Medicago Truncatula Cv Research Articles

A newly evolved chimeric lysin motif receptor-like kinase in Medicago truncatula spp. tricycla R108 extends its Rhizobia symbiotic partnership.

Rhizobial lipochitooligosaccharidic Nod factors (NFs), specified by nod genes, are the primary determinants of host specificity in the legume-Rhizobia symbiosis. We examined the nodulation ability of Medicago truncatula cv Jemalong A17 and M. truncatula ssp. tricycla R108 with the Sinorhizobium meliloti nodF/nodL mutant, which produces modified NFs. We then applied genetic and functional approaches to study the genetic basis and mechanism of nodulation of R108 by this mutant. We show that the nodF/nodL mutant can nodulate R108 but not A17. Using genomics and reverse genetics, we identified a newly evolved, chimeric LysM receptor-like kinase gene in R108, LYK2bis, which is responsible for the phenotype and can allow A17 to gain nodulation with the nodF/nodL mutant. We found that LYK2bis is involved in nodulation by mutants producing nonO-acetylated NFs and interacts with the key receptor protein NFP. Many, but not all, natural S. meliloti and S. medicae strains tested require LYK2bis for efficient nodulation of R108. Our findings reveal that a newly evolved gene in R108, LYK2bis, extends nodulation specificity to mutants producing nonO-acetylated NFs and is important for nodulation by many natural Sinorhizobia. Evolution of this gene may present an adaptive advantage to allow nodulation by a greater variety of strains.

Expression of small GTPases in the roots and nodules of Medicago truncatula cv. Jemalong

Abstract In this study we used Medicago truncatula, to identify and analyze the expression of small GTP-binding proteins (Arf1, Arl1, Sar1, Rabs, Rop/Rac) and their interacting partners in the infection process in the roots and nodules. A real-time polymerase chain reaction analysis was carried out and our results showed that Arf1 (AtArfB1c-like), MtSar1, AtRabA1e-like, AtRabC1-like, MsRab11-like and AtRop7-like genes were highly expressed in the nodules of rhizobium inoculated plants compared to the non-inoculated ones. On the contrary, AtRabA3 like, AtRab5c and MsRac1-like genes were highly expressed in non-infected nitrogen supplied roots of M. truncatula. Other Rab genes (AtRabA4a, AtRabA4c and AtRabG3a-like genes) were nearly equally expressed in both treatments. Interestingly, RbohB (a respiratory burst NADPH oxidase homologue) was more highly expressed in rhizobium infected than in non-infected roots and nodules. Our data show a differential expression pattern of small GTP-binding proteins in roots and nodules of the plants. This study demonstrates an important role of small GTP-binding proteins in symbiosome biogenesis and root nodule development in legumes.

Physical Mutagenesis in Medicago truncatula Using Fast Neutron Bombardment (FNB) for Symbiosis and Developmental Biology Studies.

Medicago truncatula has been selected as a model species for legume molecular genetics and functional genomics studies. With the completion of the Medicago truncatula cv. Jemalong A17 genome sequencing, a major challenge is to determine the function of the large number of genes in the genome. Development of diverse mutant resources is crucial for gene functional studies. In the past years, M2 seeds from over 150,000 Medicago truncatula mutant lines in the Jemalong A17 background have been generated coordinately at the Noble Research Institute, USA, and the John Innes Centre, UK, using fast neutron bombardment (FNB) mutagenesis. These mutant resources have been used in screening and characterization of different categories of mutants including symbiotic nitrogen fixation, nodule development, and growth and patterning of leaf, stem, and root system architecture in the legume system. Here, we describe the detail procedure that has been used for screening of mutants derived from fast neutron bombardment mutagenesis in Medicago truncatula.

