Characterization of salt tolerant alfalfa (Medicago sativa L.) plants regenerated from salt tolerant cell lines

Salt tolerant cell lines have been selected from Medicago sativa, by a single step selection process on tissue culture medium containing 1% NaCl. Plants regenerated from these lines show improved salt tolerance compared to parent plants. The regenerated plants are vigorous, have flowered and are self fertile. The cellular salt tolerance characteristic can be passaged through the regenerated plants, since callus cultures initiated from immature ovaries of the salt tolerant regenerated plants are salt tolerant without additional selection on 1% NaCl. Several of these second generation callus cultures have been regenerated to produce vigorous plants which maintain the salt tolerance characteristic. The tolerance phenotype appears dominant in seeds obtained from self fertilization of the tolerant plants. The regenerated salt tolerant plants are therefore a valuable source as genotypes in plant breeding for salt tolerance and isolation, identification and manipulation of genes which confer salt tolerance in alfalfa.Abbreviations SH Schenk and Hildebrandt medium - 2,4-D 2,4-dichlorophenoxyacetic acid     

Rhizobium meliloti inoculation of alfalfa selected for tolerance to acid,aluminum-rich soils

Alfalfa (Medicago sativa L.) growth and nodulation in acid soil is reduced because the plant and its bacterial symbiontRhizobium meliloti cannot tolerate acid, aluminum-rich soil. A study was conducted to determine if a relatively acid-tolerant alfalfa germplasm combined with a relatively acid-tolerantR. meliloti strain could overcome these limitations. In a light room study, an acid-tolerant alfalfa germplasm inoculated with a more acid-tolerantR. meliloti strain produced greater top growth, nodule number and weight, and acetylene reduction values in an unlimed soil (pH 4.6) than the same germplasm inoculated with a relatively acid-sensitiveR. meliloti strain or an acid-sensitive germplasm inoculated with either a relatively acid-tolerant or acid-sensitiveR. meliloti strain.     

Bacterial citrate synthase expression and soil aluminum tolerance in transgenic alfalfa

Alfalfa is very sensitive to soil acidity and its yield and stand duration are compromised due to inhibited root growth and reduced nitrogen fixation caused by Al toxicity. Soil improvement by liming is expensive and only partially effective, and conventional plant breeding for Al tolerance has had limited success. Because tobacco and papaya plants overexpressing Pseudomonas aeruginosa citrate synthase (CS) have been reported to exhibit enhanced tolerance to Al, alfalfa was engineered by introducing the CS gene controlled by the Arabidopsis Act2 constitutive promoter or the tobacco RB7 root-specific promoter. Fifteen transgenic plants were assayed for exclusion of Al from the root tip, for internal citrate content, for growth in in vitro assays, or for shoot and root growth in either hydroponics or in soil assays. Overall, only the soil assays yielded consistent results. Based on the soil assays, two transgenic events were identified that were more aluminum-tolerant than the non-transgenic control, confirming that citrate synthase overexpression can be a useful tool to help achieve aluminum tolerance. Pierluigi Barone and Daniele Rosellini contributed equally to this work.     

Abscisic acid increases anaerobic tolerance in alfalfa seedlings

In order to clarify the effect of abscisic acid (ABA) on anaerobic tolerance in alfalfa ( Medicago sativa L.), the seedlings were subjected to anaerobic stress after pretreatment with ABA. At concentrations> 1 μ M , ABA pretreatment increased the root viability of the seedlings to anaerobic stress and the viability increased with increasing ABA doses. At 100 μ M ABA, the viability was 2.5-fold greater as compared with that of control seedlings. Roots of the seedlings rapidly lost ATP under the anaerobic stress; however, the decrease in ATP was much slower in the ABA-pretreated seedlings than the control seedlings. At 12 h after onset of the stress, ATP concentrations in the roots of 100 and 10 μ M ABA-pretreated seedlings were 2.7- and 2.0-fold that of the control seedlings, respectively. During the period of ABA pretreatment under aerobic condition, ABA increased alcohol dehydrogenase (ADH, EC 1.1.1.1) activity in the roots until 12 h and then leveled off. The maximum ADH activities were 4.3- and 2.8-fold that in the roots of the control seedlings for 100 and 10 μ M ABA-pretreated seedlings, respectively. After being subjected to the anaerobic stress, both ADH activities in the roots of the ABA-pretreated and the control seedlings increased but the differences in their activity remained. These results suggest that ABA pretreatment may maintain ATP level due to induction of ADH activity, which may be one of the causes of increasing anaerobic tolerance in the seedlings.     

