Growth of Lucerne (Medicago sativa L.) Exposed to Sulphur Dioxide

The effect of exposure to sulphur dioxide (0 or 96 µgm^–3 SO₂) on the growth and sulphur content of lucerne(Medicago sativa L.) was examined in a period of 135 d duringwhich the plants were harvested four times. The lucerne wasgrown in pots of soil with and without the addition of sulphateand of nitrate. Evidence of sulphur deficiency, including areduction in the weight, the number and the sulphur contentof shoots, was found in plants grown without added sulphate.Deficiency was alleviated through exposure of plants to SO₂.Apart from reducing shoot weight at one harvest and generallyincreasimg the concentration of sulphur in the shoots, exposureto SO₂ had no significant effect on plants grown with addedsulphate. The yield of shoots was greater, and was reduced toa lesser extent with sulphur deficiency, in plants grown withadded nitrate than in those dependent on rhizobia. Whilst thetranspiration coefficient increased greatly in sulphur-deficientplants, it was reduced where otherwise similarly treated plantswere exposed to SO₂; this treatment did not alter the coefficientin plants with an adequate supply of sulphur from the soil.     

根癌农杆菌介导的苜蓿体胚转化

以苜蓿体细胞胚胎作为根癌农杆菌介导转化的受体,通过对GUS基因瞬时表达率的分析,研究该转化体系的最佳实验参数。实验结果显示,负压处理10min和共培养5d时表达率最高(可达17．4％)。以这一转化方法分别对带有3种不同启动于的表达载体进行比较,发现由CMV35S启动于驱动的GUS基因的瞬时表达率可达82．7％,Ubil启动于驱动的可达57．8％,而Actl启动于驱动的则未见表达。     

Liming of alfalfa (Medicago sativa L.)

Summary A growth-chamber experiment was conducted to study the effect of liming upon growth of alfalfa. The beneficial effects observed were related to changes in soil properties brought about by lime application. Reductions of aluminum and manganese toxicities were the major factors responsible for the increased yields and the decreased growth period required to reach harvest stage. Significant correlations between plant growth parameters and various measures of extractable aluminum were found.     

The Role of Different Daily Irradiations on Shoot Growth and Root/Shoot Ratio in Lucerne (Medicago sativa L.)

The effect of two levels of daily irradiation on shoot growthand root/shoot partitioning was investigated on two lucernevarieties (Medicago sativa L.). Individual plants were studiedunder constant temperature and optimal water and mineral nutritionconditions. For both lucerne varieties, daily irradiation did not changebiomass partitioning between shoots and roots. It can be shownthat leaf area expansion occurs independently of daily irradiationand that the process of shoot dry matter production is strictlyproportional to daily irradiation, since the ratio leaf area/shootdry matter is inversely proportional to it. From a model of shoot production of the isolated plant, we showthat the relative shoot specific activity is not affected bydaily irradiation. A simple model of partitioning leads us todetermine why partitioning remains the same for the two at thelevels of daily irradiation. Finally, the allocation between shoot and root turns out tobe independent of the main stem extension rate, which is fasterat the higher daily irradiation. Medicago sativa L., lucerne, daily irradiation, shoot production, root/shoot ratio, leaf area expansion, partitioning model     

Induced androgenesis in alfalfa (Medicago sativa L.)

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

Beta-Amylases from Alfalfa (Medicago sativa L.) Roots

Amylase was found in high activity (193 international units per milligram protein) in the tap root of alfalfa (Medicago sativa L. cv. Sonora). The activity was separated by gel filtration chromatography into two fractions with molecular weights of 65,700 (heavy amylase) and 41,700 (light amylase). Activity staining of electrophoretic gels indicated the presence of one isozyme in the heavy amylase fraction and two in the light amylase fraction. Three amylase isozymes with electrophoretic mobilities identical to those in the heavy and the light amylase fractions were the only amylases identified in crude root preparations. Both heavy and light amylases hydrolyzed amylopectin, soluble starch, and amylose but did not hydrolyze pullulan or β-limit dextrin. The ratio of viscosity change to reducing power production during starch hydrolysis was identical for both alfalfa amylase fractions and sweet potato β-amylase, while that of bacterial α-amylase was considerably higher. The identification of maltose and β-limit dextrin as hydrolytic end-products confirmed that these alfalfa root amylases are all β-amylases.     

