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).     

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.     

紫花苜蓿种子高产灌溉技术研究进展

本文回顾了20世纪50年代以来国外紫花苜蓿(Medicago sativa L.)种子生产灌溉技术的研究进展,从灌溉制度的制定依据、灌溉对种子产量和产量构成因素的影响、不同土壤质地条件的高产灌溉制度以及灌溉制度的水分预算法等方面,讨论了紫花苜蓿种子高产灌溉管理的原理和基本方法.     

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.     

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     

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.     

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.     

Detection of QTL for forage yield,lodging resistance and spring vigor traits in alfalfa (Medicago sativa L.)

Alfalfa (Medicago sativa L.) is an internationally significant forage crop. Forage yield, lodging resistance and spring vigor are important agronomic traits conditioned by quantitative genetic and environmental effects. The objective of this study was to identify quantitative trait loci (QTL) and molecular markers associated with increased forage yield, resistance to lodging, and spring vigor. A backcross population composed of 128 progeny was developed by crossing the breeding parents DW000577 (lodging susceptible) and NL002724 (lodging-resistant) and back-crossing an individual F₁ plant to the maternal parent (i.e. DW000577). A linkage map of NL002724 was developed based upon the segregation of 236 AFLP, SRAP, and SSR markers among the backcross progeny. The markers were distributed among 14 linkage groups, covering an estimated recombination distance of 1497.6 centiMorgans (cM). Replicated clones of both parents and backcross progeny were evaluated in the field for estimated forage yield, lodging, and spring vigor in Washington and Wisconsin during 2007 and 2008. Significant QTL were found for all three traits. In particular, two QTL for lodging resistance were identified that explained ≥14 % of trait variation, and were significant in all years and locations. Major QTL explaining over 25 % of trait variation for forage yield were detected in multiple environments at two separate locations on chromosome III. Several QTL for spring vigor were located in the same or similar positions as QTL for forage yield, possibly explaining the significant correlation between these traits. Molecular markers associated with the aforementioned QTL were also identified.     

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     

Karyotype and C-banding pattern of mitotic chromosomes in alfalfa, Medicago sativa L.

Numerous cytogenetic studies have been performed on alfalfa (Medicago sativa L. 2n= 4x= 32), but only a few have examined the somatic chromosomes. Because the major factor limiting the study of somatic chromosomes of M. sativa is their rather uniform morphology, characterization was attempted using the C-banding technique instead of the traditional Feulgen method. Chromosome morphology and position of the heterochromatic bands resulted in satisfactory characterization of the chromosomes and provided further evidence of the autotetraploid origin of the species. It was possible to identify the homologous chromosomes and arrange them into eight groups of four. The ideogram of the basic genome of eight chromosomes was constructed. The impact of these results on cytogenetic studies and breeding research in alfalfa is discussed.     

Water relations and use-efficiency,plant survival and productivity of nine alfalfa (Medicago sativa L.) cultivars in dryland Mediterranean conditions

The potential for alfalfa to be a valuable rainfed pasture option that can be used to increase livestock and crop production in central-south Chile is demonstrated for the first time. The persistence, productivity and water use efficiency of alfalfa (Medicago sativa) cultivars was evaluated at four sites in dryland Mediterranean environments of Chile between 2012 and 2015. Nine alfalfa cultivars were evaluated for plant persistence, winter growth, forage yield, shoot water potential (Ψ), stomatal conductance (gs), and carbon (δ¹³C) and oxygen (δ¹⁸O) compositions. The percentage of plant survival after four years varied between 69 and 97% of the established plants. Alfalfa forage yield was >9 t ha⁻¹yr⁻¹ at Yungay (Andes foothills) in year two, and at Cauquenes (interior dryland site) in years three and four. The highest forage yield was obtained in cultivars SARDI Ten and Aquarius and the lowest in WL326HQ. In 2015, the forage yield during late spring and early summer (November 2015–January 2016) ranged from 3170 to 3570 kg ha⁻¹ (average of the nine cultivars), which represented between 29% and 38% of the total production. The response of gs to Ψ was different among cultivars but all cultivars exhibited a strong decline when Ψ was <−1.0 MP. Shoot water potential evaluated in October–November was negatively correlated with δ¹³C and δ¹⁸O in shoot biomass. The apparent water use efficiency (kg DM/ha/mm of rainfall) across the nine cultivars was highest in Cauquenes in the three years evaluated (>16 kg mm⁻¹). It is concluded that alfalfa is a high yielding forage that can extend the growing season into early summer and autumn in dryland Mediterranean environments.     

