant source. The hypocotyl-derived-calli were the best regenerating tissues. Regenerated plantlets were difficult to root and it was possible to obtain plants with a well developed root system only after 5–7 weeks of culture on media containing 2iP and IAA both at 0.2 mg l-1. Mesophyll cells were the best protoplast yielding source but only those isolated from roots were able to divide and to regenerate plants. Results are discussed in relation to the genotype specificity for the morphogenetic response and the feasibility of using M. polymorpha in the somatic hybridization with M. sativa.

Proanthocyanidins (PAs) are agronomically important biopolymers in higher plants composed primarily of catechin and epicatechin units. The biosynthesis of these natural products is regulated by transcription factors including proteins of the R2R3MYB class. To gain insight into the genetic control of the catechin and epicatechin branches of the PA pathway in forage legumes, here the effects of the expression of FaMYB1, a flavonoid R2R3MYB repressor from strawberry, in Lotus corniculatus (birdsfoot trefoil), were tested. It was found that in leaves of T(0) transgenic lines the degree of PA inhibition correlated with the level of FaMYB1 expression. These effects were heritable in the transgene-positive plant T(1) generation and were tissue specific as the suppression of proanthocyanidin biosynthesis was most pronounced in mesophyll cells within the leaf, whereas other flavonoid and phenolic compounds were substantially unaltered. The data suggest that FaMYB1 may counter-balance the activity of the endogenous transcriptional MYB-bHLH-WD40 (MBW) complex promoting proanthocyanidin biosynthesis via the catechin and epicatechin branches and that FaMYB1 does not interfere with the expression levels of a resident R2R3MYB activator of PAs. It is proposed that in forage legumes leaf cell commitment to synthesize proanthocyanidins relies on the balance between the activity of activator and repressor MYBs operating within the MBW complex.

Apomixis is defined as clonal reproduction by seed. A comparative transcriptomic analysis was undertaken between apomictic and sexual genotypes of Paspalum simplex Morong to identify apomixis-related polymorphisms at the level of mRNA. cDNA-AFLP (amplified fragment length polymorphism) profiling of apomictic and sexual flowers at several stages of development yielded 202 amplicons that showed several kinds of expression specificities. Among these, the large majority consisted of amplicons that were present only in specific stages of development of the apomictic flowers. Ten percent of polymorphic amplicons were present with almost identical intensity in all stages of the apomictic flowers and never in the sexual flowers. Reverse transcription-PCR (RT-PCR) and Southern analyses of these amplicons showed that they belong to constitutively expressed alleles that are specifically present on the apomixis-controlling locus of P. simplex. The most frequent biological functions inferred from the sequence homology of the apomixis-linked alleles were related to signal transduction and nucleic acid/protein-binding activities. Most of these apomixis-linked alleles showed nonsense and frameshift mutations, revealing their probable pseudogene nature. None of the amplicons that were present only in specific stages of development of the apomictic flowers co-segregated with apomixis, indicating they did not originate from additional apomictic alleles but more probably from differential regulation of the same allele in apomictic and sexual flowers. The molecular functions inferred from sequence analysis of these latter amplicons were related to seed storage protein and regulatory genes of various types. The results are discussed regarding the possible role in apomictic reproduction of the differentially expressed genes in relation to their specificity of expression and inferred molecular functions.

Seed storage proteins accumulate either in the endoplasmic reticulum (ER) or in vacuoles, and it would appear that polymerization events play a fundamental role in regulating the choice between the two destinies of these proteins. We previously showed that a fusion between the Phaseolus vulgaris vacuolar storage protein phaseolin and the N-terminal half of the Zea mays prolamin gamma-zein forms interchain disulfide bonds that facilitate the formation of ER-located protein bodies. Wild-type phaseolin does not contain cysteine residues, and assembles into soluble trimers that transiently polymerize before sorting to the vacuole. These transient interactions are abolished when the C-terminal vacuolar sorting signal AFVY is deleted, indicating that they play a role in vacuolar sorting. We reasoned that if the phaseolin interactions directly involve the C terminus of the polypeptide, a cysteine residue introduced into this region could stabilize these transient interactions. Biochemical studies of two mutated phaseolin proteins in which a single cysteine residue was inserted at the C terminus, in the presence (PHSL*) or absence (Delta 418*) of the vacuolar signal AFVY, revealed that these mutated proteins form disulphide bonds. PHSL* had reduced protein solubility and a vacuolar trafficking delay with respect to wild-type protein. Moreover, Delta 418* was in part redirected to the vacuole. Our experiments strongly support the idea that vacuolar delivery of phaseolin is promoted very early in the sorting process, when polypeptides are still contained within the ER, by homotypic interactions.