Fine root demography in alfalfa (Medicago sativa L.)

In perennial forages like alfalfa (Medicago sativa L.), repeated herbage removal may alter root production and mortality which, in turn, could affect deposition of fixed N in soil. Our objective was to determine the extent and patterns of fine-diameter root production and loss during the year of alfalfa stand establishment. The experiment was conducted on a loamy sand soil (Udorthentic Haploboroll) in Minnesota, USA, using horizontally installed minirhizotrons placed directly under the seeded rows at 10, 20, and 40 cm depths in four replicate blocks. We seeded four alfalfa germplasms that differed in N₂ fixation capacity and root system architecture: Agate alfalfa, a winter hardy commercially-available cultivar; Ineffective Agate, which is a non-N₂-fixing near isoline of Agate; a new germplasm that has few fibrous roots and strong tap-rooted traits; and a new germplasm that has many fibrous roots and a strongly branched root system architecture. Video images collected biweekly throughout the initial growing season were processed using C-MAP-ROOTS software.More than one-half of all fine roots in the upper 20 cm were produced during the first 7 weeks of growth. Root production was similar among germplasms, except that the highly fibrous, branch-rooted germplasm produced 29% more fine roots at 20 cm than other germplasms. In all germplasms, about 7% of the fine roots at each depth developed into secondarily thickened roots. By the end of the first growing season, greatest fine root mortality had occurred in the uppermost depth (48%), and least occurred at 40 cm (36%). Survival of contemporaneous root cohorts was not related to soil depth in a simple fashion, although all survivorship curves could be described using only five rates of exponential decline. There was a significant reduction in fine root mortality before the first herbage harvest, followed by a pronounced loss (average 22%) of fine roots at the 10- and 20-cm depths in the 2-week period following herbage removal. Median life spans of these early-season cohorts ranged from 58 to 131 days, based on fitted exponential equations. At all depths, fine roots produced in the 4 weeks before harvest (early- to mid-August) tended to have shorter median life spans than early-season cohorts. Similar patterns of fine root mortality did not occur at the second harvest. Germplasms differed in the pattern, but not the ultimate extent, of fine root mortality. Fine root turnover during the first year of alfalfa establishment in this experiment released an estimated 830 kg C ha^–1 and 60 kg N ha^–1, with no differences due to N₂ fixation capacity or root system architecture.     

Changes in Growth and Quality Characteristics of Lucerne (Medicago sativa L.) in Response to Sulphur Dioxide Exposure under Field Conditions

The effects of SO₂ on some growth and quality characteristicsof lucerne (Medicago sativa L.) were investigated by exposingplants to mean SO₂ concentrations of 215, 78 or 2.8 µgm_–3 in open-top chambers for 166 d. Plants exposed to215 µg m_–3 had significantly lower shoot and rootweights compared with plants exposed to 78 µg m^–3,but not compared with control plants. Exposure to 215 or 78µg m ^–3 increased the plant shoot: root ratio, buthad no effect on leaf area. During the middle of the fumigationperiod, relative growth rate and net assimilation rate werehighest in plants exposed to 215 fig m, but these later fellbelow control values, and plants exposed to 78 µg m^–3had the highest relative growth rate and net assimilation rate.As the duration of exposure increased, an initial SO₂-inducedstimulation of growth may have developed to toxicity at thehighest SO₂ exposure. Exposure to SO₂ depressed L-ascorbic acid concentrations inleaves, had no effect on foliar protein or starch concentrations,and increased the specific energy of shoots and plant sulphurconcentrations. The effect of SO₂ on L-ascorbic acid concentrationsmay suggest a mechanism for reduced freezing tolerance of plantsafter exposure to SO₂. Key words: SO₂, Medicago sativa L., Growth     