Identification of LEC1, L1L and Polycomb Repressive Complex 2 genes and their expression during the induction phase of Medicago truncatula Gaertn. somatic embryogenesis

Although somatic embryogenesis (SE), a multi-step process starting from somatic tissues and ending with somatic embryos, has been applied to numerous plants including Medicago sp., the molecular basis of development reprograming in somatic cells toward the embryogenic pathway is still incompletely known. Though recent analysis of the proteome and transcriptome has led to the identification and characterization of new genes involved in SE, lot of these genes are up-regulated only in the late developmental stages. Consequently, this work was aimed at finding out if and when the genetic program changed during the SE induction phase in both the highly embryogenic line M9-10a of Medicago truncatula cv. Jemalong and its non-embryogenic predecessor line, M9. Based on multipoint (day 0, 2, 7, 14 and 21) gene expression qPCR analysis of two embryogenesis marker genes LEC1 and L1L and selected genes encoding proteins of PRC2 complex (CLF, SWN, FIE, MSI1, VRN2) it was possible to distinguish two periods during the induction phase. The first week was related to dedifferentiation with no visible changes in explant morphology and lack of transcripts of LEC1 and L1L; the expression of PRC2 members, however, increased in the embryogenic line. The next two weeks were regarded as the expression phase involving the beginning of a rapid callus growth and the appearance of products for LEC genes, which was observed in the embryogenic line only. However, the callus formation was observed in the non-embryogenic line as well, but the LEC1 and L1L genes were not expressed and the transcription of PRC2 genes was at stable level. It was only the SWN expression that decreased at the beginning of induction and did not change in the subsequent days in both lines. Our results indicate that LEC1 and L1L, known as marker genes of the late developmental stages, may be associated with the acquisition of embryogenic competency by somatic cells (prime events in the SE induction). The PRC2 complex genes are also expressed during embryogenic callus tissue formation on Medicago tuncatula leaf explants.

Comparative Proteomic Analysis Reveals Differential Root Proteins in Medicago sativa and Medicago truncatula in Response to Salt Stress.

Salt stress is an important abiotic stress that causes decreased crop yields. Root growth and plant activities are affected by salt stress through the actions of specific genes that help roots adapt to adverse environmental conditions. For a more comprehensive understanding of proteins affected by salinity, we used two-dimensional gel electrophoresis and mass spectrometry to characterize the proteome-level changes associated with salt stress response in Medicago sativa cv. Zhongmu-1 and Medicago truncatula cv. Jemalong A17 roots. Our physiological and phenotypic observations indicated that Zhongmu-1 was more salt tolerant than Jemalong A17. We identified 93 and 30 proteins whose abundance was significantly affected by salt stress in Zhongmu-1 and Jemalong A17 roots, respectively. The tandem mass spectrometry analysis of the differentially accumulated proteins resulted in the identification of 60 and 26 proteins in Zhongmu-1 and Jemalong A17 roots, respectively. Function analyses indicated molecule binding and catalytic activity were the two primary functional categories. These proteins have known functions in various molecular processes, including defense against oxidative stress, metabolism, photosynthesis, protein synthesis and processing, and signal transduction. The transcript levels of four identified proteins were determined by quantitative reverse transcription polymerase chain reaction. Our results indicate that some of the identified proteins may play key roles in salt stress tolerance.

Single-plant, Sterile Microcosms for Nodulation and Growth of the Legume Plant <em>Medicago truncatula</em> with the Rhizobial Symbiont <em>Sinorhizobium meliloti </em>

Rhizobial bacteria form symbiotic, nitrogen-fixing nodules on the roots of compatible host legume plants. One of the most well-developed model systems for studying these interactions is the plant Medicago truncatula cv. Jemalong A17 and the rhizobial bacterium Sinorhizobium meliloti 1021. Repeated imaging of plant roots and scoring of symbiotic phenotypes requires methods that are non-destructive to either plants or bacteria. The symbiotic phenotypes of some plant and bacterial mutants become apparent after relatively short periods of growth, and do not require long-term observation of the host/symbiont interaction. However, subtle differences in symbiotic efficiency and nodule senescence phenotypes that are not apparent in the early stages of the nodulation process require relatively long growth periods before they can be scored. Several methods have been developed for long-term growth and observation of this host/symbiont pair. However, many of these methods require repeated watering, which increases the possibility of contamination by other microbes. Other methods require a relatively large space for growth of large numbers of plants. The method described here, symbiotic growth of M. truncatula/S. meliloti in sterile, single-plant microcosms, has several advantages. Plants in these microcosms have sufficient moisture and nutrients to ensure that watering is not required for up to 9 weeks, preventing cross-contamination during watering. This allows phenotypes to be quantified that might be missed in short-term growth systems, such as subtle delays in nodule development and early nodule senescence. Also, the roots and nodules in the microcosm are easily viewed through the plate lid, so up-rooting of the plants for observation is not required.

