The potential of selection and breeding for improved salt tolerance in lucerne (Medicago sativa L.)

Summary Four cultivars of Medicago sativa L. were subjected to selection for improved salt tolerance using a salinized solution culture technique at 250 mM NaCl. Selections were made after two weeks growth, based upon seedling shoot length differences. High (shoot lengths 14–31 mm) and low (7–12 mm) selection lines were established. Unselected shoot lengths ranged from 0–11 mm. Selection intensities ranged from 0.17% to 0.22% for the high selection lines, and from 0.30% to 1.05% for the low selection lines.Eleven selected plants from cv. CUF 101, and 10 from Local Syria were grown on and polycrossed. A sample of progeny seed from each was grown at 8 NaCl concentrations in solution cultures. Selected line seedlings produced shoots at 225, 250, 260, and 275 mM NaCl, whereas unselected material failed to grow above 225 mM NaCl. A second cycle of selection at 280 mM NaCl with these two cultivars achieved selection intensities of 0.21% and 0.45% for high and low selection lines, respectively. Selected lines grew and produced shoots at 300 mM NaCl.Seedlings derived from 1st and 2nd selection cycles and unselected control material were grown for 6 weeks at 0, 150, 175, and 200 mM NaCl in a sand culture experiment. The high selection line produced significantly greater shoot fresh weight, dry weight, and % live shoot weight than the unselected control material. Clearly selection as practised here can isolate individual seedlings having enhanced genetically based tolerance to NaCl, which is manifest in mature plants derived from polycrossing those selected individuals.     

Micropropagation of mother plants of lucerne (Medicago sativa L.) for somatic embryogenesis

Summary A reliable and standard method was established for micropropagation of the A70-34 selected genotype of lecerne (Medicago sativa L., genotype A70-34), aimed at reducing contamination problems, seasonal and phenological influences on regeneration and phytosanitary problems of the mother plants, while maintaining the regenerative potential for somatic embryogenesis of the plant tissues. Mother plants were routinely maintained for several subcultures and somatic embryogenesis was regularly obtained from the subcultured explants. Proliferation, rooting and embryogenetic ability of plants cultured for 30 days was greater than those cultured for 20 days. The regenerative potential of tissues from different organs and of triturated and intact whole plants was also tested. Petioles were confirmed as the best source for embryogenesis as far as efficiency and repeatability were concerned, even though regeneration from other explant types was also achieved. Production of somatic embryos through mechanical trituration of the in vitro cultured plants was obtained; the regeneration ability of the triturated plants was greater than that observed in the intact plants.     

Possibilities of family selection in a cultivar of lucerne,Medicago sativa L., for resistance to the stem nematode,Ditylenchus dipsaci (Kühn) Filipjev

Summary Half-sib families representing the lucerne cultivar Resis were tested for resistance to a stem nematode population, recovered from lucerne. The percentage resistance of the HS-families varied from 13 to 88%. The mean resistance for the cultivar was approximately 50%. Half-sib family selection would be more efficient than recurrent mass selection. The observed data suggest the action of a few genes with relatively large effects.     

High frequency somatic embryogenesis with a pampeana-derived genotype of alfalfa (Medicago sativa L.)

Summary Somatic embryos of genotype R11 of the alfalfa variety Pampeana were produced from embryogenic calli derived from leaf sections. They were induced by an auxin shock and its development was attempted on six different media. The best condition for somatic embryo production was inducing callus on MS medium plus 10 M 2,4-D and 4,6 M KIN and transferring them, after the auxin shock, to MS with 10–20 mM NH₄ ⁺ and 30 mM proline. More than 500 somatic embryos per plate were produced. Embryos were grown to plants on MS or half strength MS media and all regenerated plants resembled the original R11 genotype. This technique could be useful in alfalfa Pampeana improvement using genetic modification.     

An improved breeding strategy for autotetraploid alfalfa (Medicago sativa L.)

