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     

Single plant versus clonal line performance in alfalfa

Summary Single plants and clonal lines of fifty-six alfalfa clones were compared in a field trial. The error coefficient of variation was much higher for single plants than for clonal lines. Unreplicated single-plant and clonal-line performance were inadequately related. Replicated single-plant performance approached closely the performance of unreplicated clonal lines. Correlation between yield and height was low.     

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.     

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.     

Competition among components of synthetic varieties in alfalfa

Summary Eighteen alfalfa polycross progenies were tested for forage and seed yield in pure stand as well as in competition with the mixture of all progenies included in the test. The type of border affected significantly the forage and seed yield of individual progenies in one third of the cases. In most of these cases competition from the mixture resulted in reduction of the yield of individual progenies. There was no correlation between pure stand forage and seed yield, but there was significant positive correlation between forage and seed yield determined under conditions of competition. It is concluded that polycross progenies should be tested in plots bordered by the mixture of all progenies being tested.     

Breeding alfalfa cultivars resistant to the alfalfa weevil

A ten-year study consisting of nine field experiments was conducted to evaluate alfalfa (Medicago sativa L.) for resistance to larval feeding by the alfalfa weevil (Hypera postica Gyllenhal). The results were consistent over years and experiments. Only one source of alfalfa germplasm, designated Starnes, showed resistance to larval feeding in the field under natural infestations of the weevil. The cultivars Team, Arc, and Liberty have been developed from this germplasm source. The only effective method of selection for field resistance to the weevil was field selection under natural weevil infestations. Selection was based on low larval feeding on spaced plants. After six generations of such selection the Starnes germplasm showed only one-third as much defoliation as did the check cultivars. The heritability of the weevil resistance was moderately high. Field resistance was transferred to other strains through hybridization; hybrids were intermediate to the resistant and susceptible parents. Resistance was stable.Laboratory and greenhouse efforts to breed alfalfa weevil resistant alfalfa-using such techniques as adult cotyledon-nonpreference tests, adult leaf-feeding tests, larval-growth and survival tests, oviposition-stimulus tests, and oviposition-nonpreference tests-have failed to produce field resistance.Paper No. 5308 of the Journal series of North Carolina Agricultural Experiment Station, Raleigh, N. C., in cooperation wich ARS, USDA. Received 15 June 1977.     

Selection for increased forage yield in alfalfa at different NaCl levels

Summary Alfalfa (Medicago sativa L.) forage yield may be reduced by moderate levels of salinity. Increasing forage yield in saline environments through plant breeding could partially ameliorate this problem. However, prior indirect selection either in cell culture, at germination, or during seedling growth has not resulted in agronomically relevant levels of salt tolerance. This study was conducted to determine whether mean forage yield of alfalfa grown in saline environments could be increased. To reduce the number of possible selection schemes, forage yield data for five harvests from Sonora and its parental cultivar African grown in a greenhouse and irrigated with 0, 30, 60, or 80 mM NaCl were used to simulate one cycle of selection at each NaCl environment. Greatest gains in forage yield were expected under non-saline conditions with decreased gains under saline conditions. Selection based on yield in regrowth harvests was also predicted to be more efficient than that based on seedling harvest. Experimental populations were developed from Sonora and African at each NaCl level using phenotypic selection for increased mean forage yield for harvests 3 to 5. After each of two cycles of selection, forage yield from selected, control (plants selected randomly under non-saline conditions), and parental populations was evaluated at 0, 30, 60, and 80 mM NaCl. Gains in forage yield were observed in African when the selection and evaluation environments were the same, but in Sonora this was observed only at 0 NaCl. Selection at 0 NaCl did not increase forage yield at 60 or 80 mM NaCl in either germplasm source. This suggests that selection for increased forage yield may be successful at low to moderate NaCl levels in germplasm with ample variation for yield when grown in saline environments, while selection for increased forage yield in non-saline conditions may be ineffective.     

Diallel analysis of tolerance to aluminium in alfalfa

Summary Acid soils having high levels of aluminium (Al) can drastically reduce yields in alfalfa and the most economically viable solution to the problem appears to be the development of Al-tolerant cultivars. To assist with the choice of a breeding method, a six-parent alfalfa diallel (crosses and reciprocals included but not parents) was evaluated in Al-toxic nutrient solution in terms of height (HT) and dry weight (DW). General combining ability was significant for both traits and constituted the majority of the genetic variation. Specific combining ability was significant only for HT and reciprocal effects were significant only for DW. Tolerance appeared to be at least partially dominant to sensitivity. Results indicate that a mass selection scheme, such as recurrent phenotypic selection, may be effective in increasing levels of tolerance in at least some alfalfa populations and that minor grains may also be achieved through exploiting non-additive genetic variation.     