Plantlet Regeneration via Multiple Shoot Induction in Indian Cultivars of Lucerne (Medicago sativa L)

An efficient protocol has been developed for in vitro plant regeneration via multiple shoot induction in lucerne (Medicago sativa L). Shoot tips from in vitro grown 5–6 days old seedlings of 3 cultivars, LLC-3, Chetak and RL-88 were used as explants for multiple shoot induction on MS medium supplemented with cytokinins. Maximum of 14 shoots per apical meristem were observed in case of cv Chetak on MS medium supplemented with BAP (12.6 μM) and KN (9.3 μM). Shoot elongation on MS medium supplemented with GA (5.8 μM), while root induction was achieved on MS medium supplemented with IAA (11.4 μM) and activated charcoal (2.0 g l^?1). Tissue raised plants showed 75% survival after transfer to soil under field conditions.     

Somatic hybrid plants between the forage legumes Medicago sativa L. and Medicago arborea L.

Interspecific somatic hybrid plants were obtained by symmetrical electrofusion of mesophyll protoplasts of Medicago sativa with callus protoplasts of Medicago arborea. Somatic hybrid calli were picked manually from semi-solid culture medium after they were identified by their dual color in fluorescent light. Twelve putative hybrid calli were selected and one of them regenerated plants. The morphogenesis of the somatic hybrid calli was induced by the synthetic growth regulator 1,2 benzisoxazole-3-acetic acid. Somatic hybrid plants showed intensive genome rearrangements, as evidenced by isozyme and RFLP analysis. The morphology of somatic hybrid plants was in general intermediate between the parents. The production of hybrids by protoplast fusion between sexually incompatible Medicago species is related to the in vitro respon siveness of the parental protoplasts. The possibility of using somatic hybrid plants in alfalfa breeding is discussed.     

Microgametophytic selection in two alfalfa (Medicago sativa L.) clones

Summary Microgametophytic selection was investigated using two ecologically diverse autotetraploid clones of alfalfa. Several selection pressures (drying, aging, freezing, and high and low temperatures) were applied to microgametophytes at three stages of the life cycle, 1) during microsporogenesis, 2) post-anthesis, and 3) pollen tube growth. Pollen aging produced a progeny population with a greater mean plant size and a lower coefficient of variation than the control progeny. High temperature (29.5 °C) applied both during microsporogenesis and pollen tube growth resulted in progeny populations which were significantly taller and, in one case, had a larger leaf number than the control populations. In contrast, air dried pollen resulted in a progeny population which had significantly smaller character means and larger coefficients of variation than the control population. Also, low temperature (15 °C) during pollen tube growth yielded progeny with reduced branch number and a larger coefficient of variation than the control progeny. In cases where progeny derived from selected microgametophytes were found to differ from the control offspring, corresponding shifts in the reciprocal cross were not observed. For the temperature stress treatments, the lack of reciprocal differences may be related to the different temperature adaptations of the two ecotypes. These results suggest that microgametophytic selection can be effective in shifting the mean of the progeny generation; however, the results obtained will vary depending upon the selection pressure, stage of selection, and the parents used.     

Cryopreservation of alfalfa (Medicago sativa L.) cells by encapsulation-dehydration