Alfalfa cultivar development will be enhanced by breeding strategies whichutilize the full potential of autotetraploid population genetic structures. Thisstudy evaluates the effectiveness of an allelic selection scheme, which wasdeveloped to overcome limitations of inbreeding depression and to exploitgeneral and specific combining ability effects in autotetraploid populations.Allelic selection entails the minimization of non-additive genetic effects byselecting among full-sib families (F1) which are at uniform levels ofheterozygosity. Such F1 lines are developed by crossing individuals fromtwo unrelated random mating populations. Selected F1 lines wereintercrossed to form an improved population. Eight random matingpopulations of alfalfa were developed to study the effectiveness of allelicselection. Selection for increased dry matter yield resulted in alfalfapopulations with 38 percent greater yield than the parent populations. Twocycles of intercrossing, among selected F1 lines, did not dissipate the gainfrom selection. This result has important implications for synthetic cultivardevelopment in which a major limitation is the decline in productivity withadvancing generations of seed increase. A positively correlated response toselection for dry matter yield was observed for plant height and stemdiameter. The results of this research indicate that continued testing of theallelic selection scheme is warranted and could have a significant impact onthe breeding of autotetraploid alfalfa, particularly for synthetic cultivardevelopment.     

Influence of plant age,disease incubation time,and host plant resistance on Verticillium wilt symptom expression in lucerne (Medicago sativa)

Summary The most economical means to control Verticillium wilt in lucerne, caused by Verticillium albo-atrum, is through resistant cultivars. Most Verticillium wilt resistance evaluations use some form of root-cut-soak inoculation procedure. In Trial 1, three lucerne cultivars were inoculated at eight plant ages and plant symptoms were scored at ten successive weekly intervals. Trial 2 consisted of two cultivars inoculated at ten plant ages and symptoms were evaluated at eight weekly intervals. In an experiment to study the genetics of resistance to this pathogen, four plants showing no visual symptoms were selected from each of four germplasms. The selected populations were crossed in diallel fashion. The population crosses plus the parents were evaluated for resistance to V. albo-atrum. Results from Trial 1 and Trial 2 showed disease severity increased with increasing inoculation age and with increasing scoring age. Cultivar symptom expression interacted with inoculation age, but not with scoring age. The diallel analysis supported resistance to Verticillium wilt being conferred by additive gene action, as heterosis was not detected and general combining ability mean square was 3.75 times larger than the specific combining ability mean square.     

Inbreeding depression and heterosis in autotetraploids with application to Medicago sativa L.

Inbreeding depression observed in Medicago sativa L. is greater in the early generations of self-fertilization than is expected for an autotetraploid based upon the coefficient of inbreeding (F). Forage yield often is reduced by 30 percent or more after one generation of selfing, while F is only one-sixth, =0. A high frequency of loci containing 3 or 4 different alleles is postulated as a basis for this rapid loss of vigor. A formula is developed that permits computation of the proportions of loci with 1, 2, 3 and 4 different alleles, along with estimates of additive and non-additive values of genes.Forage yield data for backcross and sib-mated populations indicate that quantitative characters are inherited tetrasomically.Journal Paper No. J-5190 of the Iowa Agricultural and Home Economics Experiment Station. Project 1048.Graduate Assistant and Professor of Agronomy, Department of Agronomy, Iowa State University, Ames, Iowa.     

Forage Quality Components in Grazing-Type Lucerne (Medicago sativa L. complex)

Much is now known about the morphological attributes of the lucerne plant that contribute to improved tolerance to grazing. With the aim of developing grazing-tolerant varieties, a range of genotypes were preliminarily selected for the presence of desirable attributes, and categorized into four morphological models based on their top-growth morphology and vigour. Little is known about the implications for forage quality of selection of non-conventional, grazing-type lucerne. This investigation was undertaken to study aspects of the quality (crude protein, fibre, and lignin content; nutritive value; plant leafiness) of the selected material, with particular reference to the morphological models identified in the first phase and to flower colour, which has taxonomic importance within the species. The results indicate that selection of grazing-type cultivars would not produce detrimental effects in terms of forage quality relative to common, hay-type lucerne. Plants with yellow or variegated flowers, often corresponding to rhizomatous types, were characterized by high leaf percentage, which resulted in high-quality features.     

The effects of kinetin on callus characters in alfalfa (Medicago sativa L.)