Quantitative trait loci and candidate gene mapping of aluminum tolerance in diploid alfalfa

Aluminum (Al) toxicity in acid soils is a major limitation to the production of alfalfa (Medicago sativa subsp. sativa L.) in the USA. Developing Al-tolerant alfalfa cultivars is one approach to overcome this constraint. Accessions of wild diploid alfalfa (M. sativa subsp. coerulea) have been found to be a source of useful genes for Al tolerance. Previously, two genomic regions associated with Al tolerance were identified in this diploid species using restriction fragment length polymorphism (RFLP) markers and single marker analysis. This study was conducted to identify additional Al-tolerance quantitative trait loci (QTLs); to identify simple sequence repeat (SSR) markers that flank the previously identified QTLs; to map candidate genes associated with Al tolerance from other plant species; and to test for co-localization with mapped QTLs. A genetic linkage map was constructed using EST-SSR markers in a population of 130 BC₁F₁ plants derived from the cross between Al-sensitive and Al-tolerant genotypes. Three putative QTLs on linkage groups LG I, LG II and LG III, explaining 38, 16 and 27% of the phenotypic variation, respectively, were identified. Six candidate gene markers designed from Medicago truncatula ESTs that showed homology to known Al-tolerance genes identified in other plant species were placed on the QTL map. A marker designed from a candidate gene involved in malic acid release mapped near a marginally significant QTL (LOD 2.83) on LG I. The SSR markers flanking these QTLs will be useful for transferring them to cultivated alfalfa via marker-assisted selection and for pyramiding Al tolerance QTLs.     

乌兰布和沙区紫花苜蓿生长发育模拟研究

借鉴积温学的原理,结合紫花苜蓿（Meicago sativa)生理生态学特性,建立了水分限制条件下干旱沙区紫花苜蓿生长发育模拟模型,该模型主要由生长发育阶段子模型,叶面积动态子模型,干物质积累子模型和干物质分配子模型组成。模拟计算结果表明,该模型能较好地预测沙地紫花苜蓿生长发育进程,叶面积变化动态及牧草产量变化动态,具有一定的实际应用价值。     

Oxalic acid production and aluminum tolerance in Pseudomonas fluorescens

13C NMR studies on intact cells from Al-stressed Pseudomonas fluorescens incubated with citric acid or Al-citrate yielded peaks at 158 and 166 ppm that were attributable to free and complexed oxalic acid, respectively. The presence of oxalic acid was further confirmed with the aid of oxalate oxidase. These peaks were not discernable in experiments performed with cells taken from control cultures. Enzymatic analyses of cell fractions showed the highest production of oxalic acid in the inner membrane fraction of Al-stressed cells incubated with glyoxylate. There was an eight-fold increase in the synthesis of oxalic acid in the inner membrane fraction from the Al-stressed cells compared to the control cells. Although oxalic acid production was observed when citrate, Al-citrate and isocitrate were utilized as substrates, the inner membrane fraction did not mediate the formation of oxalic acid from glycine/pyruvate, glycolic acid, oxaloacetate or ascorbate. These data suggest that the increased oxalic acid production in response to Al stress is effected via the oxidation of glyoxylate.     