Letter; International audience; The Périgord black truffle ($Tuber\ melanosporum$ Vittad.) and the Piedmont white truffle dominate today's truffle market. The hypogeous fruiting body of $T.\ melanosporum$ is a gastronomic delicacy produced by an ectomycorrhizal symbiont endemic to calcareous soils in southern Europe. The worldwide demand for this truffle has fuelled intense efforts at cultivation. Identification of processes that condition and trigger fruit body and symbiosis formation, ultimately leading to efficient crop production, will be facilitated by a thorough analysis of truffle genomic traits. In the ectomycorrhizal $Laccaria\ bicolor$, the expansion of gene families may have acted as a 'symbiosis toolbox'. This feature may however reflect evolution of this particular taxon and not a general trait shared by all ectomycorrhizal species. To get a better understanding of the biology and evolution of the ectomycorrhizal symbiosis, we report here the sequence of the haploid genome of $T.\ melanosporum$, which at $\sim$125 megabases is the largest and most complex fungal genome sequenced so far. This expansion results from a proliferation of transposable elements accounting for $\sim$58% of the genome. In contrast, this genome only contains $\sim$7,500 protein-coding genes with very rare multigene families. It lacks large sets of carbohydrate cleaving enzymes, but a few of them involved in degradation of plant cell walls are induced in symbiotic tissues. The latter feature and the upregulation of genes encoding for lipases and multicopper oxidases suggest that $T.\ melanosporum$ degrades its host cell walls during colonization. Symbiosis induces an increased expression of carbohydrate and amino acid transporters in both $L.\ bicolor$ and $T.\ melanosporum$, but the comparison of genomic traits in the two ectomycorrhizal fungi showed that genetic predispositions for symbiosis $-$'the symbiosis toolbox'$-$ evolved along different ways in ascomycetes and basidiomycetes

Nodes ofMedicago sativa, Lotus corniculatus, Lotus tenuis, andLotus pedunculatus were cultured on MS basal media with different growth regulators. InM. sativa each node produced one shoot and the apical dominance was unaffected by high levels of cytokinins, and subsequent cycles of culture. Shoot development was stimulated by the presence ofN 6-isopentenyl-adenine in the culture medium and was dependent on the genotype of the explant. Shoot development was not affected by the original position of the node on the plant nor by the plant age. Shoots rooted in MS medium gelled with starch and containing 2 mg·liter−1 indol-3-acetic acid. In the threeLotus species, node culture was a more effective technique than inM. sativa. The number of shoots per node increased with the time of culture and with the presence of 0.05 mg·liter−1 of 6-benzylaminopurine. The highest number of shoots derived from one node was achieved inL. pedunculatus and inL. tenuis by culturing single nodes, whereas inL. corniculatus stem segments had to be totally covered by the medium for success. Rooting was easily achieved in MS medium with or without auxins.

Symmetric somatic hybrid plants have been produced by electrofusion of leaf protoplasts of Medicago sativa and callus protoplasts of Medicago coerulea. The selection of hybrid individuals has been performed at the cellular level by recording the positions of single heterocaryons immobilized in a semisolid culture medium. The hybrid nature of the heterokaryons was assessed in fluorescent light on the basis of their color. Hybrid minicalli were picked up manually and grown first on propagating, and then on regenerating, media. Six putative hybrid calli were selected and two of them regenerated several plants. The hybrid nature of the regenerants was confirmed by cytological and isozyme analysis. Among the several morphological traits taken into account for the characterization of somatic hybrid plants, some were intermediate, some lower, and some higher, with respect to the parents. The somatic hybrid plants were fertile and set seed. The production of somatic hybrid plants in the genus Medicago is discussed in relation to the regenerating capability of parental protoplasts.