Summary Three callus initiation media, B2-k, B2, and 7951, were used to study the effects of kinetin on callus initiation, morphology, histology, and regenerability in alfalfa (Medicago sativa L.). The presence of kinetin in callus initiation media retarded callus initiation, but enhanced division and differentiation of callus cells. Calluses induced on kinetin-containing media (B2 and 7951) had many compact cell aggregations, which were considered meristematic regions that might differentiate to plantlets on a regeneration medium. Visually, these calluses were compact and had many nodular structures. In contrast, most calluses induced on a kinetin-free medium were composed of large, individual cells and had friable structures without nodules. After transfer to a hormone-free medium, calluses induced on kinetin-containing media regenerated more frequently than those induced on a kinetin-free medium, but cytokinin (kinetin) autotrophism also occurred. Autotrophism was sexually transmissable and especially affected by the female parent.     

Genetic control of somatic embryogenesis in alfalfa (Medicago sativa L. cv. Adriana)

Summary In alfalfa (Medicago sativa) regeneration is genotype-specific. In order to study the genetic control of somatic embryogenesis and to constitute a synthetic cultivar characterized by its high regeneration ability, 2 embryogenic plants selected from the cv. Adriana were selfed, intercrossed and also crossed in both directions with 5 non-embryogenic genotypes of the same cultivar.Progenies of all crosses were scored for their regeneration ability and results indicate that somatic embryogenesis is under the control of 2 dominant loci. However some non-embryogenic genotypes prevent regeneration when crossed with embryogenic ones and this characteristic is not under the control of a single dominant gene.When plants chosen for their capacity to regenerate within F₁ and S₁ progenies were freely intercrossed the regeneration efficiency dropped to 2% (1 plant out of 50). This result indicates that if the genetic background of the population is changed the regeneration is greatly affected and therefore some other mechanism could play a role in determining plant regeneration.     

Characterization of factors affecting plant regeneration frequency of Medicago lupulina L.

Summary Different hormones, length of hormone pretreatment, and developmental status of plants were found to influence the shoot and root differentiation frequencies from immature inflorescence explants of Medicago lupulina L., a diploid autogamous species. Explants of immature inflorescence pretreated with phytohormones for a short period (10 min –6 h) were placed on hormone-free medium for shoot differentiation. Cytokinins, such as BA, were found to be essential for shoot differentiation, whereas treatment with 2,4-D could only induce root differentiation. Explants with hormone pretreatment for 30 min to 1 h produced the highest shoot regeneration rate. Plants at the beginning of flowering were found to have a better regeneration ability than those at fruiting or maturation. After a 10 mg/l BA pretreatment, respective shoot differentiation frequencies of 39%, 36% and 23% were observed for explants of self-progeny plants, regenerated plants, and plants propagated by cuttings, although the three categories of plants were originally derived from the same plants.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - 2iP 2-iso-pentenyladenine     

Organelle based molecular analyses of the genetic relatedness of cultivated alfalfa (Medicago sativa L) to Medicago edgeworthii Sirjaev, and Medicago ruthenica (L.) Ledebour

Medicago edgeworthii Sirjaev and M. ruthenica (L.) Ledebour are allogamous, diploid (2n = 2x = 16) perennials with flat pods.Medicago edgeworthii is indigenous to the Himalayas and alpine areas west to Afghanistan, and Medicago ruthenica is found in Siberia, Mongolia, and Manchuria on open hillsides and mixed grass steppes. Because both species have a remarkable ability to survive extreme cold and poor soils, the possibility of hybridizing them with alfalfa (M. sativa L.) is being investigated. The objective of this research was to conduct an organelle based molecular assessment of the genetic relatedness of cultivated alfalfa (2n = 4x = 32) to M. edgeworthii and M. ruthenica. A hypervariable, intergenic region of cpDNA was amplified, and mtDNA was amplified with two primer pairs developed from soybean (Glycine max L.) mtDNA sequences. Mean Nei and Li genetic distances (GDs) between alfalfa and M. edgeworthii and alfalfa and M. ruthenica were 0.56 and 0.48 (mtDNA), and 0.33 and 0.30 (cpDNA), respectively. Intra specific GDs were 0.37 (mtDNA) and 0.25 (cpDNA) for M. edgeworthii; 0.42 (mtDNA) and 0.15 (cpDNA) for M. ruthenica; and 0 = 0.50 (mtDNA) and 0 = 0.23 (cpDNA) for alfalfa. Cluster analyses grouped someM. edgeworthii and M. ruthenica entries with alfalfa entries. There is some chance that alfalfa and M. edgeworthii entries which clustered closely could be hybridized; chances of alfalfa × M. ruthenica hybridizations appear to be more problematic. This revised version was published online in July 2006 with corrections to the Cover Date.     