Induced androgenesis in alfalfa (Medicago sativa L.)

Summary Anthers of 10 alfalfa (Medicago sativa L.) lines were used as initial material for the production of androgenic haploids. More than 30 variants of nutrient media were tested. Twenty five different treatments with low temperatures and gamma rays were tried in order to find optimal conditions for callus induction and organogenesis.The genotype, stage of microspore development, phytohormonal composition of the nutrient media and pretreatment with physical agents, alone or in combination, affected the efficiency of organogenesis and regeneration in anther cultures of alfalfa.Plants exhibited a high degree of variability in their chromosome number. Haploids, dihaploids and mixoploids were obtained.Cytological studies of in vitro pollen development revealed the origin of the regenerants from microspores.Abbreviations BAP 6-Benzylaminopurine - 2-ip 6-(,-dimethylallylamino)Purine - IAA Indolylacetic Acid - NAA Naphthaleneacetic Acid - 2,4-D Dichlorophenoxyacetic Acid - CMS Cytoplasmic Male Sterility     

Root health evaluation of three alfalfa varieties in Kerquin sandy land

Without a robust and healthy root system, establishment, productivity, and persistence are compromised. Consequently, research on alfalfa root morphology and health is very important in development of technology for efficient improvement and production of alfalfa. The objectives of this study were to evaluate the root morphology and health of three alfalfa varieties, Algonquin, Golden Queen, and Yellow Flower and to determine relationships among root morphology traits and root health. Yields from these varieties ranged from 5.83 to 43.93 t/ha, total root length ranged from 215.17 to 708.89 mm, root surface area from 124.95 to 468.37 cm², volume from 3.24 to 57.72 cm³, and forks from 1.25 × 10³ to 10.54 × 10³, and tips from 0.65 × 10³ to 3.17 × 10³. Root infestation score was negatively correlated with yield (r = ?0.997, P < 0.01), and was positively correlated with all root morphology traits (r = 0.466–0.997, P < 0.01), and yield was negatively associated with root morphology traits (r = ?0.755 to ?0.998, p < 0.01) with the exception of root tips (r = 0.448, P < 0.01). Results from these analyses indicated that root infestation score was the lowest averaged over age of alfalfa stand in Algonquin. Yield in 2-year old stands was greater in Golden Queen compared to the other two cultivars.     

Root health evaluation of three alfalfa varieties in Kerquin sandy land

Without a robust and healthy root system, establishment, productivity, and persistence are compromised. Consequently, research on alfalfa root morphology and health is very important in development of technology for efficient improvement and production of alfalfa. The objectives of this study were to evaluate the root morphology and health of three alfalfa varieties, Algonquin, Golden Queen, and Yellow Flower and to determine relationships among root morphology traits and root health. Yields from these varieties ranged from 5.83 to 43.93 t/ha, total root length ranged from 215.17 to 708.89 mm, root surface area from 124.95 to 468.37 cm², volume from 3.24 to 57.72 cm³, and forks from 1.25 × 10³ to 10.54 × 10³, and tips from 0.65 × 10³ to 3.17 × 10³. Root infestation score was negatively correlated with yield (r = ?0.997, P < 0.01), and was positively correlated with all root morphology traits (r = 0.466–0.997, P < 0.01), and yield was negatively associated with root morphology traits (r = ?0.755 to ?0.998, p < 0.01) with the exception of root tips (r = 0.448, P < 0.01). Results from these analyses indicated that root infestation score was the lowest averaged over age of alfalfa stand in Algonquin. Yield in 2-year old stands was greater in Golden Queen compared to the other two cultivars.     