The sorption of organic carbon onto differing clay minerals in the presence and absence of hydrous iron oxide

Sorption of organic carbon onto phyllosilicate clays and hydrous iron oxides influences the accumulation and stabilisation of organic carbon in soils. However, the effects of interactions between hydrous iron oxides and phyllosilicate clays on the sorption of dissolved organic carbon (DOC) are poorly understood. We carried out a batch experiment to examine the effects of goethite coatings on kaolinite, illite, and smectite on DOC sorption. The effect of coating illitic clay with different hydrous iron oxides (haematite, goethite, ferrihydrite) on DOC sorption was studied in another experiment. Organic matter extracted from dried medic (Medicago truncatula cv. Praggio) shoot residue was reacted with minerals at DOC concentrations ranging from 0 to 200mgCL−1 at pH6.0. The maximum adsorption capacity (Qmax) of phyllosilicate clays, as determined from fits to the Langmuir equation, increased in the order kaolinite<illite<smectite on a mass basis and illite<smectite<kaolinite on a surface area basis. The sorption capacity of kaolinitic clay increased significantly with goethite coating, whereas the sorption capacity of illitic and smectitic clays was not affected by goethite coating. Ferrihydrite coating increased the sorption capacity of the illitic clays, while haematite coating decreased the sorption capacity; goethite-coated illitic clays had a sorption capacity similar to pure illitic clays. Desorption experiments resulted in the removal of 6–14% of the sorbed DOC. The presence of goethite reduced desorption from kaolinitic clays but did not influence desorption from illitic and smectitic clays. The results suggest that interactions of hydrous iron oxides and phyllosilicate clays can modify DOC sorption and desorption, probably by affecting the surface charges. Therefore, sorption and desorption of organic matter from soils may vary with mineral assemblage, with increasing suppression of the contribution of hydrous oxides at circumneutral to slightly alkaline soil reaction.

Molecular cloning and characterization of ARF1 and COPI coat proteins from Medicago truncatula cv. Jemalong: Dissection of their role in vesicular transport in root cells

The integrity of the Golgi apparatus in both plant and mammalian cells is dependent upon a coordinated flow of COPII (coatomer protein) coated vesicles in anterograde (forward) and of COPI coated vesicles in retrograde (backward) direction. Although a fair amount of work on vesicular trafficking has been published in Arabidopsis thaliana not much information is available related to the secretory pathway in other higher plants. In the present study we have used Medicago truncatula, a model plant for legume species as for symbiotic and pathogenic interactions, to identify Arf1 and COPI components of the early secretory pathway. Their localisation and interaction with the Golgi apparatus in the root cells has been identified by confocal and two-photon laser microscopy. EST databases of the M. truncatula were screened and putative homologues for all seven coatomer proteins and MtArf1 were identified. Two isoforms of the ζ- COPI subunit and MtArf1 genes were isolated from M. truncatula cDNA libraries and were sequenced. GFP fusions of MtArf1, dominant active (Q71L), dominant inactive (T31N) forms of MtArf1and two isoforms of MtζCOP1 were expressed in transgenic “hairy root” and their subcellular localisation were analyzed. Our results show that MtArf111::GFP, Mtζ-COP1:: GFP and Mtζ-COP2::GFP were localized on mobile Golgi structures, streaming along the ER network and were sensitive to brefeldin A, indicating their potential association with Golgi stacks. This study demonstrates an important role of Arf1 and COP1 proteins in early secretory pathway in root cells of M. truncatula.

Osmotic shock improves Tnt1 transposition frequency in Medicago truncatula cv Jemalong during in vitro regeneration

Insertion mutant collections are powerful tools for genetic studies in plants. Although large-scale insertional mutagenesis using T-DNA is not feasible in legumes, the Tnt1 tobacco retrotransposon can be used as a very efficient mutagen in the Medicago truncatula R108 genotype. In this article, we show that Tnt1 can also be exploited to create insertional mutants via transformation and/or regeneration in the reference cultivar Jemalong. Tnt1 insertional mutagenesis in Jemalong following Agrobacterium tumefaciens-mediated transformation was found to be very efficient, with an average of greater than 15 insertions/line. In contrast, regeneration using low-copy transgenic starter lines resulted in a highly variable rate of new Tnt1 insertions. With the goal of increasing the number of additional Tnt1 insertions during regeneration of starter lines, we have compared the insertion frequencies for a number of different regeneration protocols. In addition, we have been able to show that sucrose-mediated osmotic shock preceding regeneration significantly increases the transposition frequency. Under optimal conditions, 95% of the regenerated Jemalong plants possess new insertions.