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.     

Selection of regenerative genotypes from highly productive cultivars of alfalfa

Summary Seedlings from Canadian alfalfa cultivars Algonquin and Apica were selected on the basis of ability to regenerate plants from hypocotyl callus. A total of eleven genotypes were selected, resulting in regeneration frequencies of 4.5 and 11.7% for Algonquin and Apica, respectively. Regeneration capacity within these cultivars was also compared with cultivars and breeding lines for which regeneration frequencies have previously been documented. Two genotypes (A1 93 and Ap 20) exhibited prolific embryogenesis from hypocotyl and petiole callus and also regenerated plants from suspension culture. Flowering and seed set have been observed for regenerated plants.     

Forage quality of genetically diverse alfalfa germplasms at four phenological growth stages

Summary Forage quality of various alfalfa (Medicago sativa L.) cultivars has been determined under different environmental conditions, and numerous trials with alfalfa have documented an inverse relationship between forage quality and maturity. Little information is available, however, regarding the comparative forage quality of the germplasm sources from which most USA cultivars were developed. We compared forage quality of these germplasm sources at four phenological stages under disease- and insect-free conditions in a greenhouse. Germplasm sources (cultivars) tested were: Indian (Sirsa #9), African (African), Peruvian (Hairy Peruvian), Flemish (DuPuits), Turkistan (Lahontan), Chilean (Kansas Common), M. varia (Grimm), and Ladak (Ladak). Four harvests were taken and forage was separated into four phenological stages: vegetative, early bud (1–3 buds-per-stem), late bud (>3 buds-per-stem), and bloom. The germplasm source X phenological stage interaction was significant for crude protein (CP) and in vitro digestible dry matter (IVDDM) concentrations. M. varia showed the least decline in IVDDM and CP with increasing maturity. M. varia had higher IVDDM than did African and Indian at late bud and bloom stages. Indian and Flemish had higher CP than did Turkistan and Peruvian at late bud and bloom stages. Alfalfa germplasm sources differ in forage quality when comparisions are made within similar stages of phenological development.Joint contribution of the Dept. of Agronomy and USDA-ARS, Kansas Agricultural Experiment Station, Manhattan, KS 66506, USA. Contribution no. 90-475-J.     

Thermal dependence of glutathione reductase in one tetraploid and its near-isogenic diploid alfalfa

Summary A more thorough understanding of the biochemical basis of superior performance of one alfalfa (Medicago sativa L.) genotype compared to another would expedite improvement in agronomically useful genotypes. Information on thermal dependence of glutathione reductase (GR, E.C. 1.6.4.2) in different ploidy levels of alfalfa is lacking. Our objective was to investigate the thermal dependency of GR in two genetically comparable populations of alfalfa at diploid and tetraploid levels. Thermal stability was similar for the enzyme from both populations, whereas variation was found for: i) the minimum apparent Km (0.53 and 4.14 M NADPH), ii) the temperature at which the minimum apparent Km was observed (15 and 25° C), and iii) the thermal kinetic windows (TKW) (the temperature range over which the apparent Km is within 200% of the observed minimum Km, 5 and 20° C width) in diploid and tetraploid alfalfa, respectively. The TKW data suggest that the diploid can perform optimally under a narrower (i.e. smaller TKW) and lower range (i.e. the temperature at which the lowest Km was observed) of temperatures, whereas the tetraploid can perform optimally under a wider range (i.e. larger TKW) of temperatures. This finding is in agreement with previously published growth data on near-isogenic populations of diploid and tetraploid alfalfa.Abbreviations GR glutathione reductase - TKW thermal kinetic window - Km apparent Km This work was supported by USDA Specific Cooperative Agreement No. 58-7MN1-8-143 from the Plant Stress and Water Conservation Unit, USDA-ARS, Lubbock, Texas. Joint contribution of the Texas Tech University, USDA-ARS, Lubbock, TX and University of Wisconsin, Madison, WI. TTU Journal No. T-4-306.     