RFLP variation in diploid and tetraploid alfalfa

Summary Alfalfa (Medicago sativa L.) is a major forage crop throughout the world. Although alfalfa has many desirable traits, continued breeding is required to incorporate pest resistances and other traits. We conducted this study to determine the amount of restriction fragment length polymorphism (RFLP) variability present within and between diploid and tetraploid alfalfa populations, and whether or not this variability is sufficient for construction of an RFLP map. Diploid plants from M. sativa ssp. falcata, ssp. coerulea, and ssp. sativa and tetraploid spp. sativa cultivars Apollo, Florida 77, and Spredor 2 were included. A total of 19 cDNA clones was probed onto genomic Southern blots containing DNA digested by EcoRI, HindIII, or BamHI. Phylogenetic trees were produced, based on parsimony analysis of shared restriction fragments. Evidence for extensive gene duplication was found; most probes detected complex patterns of restriction fragments. Large amounts of variation are present within all diploid subspecies. M. sativa ssp. falcata plants formed clusters distinct from ssp. sativa or ssp. coerulea plants, which were not distinctly clustered. Some M. sativa ssp. falcata plants were more similar to the other groups than to other plants within ssp. falcata. Variation among tetraploid cultivars showed that Florida 77 and Apollo had more similarities than either showed with Spredor 2. All three cultivars showed large within-population variation, with Apollo being the most diverse and Spredor 2 the least. Based on these results, development of an RFLP map at the diploid level appears possible. Also, differentiation of cultivars, particularly ones of divergent origin, seems possible based on RFLP patterns.     

Variation in vigour and in RFLP-estimated heterozygosity by selfing tetraploid alfalfa: new perspectives for the use of selfing in alfalfa breeding

The effect of selfing on vigour and heterozygosity was analysed in six independent families obtained by recurrent cycles of self-fertilization and selection until the S₃ generation. The heterozygosity level, estimated by means of 11 homologous probes, decreased from the S₀ to S₃ generations and was partially restored in S₂×S₂ polycrosses. The decreasing trend was influenced by the inbreeding level of the chosen mother plant in each self generation to advance in subsequent selfing. Plant vigour estimated by Dry Matter Yield (DMY) decreased during inbreeding, and phenotypic selection of S₂ individuals seemed to prove effective as the differences in DMY between vigorous and weak plants were maintained in S₂×S₂ crosses. No correlation between plant vigour and heterozygosity was found between subgroups of vigorous and weak plants selected within the same S₂ family. Results are discussed with a view to selecting the best performing and least heterozygous plants during inbreeding to isolate useful genes and linkats in superior partially inbred parental lines. Received: 4 August 1999 / Accepted: 6 December 1999     

Detection of new viruses in alfalfa,weeds and cultivated plants growing adjacent to alfalfa fields in Saudi Arabia

A total of 1368 symptomatic plant samples showing different virus-like symptoms such as mottling, chlorosis, mosaic, yellow mosaic, vein clearing and stunting were collected from alfalfa, weed and cultivated plant species growing in vicinity of alfalfa fields in five principal regions of alfalfa production in Saudi Arabia. DAS-ELISA test indicated occurrence of 11 different viruses in these samples, 10 of which were detected for the first time in Saudi Arabia. Eighty percent of the alfalfa samples and 97.5% of the weed and cultivated plants samples were found to be infected with one or more of these viruses. Nine weed plant species were found to harbor these viruses namely, Sonchus oleraceus, Chenopodium spp., Hibiscus spp., Cichorium intybus, Convolvulus arvensis, Malva parviflora, Rubus fruticosus, Hippuris vulgaris, and Flaveria trinervia. These viruses were also detected in seven cultivated crop plants growing adjacent to the alfalfa fields including Vigna unguiculata, Solanum tuberosum, Solanum melongena, Phaseolus vulgaris, Cucurbita maxima, Capsicum annuum, and Vicia faba. The newly reported viruses together with their respective percent of detection in alfalfa, and in both weeds and cultivated crop plant species together were as follows: Bean leaf roll virus (BLRV) {12.5 and 4.5%}, Lucerne transient streak virus (LTSV) {2.9 and 3.5%}, Bean yellow mosaic virus (BYMV) {1.4 and 4.5%}, Bean common mosaic virus (BCMV) {1.2 and 4.5%}, Red clover vein mosaic virus (RCVMV) {1.2 and 4%}, White clover mosaic virus (WCIMV) {1.0 and 5%}, Cucumber mosaic virus (CMV) {0.8 and 3%}, Pea streak virus (PeSV) {0.4 and 4.5%} and Tobacco streak virus (TSV) {0.3 and 2.5%}. Alfalfa mosaic virus (AMV), the previously reported virus in alfalfa, had the highest percentage of detection in alfalfa accounting for 58.4% and 62.8% in the weeds and cultivated plants. Peanut stunt virus (PSV) was also detected for the first time in Saudi Arabia with a 66.7% of infection in 90 alfalfa samples collected from the surveyed regions during the last visit that tested negative to all the previously detected viruses.     

