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.     

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.     

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     

Production and field performance of transgenic alfalfa (Medicago sativa L.) expressing alpha-amylase and manganese-dependent lignin peroxidase

Summary Transgenic alfalfa plants expressinBacillus licheniformis alpha-amylase and mangaese-dependent lignin peroxidase (Mn-P) from Phanerochaete chrysosporium were produced using the Agrobacterium tumefaciens transformation system. In each case, there was a range of expression of the introduced gene among independent transgenic plants. Plants producing alpha-amylase showed no alteration of phenotype. Production of Mn-P in alfalfa, howeven, in most cases adversely affected plant growth and development. Affected plants were stunted with yellowing foliage, but survived and produced seed. Results from field trials showed that Mn-P production in transgenic alfalfa reduced dry matter yield and plant height. The extent of these symptoms and yield reduction was, for the most part, related to the level of foreign protein production as estimated by Western analysis. Field data from transgenic plants expressing alpha-amylase showed that there was no effect of foreign protein production on plant performance. Expression of Mn-P was shown to segregate in sexual progeny derived from transgenic plants.Abbreviations Mn-P manganese-dependent lignin peroxidase     

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.     

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.     

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.     

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.     

Optimisation of breeding for agronomic traits in fibre hemp (Cannabis sativa L.) by study of parent-offspring relationships

Summary In the framework of a comprehensive Dutch research project the feasibility of hemp cultivation for paper pulp production is being investigated. This project also includes a hemp breeding programme which was initiated at CPRO-DLO in 1990. Hemp breeding is primarily aimed at improving bast fibre production since bast fibre is qualitatively superior to woody core fibre for paper pulp production. The progress of the ongoing breeding programme is hampered by the lack of knowledge on the inheritance of agronomic traits.The following traits were examined on 252 parental plants in 1991 and subsequently on their progenies in 1992: fibre content, plant height, date of flowering, stem diameter and cannabinoid contents. For each trait heritabilities were estimated and direct and indirect effects of artificial selection were studied.The heritability of bast fibre content was high and mass selection proved to be an efficient method causing no undesirable influence on other characters. Characters not directly related to bast fibre yield such as date of flowering, plant height and stem diameter were shown to have disadvantages as selection criteria for the improvement of bast fibre yield.The cannabinoids THC and CBD were studied, as the acceptance of hemp cultivation requires a low level. The content of THC, the psychoactive component can be successfully reduced by mass selection, but it is not certain that mass selection is the most efficient method.     

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.     

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.     

Genetic control of in vitro regeneration in alfalfa (Medicago sativa L.)

Summary Cultivars of common wheat (Triticum aestivum L.) and durum wheat (T. turgidum L. var. durum) were evaluated for epicuticular wax content and its relationship with spectral reflectance. Epicuticular wax is associated with drought tolerance. Highly significant differences (1.51 to 2.80 mg/dm²) were found in the amount of epicuticular wax (EW) among the cultivars. Water stress significantly enhanced the level of EW. EW content under stress and control conditions were positively correlated (r=0.85, p<0.01) suggesting that selection for this trait could be practised in either environment. Surface reflectance was reduced when the waxy layer from the leaf was removed with chloroform. The reduction for the abaxial surface was twice that for the adaxial surface indicating that the abaxial surface was more waxy than the adaxial one. The mean reduction (both surfaces) termed was positively correlated with the amount of EW (r=0.59, p<0.01).     

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.     

Genetics of stem elongation ability in rice (Oryza sativa L.)

Summary The genetics of stem elongation ability in rice was studied in parents, F₁, F₂ and backcross generations of six crosses. Segregation analysis indicated dominance for stem elongation ability. Estimation of genetic parameters under epistatic model indicated more than one locus control stem elongation ability and both additive and nonadditive gene effects were important. Epistatic effects were predominant over additive and dominance effects with an important role of duplicate type of epistasis. The occurrence of significant additive and additive x additive types of genetic variation and the moderately high broad sense heritability indicated the possibility of selection for an increased manifestation of stem elongation ability.     

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.     

Cytological mechanisms of 2n egg formation in a diploid genotype of Medicago sativa subsp. falcata

Summary Gametes formed without meiosis in alfalfa would be useful in basic and applied research. Therefore, the cytological analysis of macrosporogenesis of a diploid plant of Medicago sativa subsp. falcata (L.) Arcangeli (named PG-F9), previously selected as a good 2n egg producer, was conducted. A stain-clearing technique was applied which also allowed the analysis of microsporogenesis to be performed. Two mechanisms of 2n egg formation were determined: absence of the second meiotic division (mechanism of the SDR type) and apomeiosis. In particular, it is noteworthy that 2n eggs produced via apomeiosis should retain the same parental genotype, because apomeiotic cells give rise directly to the female gametophyte without undergoing meiosis. The presence of binucleate female gametophytes in the ovules at an early stage of development confirmed the presence of apomeiosis in PG-F9. These abnormalities concerned only macrosporogenesis; in fact, the analysis of microsporogenesis showed the production of normal tetrads of microspores after normal meiosis. Possible application of the discovered mechanisms of 2n egg production in breeding research are discussed.     

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.     

Relationship between genealogical distance and breeding behaviour in oats (Avena sativa L.)

Summary Eight oat cultivars and experimental lines from four germplasm sources were crossed in a diallel mating design without reciprocals. F₁ heterosis for grain yield was evaluated in two experiments, and 48 F₂-derived lines from each of the 28 matings were evaluated for bundle weight, grain yield, straw yield, harvest index, height, and heading date in two experiments. Number of transgressive segregates per trait and generalized genetic variance were calculated for each mating. Genealogical distance for each mating was obtained by using coefficient of kinship based on the pedigree of the parents. The relationship between genealogical distance and the three types of breeding behaviour was examined via correlation and regression. Significant correlations occurred only for genealogical distance with numbers of transgressive segregates for height and with generalized genetic variances. Both were positive. Significant heterosis was observed for matings of more distantly related parents. Regressions on genealogical distance, when significant, were linear. Genealogical distance between parents was positive associated with diversity on the basis of breeding behavior.Journal Paper No. J-11874 of the Iowa Agriculture and Home Economics Station, Iowa State Univ., Ames, IA 50011. Project 2447.     

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.