Our objective was to establish a cryopreservation protocol for alfalfa (Medicago sativa L.) cells and study the physiological changes occurring in cells during cryopreservation treatment. Cell cultures of Pioneer cvs. 5262 (fall-dormant, winter-hardy) and 5929 (non-dormant, non-hardy) plants initiated regrowth after cryopreservation by encapsulation-dehydration (ED). Pre-treatment of the encapsulated cells for 4 days in B5 medium containing 0.75 M sucrose and dehydration for 4 h in a laminar flow hood were necessary to achieve maximum cell viability after ED and cryopreservation in liquid N₂ (EDN). Viability (measured as triphenyl tetrazolium chloride reduction) of the cv. 5262 cells after cryopreservation was two- to three-fold greater than that of the cv. 5929 cells. Cold acclimation of the cells (10 days at 2°C) improved viability after cryopreservation. The addition of 7.6 µM ABA to the B5 medium enhanced viability in ED but did not improve cell cryopreservability. Cold-acclimated cells had higher protein concentrations, but neither ABA nor cold acclimation influenced protein composition of cold-acclimated cells determined using SDS-PAGE. Encapsulated cells pre-treated for 4 days in B5 medium containing 0.75 M sucrose showed an increased concentration of cell protein and an altered protein composition. Suspension cultures were re-initiated from both ED and EDN treatments by transferring beads sequentially to B5 media containing 0.75, 0.5, 0.25 M sucrose and then to fresh B5 medium. The ED cells resumed rapid growth after two subcultures, whereas EDN cells needed four or five subcultures to resume rapid growth.     

Purification of beta-amylase from alfalfa (Medicago sativa L.) seeds

An amylase from alfalfa (Medicago sativa L. c.v. Moapa) seeds was purified by column chromatography and gel filtration, followed by chromatofocusing on Mono P HR 5/20. The last step was effective for separation of the alfalfa amylase to a homogeneous state. The purified amylase was identified as beta-amylase from the fact that only beta-maltose was formed by the enzymatic degradation of soluble starch. The molecular weight and specific activity of the beta-amylase (E1%(280 nm) = 18.3) were determined to be 61,000 and 1,077 A.U./mg, respectively. The beta-amylase activity was inhibited by the modification of sulfhydryl groups with p-chloromercuribenzoic acid. The optimum pH and isoelectric point of alfalfa beta-amylase were 7.0 and 4.8, respectively, which were different from other plant beta-amylases.     

葡萄体细胞胚的保存

研究干化对酿酒葡萄品种'神索'体细胞胚保存效应的结果表明,葡萄体细胞胚失水量在40%～50%之间的萌发率较高,达58%左右,比未经干化的萌发率高9.4%;在较高相对湿度的情况下,体细胞胚失水速度变慢,干化处理时间延长,可以提高体细胞胚的存活率和萌发率;相对湿度70%的情况下干化15 d的效果最好,其萌发率从未经干化处理的33.5%提高到56.2%.     

Cloning and characterization of chromosomal markers in alfalfa (Medicago sativa L.)

Eleven tandemly repetitive sequences were identified from a Cot-1 library by FISH and sequence analysis of alfalfa (Medicago sativa). Five repetitive sequences (MsCR-1, MsCR-2, MsCR-3, MsCR-4, and MsCR-5) were centromeric or pericentromeric, of which three were satellite DNAs and two were minisatellite DNAs. Monomers of 144, 148, and 168 bp were identified in MsCR-1, MsCR-2, and MsCR-3, respectively, while 15 and 39 bp monomers were identified in MsCR-4 and MsCR-5, respectively. Three repetitive sequences were characterized as subtelomeric; one repetitive sequence, MsTR-1, had a 184 bp monomer, and two repetitive sequences had fragments of 204 and 327 bp. Sequence analysis revealed homology (70–80 %) between MsTR-1 and a highly repeated sequence (C300) isolated from M. ssp. caerulea. Three identified repetitive sequences produced hybridization signals at multiple sites in a few of the chromosomes; one repetitive sequence was identified as the E180 satellite DNA previously isolated from M. sativa, while the other 163 and 227 bp fragments had distinct sequences. Physical mapping of the repetitive sequences with double-target FISH revealed different patterns. Thus, nine novel tandemly repetitive sequences that can be adopted as distinct chromosome markers in alfalfa were identified in this study. Furthermore, the chromosome distribution of each sequence was well described. Though significant chromosome variations were detected within and between cultivars, a molecular karyotype of alfalfa was suggested with the chromosome markers we identified. Therefore, these novel chromosome markers will still be a powerful tool for genome composition analysis, phylogenetic studies, and breeding applications.     