Can differences of nitrogen nutrition level among Medicago truncatula genotypes be assessed non-destructively? Probing with a recombinant inbred lines population

The international consensus on Medicago truncatula as a model system has lead to the development of powerful approaches for dissecting the genetic and molecular bases of legume nitrogen nutrition. However, such approaches now come up against a poor knowledge of the phenotypic traits that should be used for the large-scale screening of the genotypic variability associated with nitrogen nutrition. This issue was unravelled in a previous report, in which an ecophysiological approach allowed a better understanding of the relationships between plant nitrogen nutrition and plant growth traits, for the model symbiotic association between M. truncatula cv. Jemalong and Rhizobium meliloti strain 2001. From this analysis, phenotypic traits were identified as potentially relevant for the large-scale screening of the genotypic variability. Here, by the phenotyping of a recombinant inbred lines population, we show that the proposed methodology provides a valuable support for assisting the detection of genetic variants affected for nitrogen uptake. Especially, the relative expansion rate of plant leaf area is identified as a good proxy for ranking genotypes according to their ability to uptake nitrogen in given environmental conditions. As leaf area can be measured non-destructively, such finding should pave the way for a more efficient evaluation of the genetic variability.Addendum to: Moreau D, Voisin AS, Salon C, Munier-Jolain N. The model symbiotic association between Medicago truncatula cv. Jemalong and Rhizobium meliloti strain 2001 leads to N-stressed plants when symbiotic N2 fixation is the main N source for plant growth. J Exp Bot 2008; 59:3509-22; PMID: 18703494; DOI:10.1093/jxb/ern203.

The model symbiotic association between Medicago truncatula cv. Jemalong and Rhizobium meliloti strain 2011 leads to N-stressed plants when symbiotic N2 fixation is the main N source for plant growth

A better knowledge of the nitrogen nutrition of Medicago truncatula at the whole plant level and its modulation by environmental factors is a crucial step to reach a complete understanding of legume nitrogen nutrition. This study was based on the symbiotic system that is the most commonly used by the research community (M. truncatula cv. Jemalong A17 x Rhizobium meliloti strain 2011). Plant nitrogen nutrition was analysed in relation to carbon nutrition, under a range of nitrate concentrations in the nutrient solution and different light conditions. This study shows that this 'model symbiotic association' does not allow the plant to meet its nitrogen requirements, when dinitrogen fixation is the main nitrogen source for plant growth. A strong interaction between nitrogen and carbon nutrition was shown: when plant nitrogen requirements were not sustained, plant leaf area was much affected whereas photosynthesis per unit leaf area remained relatively stable. Both total nitrogen uptake and leaf area increased with increasing nitrate concentration in the nutrient solution; the magnitude of these responses varied according to the light conditions. Interestingly, the plant nitrogen nutrition level remained nearly unaffected by the light conditions. The observed nitrogen-limitation in this 'model symbiotic association' is an important finding for the research community. Based on practical recommendations regarding both the experimental conditions and the phenotypic traits to consider, a methodological framework was proposed to (i) help genomicists to assess plant nitrogen nutrition better, and (ii) assist in the detection of new genetic variants affected for nitrogen uptake in large-scale phenotyping studies.

AFLP and AMP Fingerprints as Markers to Evaluate Genetic Differences between Medicago truncatula Line Jemalong and 2HA, a New Line Produced by in vitro Culture Selection

A new line, Medicago truncatula cv. Jemalong 2HA (herein known as 2HA) has been developed via repetitive regeneration and selection of M. truncatula cv. Jemalong. During somatic embryogenesis, 2HA produces 500 times more embryos than its progenitor, Jemalong. It is interesting to study if those two lines are isogenic or has genetic differences. The main objectives of the study was to evaluate the genotypic differences between Jemalong and 2HA also to evaluate the methylation event in 2HA utilized two DNA fingerprinting techniques, i.e AFLP fingerprints (Amplified Length of Polymorphism) and AMP (Amplified Methylation Polymorphism). The results showed that AFLP analysis using eight primers combinations could not detect any differences between Jemalong and 2HA. However, using AMP methylation sensitive primers it could detect 15 polymorphisms out of 840 markers. These results lead to a conclusion that Jemalong and 2HA are isogenic lines. 2HA may have higher regeneration capacities due to methylation process which occurs during the production of 2HA through repetitive regeneration cycles.