Effect of various plant factors on the expression of creeping-rootedness in lucerne (Medicago sativa L. complex)

Although agronomically interesting, selection of creeping-rootedness inlucerne, that is, the ability to form adventitious shoots on horizontal,spreading roots, remains difficult because of the complex and still unclear geneticcontrol of this character, coupled with the possible occurrence of external factorsthat affect unpredictably its expression. Two experiments were undertaken onprogenies of creeping-rooted plants, to verify previous findings and inferences,and test novel hypotheses, concerning the effect of various plant factors on theexpression of creeping-rootedness. The ultimate aim was to provide information onthe most appropriate germplasm, procedures, or indirect selection criteria, that wouldimprove the efficiency of breeding for this character. The factors here examinedincluded genotype, age, kind of progeny, vigour, spreading ability, and undergroundmorphology. Some genotypes appeared to be good donors of the character, whereasothers did not. The proportion of creeping-rooted plants increased in allcases with the plant age. Selfing consistently depressed the expression ofthe character compared with clonal propagation or crossing. It is suggestedthat the effect of age and selfing on the penetrance of creeping-rootedness ismediated through their effect on plant vigour. Both aerial and root vigour showedindeed an effect per se on the presence of the character. Beside thevigour, possible indirect selection criteria were identified in the frequenciesof swollen zones and latent stem primordia on horizontal roots. Proportion ofcreeping-rooted plants in progenies after one year of growth appeared as a simple anduseful criterion to predict future trends. Consistent with previous conclusions, theresults provided evidence that a combination of family and individualselection may enhance the character expression.     

Regenerative trait and cold hardiness in highly productive cultivars of alfalfa and red clover

Summary In earlier work on improvement of persistance in forage legumes, we selected genotypes from highly productive cultivars of alfalfa, Algonquin and Apica (Euphytica 45: 105–112, 1990) and cv. Florex red clover (Plant Cell Reports 8: 395–398, 1989) capable of in vitro regeneration from callus and cell culture. The alfalfa germplasm and its F₁ progeny as well as an F₂ red clover population were tested for cold stress tolerance. Plantlets were hardened in culture tubes at 2 or 5°C, 8h photoperiod, for at least four weeks and then subjected to freezing temperatures, –16 or –10°C for alfalfa and red clover, respectively. Survival of regenerative genotypes was significantly higher than of the non-regenerative ones in both species. A strong oositive correlation (r=0.78) between the regenerative trait and plant survival was found in alfalfa. The experiments indicate that in vitro selection for regenerative trait may improve cold stress tolerance of alfalfa and red clover.     

The potential for breeding an improved lucerne-rhizobium symbiosis. 1. Assessment of genetic variation

Summary Six cultivars of lucerne (Medicago sativa L.) were grown in all possible combinations with eight strains of Rhizobium meliloti in order to assess genetic variation in symbiotic nitrogen fixation. Host genotype and Rhizobium genotype effects on nitrogen fixation were respectively 4.8% and 21.0% of total phenotypic variance. Genetic variation due to host cultivar × Rhizobium strain interactions accounted for a further 6.0% of phenotypic variance. The results indicated low heritability of general symbiotic effectiveness in the host with interaction effects being large enough to suggest that plant performance may be unpredictable with populations of R. meliloti in field soils. Joint regression analysis showed that about 50% of the interactive variation could be explained by generalized differences in the sensitivity of cultivars to alterations in the genotype of Rhizobium strains. One cultivar, Siriver, was relatively insentitive to changes in the Rhizobium genotype whilst still maintaining high average yield. The implications of the results for lucerne breeding are discussed.     

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.     

In vitro selection of wheat callus tolerant to high levels of salt and plant regeneration

Summary Embryogenic calli isolated from immature embryos of four wheat cultivars were subjected to three in vitro selection methods for salt tolerance. The effect of NaCl on the selected and unselected cell lines has been investigated. The results indicated that the relative growth rate of callus decreased as the concentration of NaCl increased in both callus lines. The selected callus line gave a higher growth weight in the presence of NaCl in the medium and was highly significant as compared with unselected callus line across medium protocols in all wheat cultivars. The dry weight of both kinds of callus lines of all wheat cultivars increased markedly with increasing NaCl concentration in most cases. The Na⁺ and Cl^- contents of both callus lines were increased with increasing salinity levels while K⁺ content was decreased. The selected callus line of each cultivar at the same salinity level produced significant amounts of Na⁺, K⁺ and Cl^- higher than the unselected callus line in most salinity levels. However, the unselected callus lines of the cultivars Giza-157 and Sakha-90 at the same salinity level produced significant amounts of K⁺ higher than the selected callus line in most salinity levels. The proline content of both kinds of callus lines for all wheat cultivars was increased with increasing salinity level. However, the selected callus line gave a significantly higher proline content than the unselected callus line in all wheat cultivars at the same Salinity level. Results from the in vitro selection for NaCl tolerance showed that the stepwise method of increasing NaCl in the medium was more effective for plant regeneration than other methods.     

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.     

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.