8种微肥配施在紫花苜蓿优质生产中的应用

针对河南省土壤微量元素的含量以及紫花苜蓿(MedicagosativaL.)对微量元素需要的特点,该文对锌、铁、铜、锰、硼、钼、钴、硒等八大微肥在紫花苜蓿优质生产中的应用进行了定量的研究,分析和比较不同的微肥配施技术对紫花苜蓿优质生产的关键性的影响,以达到用不同的配施方案,解决不同生产要求的目的。     

Effect of Boron on Seed Yield in Lucerne (Medicago sativa L.)

The present study aimed at studying the effect of boron on pollen characteristics, translocation of water soluble sugars and its importance as a specific nutrient for seed production in lucerne. Aquous solutions of borax (0, 2500, 5000, 7500, 10 000 and 20 000 ppm) were sprayed at flowering stage. After 20 days, the fresh flowers were collected from the plants of all the treatments separately. The pollen was dusted on glass slides containing Muntzing's mixture for staining. After 6 hours, the data on pollen size, stainability and germination were recorded in 10 randomly selected microscopic fields. The estimation of water soluble sugars and boron content in flowers was done by standard methods. Data on grains per pod, grains per inflorescence and 1000 seed weight were recorded at seed maturity. With increasing rates of applied B, pollen germination, water soluble sugars, boron content and number of seeds per pod and per inflorescence increased but pollen fertility decreased. The pollen size and 1000 seed weight was not affected by boron concentrations. The results indicate that boron plays a definite role in increasing the seed yields through stimulating the physiological processes during reproductive phase.     

紫花苜蓿生产性能构成因子研究

对3种不同秋眠类型9个不同秋眠级别的苜蓿品种进行生产性能构成因子以及构成因子间的相互关系的研究,结果表明,植株高度与节间长和再生速度达显著相关(P<0.05);生物量与植株高度和再生速度达显著相关(P<0.05);关于再生速度与植株高度、节间长和生物量的影响程度拟合方程分别如下:Y1=37.165+8.17x1;Y2=2.28+1.13x1;Y=3.03x1.再生速度与植株高度、节间长和地上生物量有着很高的相关性.植株高度是苜蓿产量一个很好的预测指标;决定苜蓿地上生物量的最终决定因子是再生速度,所以苜蓿的再生性是苜蓿高产育种首先考虑的因子.     

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.     

不同品种紫花苜蓿中总皂苷含量的比较研究

采用比色法测定了北京地区栽培不同年限的45个紫花苜蓿品种茎、叶中总皂苷的含量。结果表明，各品种苜蓿叶片中的皂苷含量均高于茎，在不同品种之间存在明显差异。4年生植株中皂苷含量最高的品种“Vernal”为1.174%，最低的品种“Empress”为0.606%；3年生植株中最高的品种“WL442”为1.141%，最低的品种“农保”为0.788%；2年生植株中最高的品种“胜利者”为1.293%，最低的品种“全能”为0.722%。综合比较，含量最低品种与最高的品种相差2.13倍。栽培年限对紫花苜蓿皂苷含量具有一定影响。     

Extent of RFLP variability in tetraploid populations of alfalfa, Medicago sativa

Seven widely-cultivated alfalfa varieties and three ecotypes adapted to Central Italy were used to evaluate the extent of polymorphism in that species. Twenty plants per accession were analysed with 16 RFLP probes combined with three restriction enzymes (48 probe/enzyme combination in total) and the data were used to compute the Nei's similarity index taken as a measure of inter- and intra-population RFLP variability. The varieties were, in general, more homogeneous than the ecotypes and the cultivars ‘Adriana’ and ‘Florida’ could be differentiated more easily than the others. Few accession-specific hybridizing fragments were scored and seven populations could be distinguished from the others on the basis of significant differences in the frequencies of specific fragments. The DNA of plant populations of several sizes was bulked and the ability to detect a given fragment in pooled samples was related to the fraction of plants having that fragment among the plants forming the bulk. The results are discussed with special emphasis on the practical utilization of RFLPs for varietal identification.     

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.     

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.