Use of fused gfp and gus reporters for the recovery of transformed Medicago truncatula somatic embryos without selective pressure

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.

Genetic variation and pathogenicity of anastomosis group 2 isolates of Rhizoctonia solani in Australia

A collection of isolates of Rhizoctonia solani anastomosis group (AG) 2 was examined for genetic diversity and pathogenicity. Anastomosis reactions classified the majority of isolates into the known subgroups of AG 2-1 and AG 2-2 but the classification of several isolates was ambiguous. Morphological characters were consistent with the species, with no discriminating characters existing between subgroups. Vertical PAGE of pectic enzymes enabled the separation of zymogram group (ZG) 5 and 6 within AG 2-1, but not the separation of ZG 4 and 10 within AG 2-2. PCR analysis using inter-simple sequence repeats (ISSR) and the intron-splice junction (ISJ) region supported the separation of ZG 5 and 6, while the AG 2-2 isolates were separated by geographic region. A comparison of distance matrices produced by the zymogram analysis and PCR indicated a strong correlation between the marker types. Pathogenicity studies suggested canola ( Brassica napus) cultivars were most severely affected by AG 2-1, while cultivars of two species of medic ( Medicago truncatula cv. Caliph and M. littoralis cv. Herald) were susceptible to both AG 2-1 and 2-2. The results indicate that AG 2 is a polyphyletic group in which the classification of subtypes is sometimes difficult. Further investigation of the population structure within Australia is required to determine the extent and origin of the observed diversity.

Determination of Saponins in Aerial Parts of Barrel Medic (Medicago truncatula) by Liquid Chromatography−Electrospray Ionization/Mass Spectrometry

Triterpene saponins from aerial parts of Medicago truncatula cv. Jemalong A-17, M. truncatula Gaertn.var. longispina Urb., and M. truncatula Gaertn. var. truncatula were profiled and quantified using reverse-phase liquid chromatography with on-line photodiode array detection and electrospray ionization mass spectrometry (LC-PDA/ESI/MS/MS). The determination was based on standard curves obtained for the 18 available saponin standards, previously isolated from Jemalong A-17. Aerial parts of all three subspecies contained 17 saponins previously identified and also a substantial amount of astragaloside VIII (3-GlcA-Xyl-Rha soyasapogenol B), not previously reported in M. truncatula. The compositions of saponin mixtures were very similar in the three subspecies with three dominant groups, recognized as zanhic acid, medicagenic acid, and soyasapogenol glycosides. Relative proportions of these three groups were also similar in the three subspecies: var. longispina had 49.5, 48.1, and 2.4%; var. truncatula, 41.5, 53.4, and 5.1%; and Jemalong A-17, 42.1, 56.6, and 1.3% of zanhic acid, medicagenic acid, and soyasapogenol glycosides, respectively. Jemalong A-17 had 30% lower total content of saponins as compared to M. truncatula var. longispina and M. truncatula var. truncatula; in relation to the dry matter, var. longispina contained 0.22%, var. truncatula, 0.22%, and Jemalong A-17, 0.15% dry matter of saponins. If one takes into consideration that this determination was performed on spring-collected samples, it can be concluded that the concentration of saponins in M. truncatula is similar to the concentration in alfalfa (Medicago sativa); the proportions of the three groups of saponins in these species are slightly different from those found in alfalfa, having a higher content of zanhic acid glycosides.

Quantification of Saponins in Aerial and Subterranean Tissues of Medicago truncatula

Triterpene saponins from aerial and subterranean organs of Medicago truncatula cv. Jemalong A-17 were qualitatively profiled and quantified using reverse-phase HPLC with on-line photodiode array detection and electrospray-ionization mass spectrometry (HPLC/PDA/ESI/MS). Absolute quantifications were performed for 3-O-beta-D-glucopyranosyl-medicagenic acid and soyasaponin 1 (3-O-[alpha-L-rhamnopyranosyl(1-->2)-beta-D-galactopyranosyl(1-->2)-beta-D-glucuronopyranoside] soyasapogenol B), whereas relative quantifications were determined for 29 other saponins in root, stem, leaf, seedpod, and seed. Roots contained the greatest total amount of saponins followed by leaf and seed, respectively. The quantitative data also reveal the differential accumulation of triterpene saponins in the various organs of M. truncatula. Specifically, relatively higher quantities of medicagenic acid conjugates accumulated in leaf and seed, whereas relatively higher levels of soysapogenol conjugates were observed in root. The differential accumulation of specific triterpene saponins is suggestive of spatially differentiated biosynthesis and/or biological function.