The correct folding and assembly of newly synthesized secretory proteins are monitored by the protein quality control system of the endoplasmic reticulum (ER). Through interactions with chaperones such as the binding protein (BiP) and other folding helpers, quality control favors productive folding and sorts for degradation defective proteins. A major route for quality control degradation identified in yeast, plants, and animals is constituted by retrotranslocation from the ER to the cytosol and subsequent disposal by the ubiquitin/proteasome system, but alternative routes involving the vacuole have been identified in yeast. In this study, we have studied the destiny of sGFP418, a fusion between a secretory form of GFP and a domain of the vacuolar protein phaseolin that is involved in the correct assembly of phaseolin and in BiP recognition of unassembled subunits. We show that sGFP418, despite lacking the phaseolin vacuolar sorting signal, is delivered to the vacuole and fragmented, in a process that is inhibited by the secretory traffic inhibitor brefeldin A. Moreover, a fusion between GFP and a domain of the maize storage protein gamma-zein involved in zein polymerization also undergoes post-translational fragmentation similar to that of sGFP418. These results show that defective secretory proteins with permanently exposed sequences normally involved in oligomerization can be delivered to the vacuole by secretory traffic. This strongly suggests the existence of a plant vacuolar sorting mechanism devoted to the disposal of defective secretory proteins.

It is very important for the application of chloroplast engineering to extend the range of species in which this technology can be achieved. Here, we describe the development of a chloroplast transformation system for the sugar beet (Beta vulgaris L. ssp. vulgaris, Sugar Beet Group) by biolistic bombardment of leaf petioles. Homoplasmic plastid-transformed plants of breeding line Z025 were obtained. Transformation was achieved using a vector that targets genes to the rrn16/rps12 intergenic region of the sugar beet plastome, employing the aadA gene as a selectable marker against spectinomycin and the gfp gene for visual screening of plastid transformants. gfp gene transcription and protein expression were shown in transplastomic plants. Detection of GFP in Comassie blue-stained gels suggested high GFP levels. Microscopy revealed GFP fluorescence within the chloroplasts. Our results demonstrate the feasibility of engineering the sugar beet chloroplast genome; this technology provides new opportunities for the genetic improvement of this crop and for social acceptance of genetically modified sugar beet plants.

Leaf mesophyll protoplasts of Medicago arborea (2n = 32) were electrofused with cell suspension and/or callus protoplasts of Medicago sativa. Heterokaryons were identified in agarose beds by their dual fluorescein isothiocianate—chlorophyll fluorescence and their coordinates were recorded. Hybrid minicalli were manually picked up and grown first in nurse culture and then in callus induction medium. Hybrid nature of the selected calli was confirmed by isoenzyme analyses. In order to verify whether morphogenesis of somatic hybrid calli was affected by cell incompatibility, mesophyll and cell suspension protoplasts, derived from the same plant of M. sativa with high embryogenic capacity, were fused. Only callus tissues derived from mesophyll protoplasts retained the highly embryogenic character of the M. sativa genotype, while hybrid cell lines were non-morphogenic and showed isoenzyme patterns similar to tissues derived from cell suspension protoplasts. The achievement of somatic hybrid plants in the genus Medicago is discussed.

A cultivar of Medicago sativa was transformed with Agrobacterium tumefaciens strain At81 binary-vector carrying the plasmid pKan3A, with the neomycin phosphotransferase gene under the control of the mannopine biosynthesis promoter. Stem segments were infected with the bacterium and kanamycin resistant calli were obtained; plant regeneration via somatic embryogenesis and polyembryogenesis was achieved only in media devoid of the antibiotic. Genetic transformation was confirmed by the presence of the structural gene through DNA-DNA hybridization and the enzymatic assay showed its functional expression. Mesophyll protoplasts, leaf calli and S1 progeny of transformed plants grew in the presence of kanamycin (100 μgml-1). Results are discussed in relation to the use of kanamycin-resistant plants in somatic hybridization in the genus Medicago.