Photosynthesis, Water Relations, Temperature and Canopy Structure as Factors Influencing the Growth of Sainfoin (Onobrychis viciifolia Scop.) and Lucerne (Medicago sativa L.)

Measurements of the growth of sainfoin and lucerne were madein the field after cutting on 31 May 1977. Sainfoin reacheda total above-ground dry weight of 408 g m^–2 over thegrowing period of 48 days compared with 598 g m^–2 in lucerne.Final leaf area indices (LAIs) were 2.8 in sainfoin and 6.1in lucerne. The specific leaf areas (SLAs) for sainfoin wereapproximately half those of lucerne throughout the regrowthperiod. The maximum rates of leaf appearance were 0.12 leavesper day in sainfoin and 0.85 leaves per day in lucerne. Themaximum mean rate of plant extension growth for lucerne of 2.12mm h^–1 occurred during the night, whereas, in sainfointhe maximum rate of 1.72 mm h^–1 occurred during the day. Measurements of extinction coefficients for PAR ranged from0.45 to 0.89 in sainfoin and from 0 42 to 0.57 in lucerne. Asthe lucerne crop increased in size leaf water potentials andsolute potentials became more negative. In sainfoin leaf waterpotentials remained remarkably high throughout the growth period,solute potentials decreased and turgor potentials increased.The stomatal conductances of the two species were similar. The photosynthetic capacities and rates of dark respirationper unit leaf area in both species were similar. The rate ofcanopy ‘gross’ photosynthesis at 295 W m^–2was always greater in lucerne than in sainfoin. This was largelya matter of differences between the species in LAI, althoughat higher LAIs the more erect structure of lucerne leads toa better utilization of photosynthetically active radiation. Onobrychis vicifolia Scop, sainfoin, Medicago sativa L., lucerne, photosynthesis, water relations, temperature, canopy structure     

Non-additive gene effects for creeping-root in lucerne (Medicago sativa L.)

Summary Both general and specific combining abilities for creeping-rootedness of lucerne were found to be highly significant although there were substantial differences among genotypes for both parameters. These results indicate that both additive and non-additive gene effects are involved in the genetic substrate of creeping-rootedness; hence utilization of heterosis would seem to be the most appropriate procedure for further improvement in this trait.     

Stable transformation of alfalfa (Medicago sativa L.) by particle bombardment

Summary Stable transformation of alfalfa (Medicago sativa L.) was obtained by particle bombardment of calli (derived from petiole and stem sections cultured for three weeks on SH medium), followed by delayed selection with 50 mg/l kanamycin on BOi2Y medium. Selection at a lower level of kanamycin (25 mg/l) in late stages of culture resulted only in escapes. Analysis of seven transgenic plants revealed that all were derived from one transformation event. Segregation of the neomycin phosphotransferase gene in test cross progeny followed a 11 Mendelian ratio for a single locus insertion in a heterozygous state.     

The response of lucerne (Medicago sativa L.) to sodium sulphate and chloride salinity

Sodium and sulphate-dominated salinity is a serious environmental problem occurring in soils and groundwater in many parts of the world. The effect of Na2SO4 and NaCl, at electrical conductivity levels ranging from 2 to 17 dS m-1, on the growth and tissue ion concentrations of 16 lines of lucerne (Medicago sativa L.) was examined in the greenhouse over a 2 month period. Averaged across all lines, plants grown at 17 dS m-1 produced 66% of the dry matter of plants grown at 2 dS m-1. However there were significant differences among lines in relative salt tolerance (as defined by the slope of the reduction in dry matter) versus electrical conductivity. Dry matter production was negatively correlated with shoot concentrations of Na+, Cl- and S2- and generally lines that were more tolerant to salinity had lower concentrations of those ions in the shoots. We conclude that lucerne is moderately tolerant to Na2SO4 -predominated salinity, and that the degree of intraspecific variation that exists within this species will allow more tolerant lines to be selected for establishment in conditions where sulphate salinity is a problem.Collaborator