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.

Seeds of Medicago truncatula, a genomic model species for legumes, exhibit physiological and physical dormancy. Here, the factors influencing the germination behaviour of freshly harvested and stored seeds were investigated using several genotypes. Hardseededness is promoted when mature seeds are equilibrated at relative humidities (RH) below 75%. The release of physical dormancy during imbibition was dependent on the initial water content/RH that the seeds were dried to: the drier the seeds, the longer the imbibition time needed to break physical dormancy. The kinetics of physical dormancy release was slower than that of physiological dormancy, making it possible to uncouple both phenomena. Freshly harvested embryos without seed coverings germinated at the same speed as afterripened seeds. The depth of dormancy varied between different M. truncatula genotypes, from more to less dormant: DZA315.16>A17 (Jemalong)>R108>DZA45.5. This difference was eliminated by removing the endosperm. Collectively, these observations indicate that the endosperm is likely the main factor in the reduced germination of freshly harvested seeds. White light decreased germination speed of dormant seeds whereas it had no effect on non-dormant seeds. Recently harvested seeds were most dormant at temperatures above 17°C, whereas afterripened seeds germinated over a wider range of temperature. Fluridone could efficiently break dormancy, reinforcing the role of abscisic acid (ABA) synthesis. However, dormancy was not affected by gibberellic acid (100 μM GA3) or nitrate. The particular dormancy features unravelled here for M. truncatula, combined with the available genomic resources, make it a new, useful model for genetic and molecular studies which can complement those developed for Arabidopsis.

Molecular markers have been used to study genetic diversity within a set of Lablab purpureus accessions collected from the southern states of India. Amplified fragment length polymorphism (AFLP) molecular marker studies using a total of 78 L. purpureus accessions with nine primer combinations showed there was very little genetic diversity within the L. purpureus accessions from the southern Indian germplasm collection as compared to a set of 15 accessions from other international germplasm collections that included African accessions. The set of 15 were selected from a random amplified length polymorphism (RAPD) marker study and chosen on the basis of widest genetic distance. Further molecular analysis with polymerase chain reaction (PCR) markers from 97 expressed sequence tag (EST) and gene-specific primer pairs, designed from a range of legume sequences, concurred with the AFLP analyses. Both of these approaches provide a wealth of markers for diversity and mapping studies. The 97 sequence-specific primer pairs tested in L. purpureus resulted in 70% amplification success, with 44% of primer pairs amplifying single bands and 10% double bands. Markers generated from these EST and genomic sequences provide useful cross-reference to comparative legume genomics that will potentially have long-term benefit to legume plant breeding.

Germinated seeds of Medicago truncatula Gaertn. with a protruded radicle length of 2.7 mm did not survive drying below 0.2 g H2O g–1 dw, as indicated by vital stain assays and the absence of growth resumption after rehydration. The re-establishment of desiccation tolerance was achieved using an osmotic treatment with polyethylene glycol (PEG), combined with a cold treatment. The ability to regain desiccation tolerance after germination was restricted to a period of growth characterized by radicle lengths between 1 and 3 mm. After PEG treatment of germinated seeds with 2.7 mm long radicles at –1.7 MPa at 10°C for 3 d and subsequent drying to 0.04 g H2O g–1 dw, 90% survived and developed into normal seedlings after rehydration. Desiccation tolerance could also be re-established in excised radicles, demonstrating that cotyledons were not essential for this process. Upon PEG incubation, sucrose accumulated rapidly prior to the re-establishment of desiccation tolerance in germinated radicles, regardless of the presence of cotyledons. Induction of MtDHN (a dehydrin) gene expression was correlated with the re-establishment of desiccation tolerance. Furthermore, the PEG-induced expression of MtDHN was repressed when fluridone was added to the PEG solution.

Fifty lines of Medicago truncatula, derived from ecotypes or cultivars of diverse geographical origin, were grown under uniform conditions, and variation in seed protein composition and quantity was investigated. One-dimensional electrophoretic profiles revealed 46 major seed polypeptides, of which 26 were polymorphic within the collection. The vicilin/convicilin (7S) and the legumin (11S) type proteins were identified by immunoblotting using antibodies raised against the homologous pea proteins. The polymorphism for the major seed protein classes allowed the clustering of the genotypes into four groups. There was no evidence of clustering according to geographical origin of the lines. However, all lines not belonging to either M. truncatula ssp. truncatula or ssp. longispina were clustered in a single group, demonstrating the value of seed protein profiles in delimiting species boundaries. The Jemalong line group was differentiated early in the dendrogram, and thus represents an ancient clade in the seed diversity of M. truncatula. Within-accession variation was investigated for one-dimensional seed profiles, with additional lines obtained from the same ecotypes. As expected for an autogamous species, within-accession variation was low. Seed protein content was highly variable among the 50 lines examined. Lines contrasting for qualitative traits and seed protein content were identified to allow for the genetic determination of these characters.

Responses of Medicago truncatula to colonization by two arbuscular mycorrhizal fungi, Scutellospora calospora isolate WUM 12(2) and Glomus caledonium isolate RIS 42, were compared in the light of previous findings that the former fungus can be ineffective as a beneficial microsymbiont with some host plants. The plants were grown individually in two-compartment systems in which a lateral side arm containing soil labelled with 33P was separated from the main soil compartment by a nylon mesh that prevented penetration by roots but not fungal hyphae. Fungal inoculum was applied as a root–soil mixture in a band opposite the side arm. Nonmycorrhizal controls were set up similarly, without inoculum. There were harvests at 28, 35, 42 and 49 d. Both sets of mycorrhizal plants grew better than nonmycorrhizal plants and initially had higher concentrations of P in shoots and roots. Plants grown with S. calospora grew better than plants grown with G. caledonium, and this was associated with somewhat greater fungal colonization in terms of intraradical hyphae and numbers of arbuscules. Scutellospora calospora formed denser hyphae at root surfaces than G. caledonium. By 28 d there were extensive hyphae of both fungi in the side arms, and after 35 d S. calospora produced denser hyphae there than G. caledonium. Nevertheless, there was very little transfer of 33P via S. calospora to the plant at 28 d, and thereafter its transfer increased at a rate only c. 33% of that via G. caledonium. The results showed that plants colonized by S. calospora preferentially obtained P from sites in the main soil chamber relatively close to the roots, compared with plants colonized by G. caledonium. Hence formation of a highly beneficial arbuscular mycorrhizal symbiosis does not necessarily depend on development of hyphae at a distance from the roots or on large-scale translocation of P from distant sites. The results are discussed in relation to previous studies with compartmented systems that have involved the same fungi. Possible causes of the variable effects of S. calospora in symbiosis with different host plants are briefly assessed. Differences in spatial abilities of individual arbuscular mycorrhizal fungi to acquire P might have strong ecological implications for plant growth in soils low in P.