Resistance to Verticillium albo-atrum in lucerne (Medicago sativa L.) to distinguish between varieties

France is responsible for DUS (Distinction–Uniformity–Stability) testing of new varieties of lucerne (Medicago sativa L.) for several European countries. Many varieties fail to be registered because of a lack of distinctness from the already registered varieties. The addition of the other traits such as disease or pest resistance could help in distinction. The aim of the present study was to optimize measurement of the resistance of lucerne to Verticillium albo-atrum. Although the resistance to V. albo-atrum is a qualitative trait for each plant, varietal resistance is a continuous variable because each variety contains a variable percentage of resistant plants. Fifty cultivars were tested for V. albo-atrum in two laboratories at random from among five private and public laboratories. Five check varieties were tested in the five laboratories in two tests. A score of resistance, corrected from the effect of the test, was given for each cultivar. A method to validate any new test is proposed. This data set can now be incremented by the resistance scores to V. albo-atrum of new varieties. It will help in distinguishing between lucerne varieties.     

Characterization of alfalfa (Medicago sativa L.) following in vitro selection for salt tolerance

Summary Alfalfa (Medicago sativa L.) is a valuable forage crop which is grown in areas of limited rainfall, high temperature and where the land is often salt affected. Seedlings of the commercial variety CUF 101 and the more salt tolerant breeding line CUF101-1S were used as explant material to produce tissue cultures for an in vitro screen for salt tolerance. Callus cultures were placed on a regeneration medium containing 0–350 mol m^-3 NaCl to establish the highest possible salt concentration that would allow buds to regenerate and survive as plantlets. Buds were regenerated in 250 mol m^-3 NaCl then isolated, grown to maturity and set seed. The response to salt tolerance in the short term was investigated by exposing 14-day-old seedlings to 200 mol m^-3 NaCl. Measurement of growth, survival, proline concentration and the activity of antioxidant enzymes were made after 14 days exposure to NaCl. One somaclone in particular (6R2IV) compared with the parent line showed increased salt tolerance, greater accumulation of proline and a greater increase in the antioxidant enzyme, glutathione reductase. A potentially important gene is pA9 which is known to be responsible for the production of a proline rich cell wall protein. Polymerase Chain Reaction (PCR) amplification of a region of the pA9 gene indicated that it was present in both tolerant and sensitive lines. Southern blotting has shown that the copy number of the pA9 was present as multiple copies in the tolerant clone compared with a single copy in the parents.Abbreviations GR glutathione reductase     

Evaluation of a lucerne germplasm collection in relation to traits conferring grazing tolerance

Summary A collection of 90 accessions of different germplasm groups from the Medicago sativa complex was evaluated at Lodi, Italy, for dry-matter yield, time to anthesis, height of regrowth, plant diameter, stem density, vegetative recovery after winter, and presence of deep-set crown and ability to spread with creeping roots or rhizomes, these latter characters possibly contributing to grazing tolerance. The germplasm groups proved rather distinct for these traits, but all included types potentially suitable for grazing. Interesting types combining deep-set crown and high yield occurred mainly in subsp. x varia and materials originated from artificial crosses involving at various extent the subspecies falcata, sativa and x varia. Though belonging to subsp. sativa, mielga wild populations from Spain had aerial traits similar to those of the long-dormant, low-yielding subsp. falcata. Deep-crowned plants had greater plant diameter than shallow-crowned plants, and could be categorised into four distinct top-growth models according to their habit and vigour. One of these models combined spreading ability with high yield. The about 150 plants selected for their combination of deep crown, wide diameter, and good yield were characterised by a branch-rooted system. There was general correspondence between top-growth and root morphology of each model. Of the four deep-crowned models, three had rhizomes, with wide range of underground development and morphology, while the fourth had creeping roots. The latter model was the most rare among the examined accessions, never appearing in either cultivated or wild subsp. sativa. The implications of the results on the development of grazing-tolerant lucerne varieties are discussed.     