Development of an RFLP map in diploid alfalfa

Summary We have developed a restriction fragment length polymorphism (RFLP) linkage map in diploid alfalfa (Medicago sativa L.) to be used as a tool in alfalfa improvement programs. An F₂ mapping population of 86 individuals was produced from a cross between a plant of the W2xiso population (M. sativa ssp. sativa) and a plant from USDA PI440501 (M. sativa ssp. coerulea). The current map contains 108 cDNA markers covering 467.5 centimorgans. The short length of the map is probably due to low recombination in this cross. Marker order may be maintained in other populations even though the distance between clones may change. About 50% of the mapped loci showed segregation distortion, mostly toward excess heterozygotes. This is circumstantial evidence supporting the maximum heterozygote theory which states that relative vigor is dependent on maximizing the number of loci with multiple alleles. The application of the map to tetraploid populations is discussed.     

Role of plant defence in alfalfa during symbiosis

During effective symbiosis, rhizobia colonize their hosts, and avoid plant defence mechanisms. To determine whether the host defence responses can be elicited by the symbiotic bacteria, specific markers involved in incompatible pathogenic interactions are required. The available markers of alfalfa defence mechanisms are described and their use in the study of the symbiotic interaction discussed. As defence-related gene expression in roots is not always related to defence mechanisms, other model systems have been established allowing confirmation of an important role of bacterial surface components in alfalfa-Rhizobium meliloti interactions. Nod factors at high concentrations have been shown to elicit defence-like responses in Medicago cell suspensions and roots. Elicitation of defence mechanisms by high levels of Nod factors in Rhizobium-infected roots may be a part of the mechanism by which nodulation is feed-back regulated.The authors are with the Institut des Sciences Végétales, CNRS, F-91198 Gif-sur-Yvette cédex, France.     

Genetic diversity for aluminum tolerance in sorghum

Genetic variation for aluminum (Al) tolerance in plants has allowed the development of cultivars that are high yielding on acidic, Al toxic soils. However, knowledge of intraspecific variation for Al tolerance control is needed in order to assess the potential for further Al tolerance improvement. Here we focused on the major sorghum Al tolerance gene, Alt _SB , from the highly Al tolerant standard SC283 to investigate the range of genetic diversity for Al tolerance control in sorghum accessions from diverse origins. Two tightly linked STS markers flanking Alt _SB were used to study the role of this locus in the segregation for Al tolerance in mapping populations derived from different sources of Al tolerance crossed with a common Al sensitive tester, BR012, as well as to isolate the allelic effects of Alt _SB in near-isogenic lines. The results indicated the existence not only of multiple alleles at the Alt _SB locus, which conditioned a wide range of tolerance levels, but also of novel sorghum Al tolerance genes. Transgressive segregation was observed in a highly Al tolerant breeding line, indicating that potential exists to exploit the additive or codominant effects of distinct Al tolerance loci. A global, SSR-based, genetic diversity analysis using a broader sorghum set revealed the presence of both multiple Alt _SB alleles and different Al tolerance genes within highly related accessions. This suggests that efforts toward broadening the genetic basis for Al tolerance in sorghum may benefit from a detailed analysis of Al tolerance gene diversity within subgroups across a target population. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Enhancing alfalfa conversion efficiencies for sugar recovery and ethanol production by altering lignin composition