Shape of Pods of Medicago Truncatula as a Promising Morphological Marker for Embryogenic Capacity

ABSTRACTThree different types of pods (N- normal with spines, LS- less spines and WS-without spines) were harvested from Medicago truncatula cv. R 108 1 plants grown in greenhouse. The pods with changed shape LS and WS were developed at the end of period of plant maturity at the upper part of plants. In vitro plants originated from seeds from three types of pods were obtained. Callus tissue produced from leaves and petioles of plants from type N, LS and WS showed different embryogenic potential. Callus tissue of type N formed embryos from both -leaves and petioles, while the callus from type WS was able to form embryos only from petioles. Regardless of the initial explants callus from LS plants did not show any embryogenic ability. In contrast to the regenerants from N type capable to form normal root system, those from WS type possess poor developed root system.

Genetic Transformation of Medicago Truncatula using System for Direct Somatic Embryogenesis Promoted By TDZ

ABSTRACTThe efficient procedure for Agrobacterium mediated transformation of Medicago truncatula cv. R 108 1 was developed. Using the advantage of recycling embryo formation of system for direct somatic embryogenesis promoted by TDZ, transgenic plantlets were obtained for a short period of 60 days. Transformation was performed with bacterial strain—LBA 4404, containing binary vector pBI 121 carries two genes—uid A gene exprecing β- glucoronidase (GUS) and npt II gene for resistance to kanamicin. Different parameters of the gene transfer—pre-treatment of the embryo clusters, density of the bacterial suspension, time for inoculation and co-cultivation, type of selection were optimized. For evaluation of the transformation efficiency, histochemical GUS assay was performed and glucoronidase activity was detected in embryos, plantlets and seedlings of T1 progeny. Transgenic nature of plantlets was confirmed by npt II-specific PCR amplification and Southern hybridization. In the progeny transgenes segregated in Mendelian manner.

Pharmacological evidence that multiple phospholipid signaling pathways link Rhizobium nodulation factor perception in Medicago truncatula root hairs to intracellular responses, including Ca2+ spiking and specific ENOD gene expression.

Rhizobium nodulation (Nod) factors are specific lipochito-oligosaccharide signals essential for initiating in root hairs of the host legume developmental responses that are required for controlled entry of the microsymbiont. In this article, we focus on the Nod factor signal transduction pathway leading to specific and cell autonomous gene activation in Medicago truncatula cv Jemalong in a study making use of the Nod factor-inducible MtENOD11 gene. First, we show that pharmacological antagonists that interfere with intracellular ion channel and Ca2+ pump activities are efficient blockers of Nod factor-elicited pMtENOD11-beta-glucuronidase (GUS) expression in root hairs of transgenic M. truncatula. These results indicate that intracellular Ca2+ release and recycling activities, essential for Ca2+ spiking, are also required for specific gene activation. Second, pharmacological effectors that inhibit phospholipase D and phosphoinositide-dependent phospholipase C activities are also able to block pMtENOD11-GUS activation, thus underlining a central role for multiple phospholipid signaling pathways in Nod factor signal transduction. Finally, pMtENOD11-GUS was introduced into all three Nod-/Myc- dmi M. truncatula mutant backgrounds, and gene expression was evaluated in response to the mastoparan peptide agonist Mas7. We found that Mas7 elicits root hair MtENOD11 expression in dmi1 and dmi2 mutants, but not in the dmi3 mutant, suggesting that the agonist acts downstream of DMI1/DMI2 and upstream of DMI3. In light of these results and the recently discovered identities of the DMI gene products, we propose an integrated cellular model for Nod factor signaling in legume root hairs in which phospholipids play a key role in linking the Nod factor perception apparatus to downstream components such as Ca2+ spiking and ENOD gene expression.

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.