Traditional seed maintenance and origins of the French lucerne landraces

Summary As for other forage crops the notion of variety is quite recent in lucerne (Medicago sativa L.). Up to 1950 in France, farmers cultivated landraces whose origins were geographically defined. Seed production was a by-product of forage production. Usually, seeds were harvested on the second or third crop, in old lucerne fields. Natural selection created landraces adapted to local conditions. In some years, climatic conditions, especially in northern regions, were unfavourable to seed production. In the exchanges and trades of seeds between French regions, the genetic origin of the seeds was omitted, the geographical origin was only mentioned. Even if France usually exported lucerne seeds, imports occurred in bad years. Seeds were imported from European countries and from North and South America. Varieties from America were poorly adapted to the French conditions for forage production. These foreign varieties have probably intercrossed with the local landraces but no data is available to know to what extent. Among the various French landraces, five main types were defined using morphological characters: Flamande in the north, three types (Poitou, Marais de Luçon, Marais de Challans) in the west, and Provence in the south. These landraces have been widely used in breeding since 1950. Even though these five landraces are, up to now, maintained, the other landraces have probably been lost (no more cultivated and not in genebanks), replaced by registered varieties.     

接种不同根瘤菌对紫花苜蓿固氮效能及生物量的影响

为了在晋北生态区给主要推广品种筛选适宜的高效根瘤菌,使紫花苜蓿-根瘤菌共生体充分发挥共生固氮作用,建立高效、高产、优质紫花苜蓿生产体系。将中国农业科学院菌种保藏中心筛选出的9株紫花苜蓿根瘤菌作为试验材料,以山西大同地区的土壤为基质,结合温室盆栽和田间试验,比较供试材料的结瘤数、固氮酶活性、株高、生物量等指标的变化情况。筛选出适合供试材料巨能耐盐紫花苜蓿品种的4株高效根瘤菌CCBAU30015、CCBAU NMC024-1、HBU07004和CCBAU30198,并将这4株菌进行田间接种效果验证。结果表明,接种CCBAU30015、CCBAU NMC024-1、HBU07004和CCBAU30198后,全年干草产量分别比对照增加18.7%,15.8%,11.3%和13.3%,单位面积蛋白质产量分别比对照增加10.9%,10.2%,9.7%和10.1%,其中,CCBAU30015表现最为突出,在各个指标上与对照间差异均显著(P0.05)。说明通过土壤筛选的高效根瘤菌可以应用到实际生产中。     

紫花苜蓿愈伤组织诱导和分化研究

以紫花苜蓿SANDITI无菌苗下胚轴、茎、叶3种不同外植体,在附加不同激素浓度的MS培养基上诱导愈伤组织。结果表明,SANDIT最佳外植体为下胚轴,最佳下胚轴愈伤组织诱导培养基为MS+2,4-D 2.0 mg/L+6-BA 0.5mg/L。15 d继代1次,或适当降低2,4-D浓度,提高6-BA或KT浓度,可以降低愈伤组织褐化率。愈伤组织在6-BA0.5 mg/L,NAA 0.1 mg/L,GA31.0 mg/L,YE 250 mg/L,CH 250 mg/L的MS培养基上,可获得较高的分化率。     

盐胁迫下金花菜和紫花苜蓿试管苗的转录组分析及其耐盐基因筛选

为筛选金花菜和紫花苜蓿的耐盐基因,本研究以盐胁迫下的金花菜(JHC)和紫花苜蓿(ZHMX)试管苗为试验材料进行转录组分析。结果表明：金花菜组和紫花苜蓿组差异基因数为26722,金花菜组vs紫花苜蓿组有15850个基因下调,有10872个基因上调。金花菜组和紫花苜蓿组差异基因KEGG富集显著的Pathways有核糖体、光合作用天线蛋白、糖酵解/糖异生、光合作用、苯丙烷生物合成等。金花菜组耐盐基因有碱性亮氨酸拉链43、NAC转录因子47、ABC转运A家族成员7、晚期胚胎发生丰富蛋白2、乙烯反应转录因子ERF110等基因;紫花苜蓿组耐盐基因有WRKY转录因子、蛋白TIFY 8、转录因子MYB13、核运输因子2A、低温盐响应蛋白等基因。本试验结果可为了解金花菜和紫花苜蓿的耐盐分子机制及选育金花菜和紫花苜蓿耐盐新品种提供理论依据。