Alfalfa (Medicago sativa L.) biomass was evaluated for biochemical conversion into ethanol using dilute-acid and ammonia pretreatments. The two alfalfa lines compared were a reduced S-lignin transgenic cultivar generated through down regulation of the caffeic acid O-methyltransferase gene and a wild-type control. Both were harvested at two maturities. All the samples had similar carbohydrate contents including a mean composition of 316 g glucan and 497 g total neutral carbohydrates per kg dry biomass, which corresponds to a theoretic ethanol yield of 382 l/ton. Ethanol yields for alfalfa stems pretreated with dilute-acid were significantly impacted by harvest maturity and lignin composition, whereas when pretreated with dilute-ammonia, yield was solely affected by lignin composition. Use of a recombinant xylose-fermenting Saccharomyces strain, for converting the ammonia pretreated alfalfa samples, further increased ethanol yields. Ethanol yields for the xylose-fermenting yeast were 232-278 l/ton and were significantly enhanced for the reduced S lignin cultivars.     

De novo hydroponics system efficiency for the cuttings of alfalfa (Medicago sativa L.)

The legume plant alfalfa (Medicago sativa L.) is a widely cultivated perennial forage due to its high protein content, palatability, and strong adaptability to diverse agro-ecological zones. Alfalfa is a self-incompatible cross-pollinated autotetraploid species with tetrasomic inheritance. Therefore, maintaining excellent traits through seed reproduction is a prime challenge in alfalfa. However, the cutting propagation technology could enable consistent multiplication of quality plants that are genetically identical to the parent plant. The current study aimed to develop a simple, cost-effective, reproducible, and efficient hydroponic cutting method to preserve alfalfa plants and for molecular research. In this study, alfalfa landrace ‘Wudi’ was grown in hydroponics for 30 days and used as source material for cuttings. The top, middle and bottom sections of its stem were used as cuttings. The rooting rate, root length, and stem height of the different stem sections were compared to determine the best segment for alfalfa propagation in four nutrient treatments (H^M, H^M + 1/500H, H^M + 1/1000H and d H^M + 1/2000H). After 21 days of culture, the rooting rates of all the three stem types under four cutting nutrient solutions were above 78%. The rooting rate of the middle and bottom parts in H^M + 1/1000 H and H^M + 1/2000 H nutrient solutions reached more than 93%, with a higher health survey score (> 4.70). In conclusion, this study developed a de novo cutting propagation method that can be used to conserve and propagate germplasm in breeding programs and research. This method is a new report on the cutting propagation of alfalfa by hydroponics, which could supplement the existing cutting propagation methods.     

Quantitative genetic studies at the tetraploid and hexaploid levels in alfalfa

Summary Two sets of diallel crosses involving seven tetraploid and five hexaploid alfalfa lines, previously selected for seed and forage yield, are studied. On the basis of combining ability and heritability estimates the following conclusion is drawn. The dominant and epistatic interactions (heterozygosity) are the major components in the expression of variability for yield and characters associated with yield in tetraploid populations. In hexaploid populations, however, there is an appreciable amount of additive variance, and the additive effect of genes along with interaction of various forms may account for the expressed variability for all the characters studied. Better genes and desirable interactions (possibly through heterozygosity) are the two essential genetic components of yield. The selection of better genes is feasible, but by itself will not produce the desired results. Future improvement may therefore depend on the utilization of heterosis and other forms of interactions. There is, in general, a very strong genetic correlation between yield and its components, and therefore, when a direct selection for yield is not feasible, the selection program may rely on a number of closely related characters.