Root and Nodule Enzymes of Ammonia Assimilation in Two Plant-Conditioned Symbiotically Ineffective Genotypes of Alfalfa (Medicago sativa L.)

Biochemical and physiological parameters associated with nitrogen metabolism were measured in nodules and roots of glasshouse-grown clones of two symbiotically ineffective alfalfa (Medicago sativa L.) genotypes supplied with either NO₃⁻ or NH₄⁺. Significant differences were observed between genotypes for nodule soluble protein concentrations and glutamine synthetase (GS) and glutamate synthase (GOGAT) specific activities, both in untreated controls and in response to applied N. Nodule soluble protein of both genotypes declined in response to applied N, while nodule GS, GOGAT, and glutamate dehydrogenase (GDH) specific activities either decreased or remained relatively constant. In contrast, no genotype differences were observed in roots for soluble protein concentrations and GS, GOGAT, and GDH specific activities, either in untreated controls or in response to applied N. Root soluble protein levels and GS and GOGAT specific activities of N-treated plants increased 2- to 4-fold within 4 days and then decreased between days 13 and 24. Root GDH specific activity of NH₄⁺-treated plants increased steadily throughout the experiment and was 50 times greater than root GS or GOGAT specific activities by day 24.     

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.     

Nitrogen Fixation, Nodule Development, and Vegetative Regrowth of Alfalfa (Medicago sativa L.) following Harvest

Nitrogenase-dependent acetylene reduction, nodule function, and nodule regrowth were studied during vegetative regrowth of harvested (detopped) alfalfa (Medicago sativa L.) seedlings grown in the glasshouse. Compared with controls, harvesting caused an 88% decline in acetylene reduction capacity of detached root systems within 24 hours. Acetylene reduction in harvested plants remained low for 13 days, then increased to a level comparable to the controls by day 18.     

Alfalfa (Medicago sativa L.) clones tolerant to salt stress: in vitro selection

In order to quickly and efficiently evaluate the salt tolerance of alfalfa, salinity tests were conducted on Medicago sativa L. var. australis, var. icon, var. loi, and var. gea, under in vitro conditions. Pregerminated seeds of four varieties were subjected to five different NaCl concentrations (0, 50, 100, 150, 200 mM). The influence of saline stress was estimated on the basis of survival percentage, growth parameters, and electrolyte leakage. The seedlings surviving on the medium enriched with salt at the highest concentration were presumed to be tolerant and represented the mother plants for the production of in vitro clones. In the following step, the clones were evaluated in vitro to confirm the salt tolerance. The influence of mild salt stress (75 mM NaCl) on the growth parameters of selected clones was examined. At the end of this trial, the proline accumulation and sodium content in alfalfa shoots were also quantified. The results suggest an increased level of proline promotes salt tolerance. Medicago sativa L. var. icon is highly tolerant in comparison with the other varieties tested. In vitro selection of M. sativa L. varieties on salt-containing media allowed us to obtain clones with increased salinity tolerance.     

大孔吸附树脂对苜蓿皂苷的吸附和解吸附作用(英文)

采用5种不同极性的树脂(AB-8、S-8、NKA-9、D-101和X-5)来评价对苜蓿皂苷的吸附和解吸附作用,其中中等极性的AB-8树脂对苜蓿皂苷具有最大的吸附量,用55%~65%的乙醇溶液能有效地将吸附的皂苷洗脱下来。当苜蓿粗提物量和AB-8树脂量为1∶1时,树脂的吸附量达到饱和。采用AB-8树脂,用90%乙醇洗脱,苜蓿提取物的最大解吸附量为108.4 mg/g干重树脂。通过大孔树脂吸附和解吸附,将90%乙醇洗脱液浓缩,皂苷含量(53%)是苜蓿粗提物含量(5.68%)的9倍。结果表明,AB-8大孔吸附树脂可用于苜蓿皂苷的大规模制备。     

Occurrence of Transgenic Feral Alfalfa (Medicago sativa subsp. sativa L.) in Alfalfa Seed Production Areas in the United States

The potential environmental risks of transgene exposure are not clear for alfalfa (Medicago sativa subsp. sativa), a perennial crop that is cross-pollinated by insects. We gathered data on feral alfalfa in major alfalfa seed-production areas in the western United States to (1) evaluate evidence that feral transgenic plants spread transgenes and (2) determine environmental and agricultural production factors influencing the location of feral alfalfa, especially transgenic plants. Road verges in Fresno, California; Canyon, Idaho; and Walla Walla, Washington were surveyed in 2011 and 2012 for feral plants, and samples were tested for the CP4 EPSPS protein that conveys resistance to glyphosate. Of 4580 sites surveyed, feral plants were observed at 404 sites. Twenty-seven percent of these sites had transgenic plants. The frequency of sites having transgenic feral plants varied among our study areas. Transgenic plants were found in 32.7%, 21.4.7% and 8.3% of feral plant sites in Fresno, Canyon and Walla Walla, respectively. Spatial analysis suggested that feral populations started independently and tended to cluster in seed and hay production areas, places where seed tended to drop. Significant but low spatial auto correlation suggested that in some instances, plants colonized nearby locations. Neighboring feral plants were frequently within pollinator foraging range; however, further research is needed to confirm transgene flow. Locations of feral plant clusters were not well predicted by environmental and production variables. However, the likelihood of seed spillage during production and transport had predictive value in explaining the occurrence of transgenic feral populations. Our study confirms that genetically engineered alfalfa has dispersed into the environment, and suggests that minimizing seed spillage and eradicating feral alfalfa along road sides would be effective strategies to minimize transgene dispersal.     

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。     

Stress Responses in Alfalfa (Medicago sativa L.): I. Induction of Phenylpropanoid Biosynthesis and Hydrolytic Enzymes in Elicitor-Treated Cell Suspension Cultures

Alfalfa (Medicago sativa L.) cell suspension cultures accumulated high concentrations of the pterocarpan phytoalexin medicarpin, reaching a maximum within 24 hours after exposure to an elicitor preparation from cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum. This was preceded by increases in the extractable activities of the isoflavonoid biosynthetic enzymes l-phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, 4-coumarate coenzyme A-ligase, chalcone synthase, chalcone isomerase, and isoflavone O-methyltransferase. Pectic polysaccharides were weak elicitors of phenylalanine ammonia-lyase activity but did not induce medicarpin accumulation, whereas reduced glutathione was totally inactive as an elicitor in this system. The fungal cell wall extract was a weak elicitor of the lignin biosynthetic enzymes, caffeic acid O-methyltransferase and coniferyl alcohol dehydrogenase, but did not induce appreciable increases in the activities of the hydrolytic enzymes chitinase and 1,3-β-d-glucanase. The results are discussed in relation to the activation of isoflavonoid biosynthesis in other legumes and the development of the alfalfa cell culture system as a model for studying the enzymology and molecular biology of plant defense expression.     

Photoperiodic Effects on the Emanation of Volatiles from Alfalfa (Medicago sativa L.) Florets

Alfalfa (Medicago sativa L.) plants acclimated to photoperiods of 18 hours light, 6 hour dark in plant growth chambers exhibited a daily cyclic pattern of floret volatile emanation with a maximum emanation of about 6.5 nanograms of hydrocarbons/floret·30 minutes. This maximum was reached about 6 to 8 hours into the light period. After 8 hours of light, emanation of volatiles decreased rapidly to less than 0.1 ng/floret·30 min even though light and temperature remained constant. Under continuous illumination, only a small increase of volatile emanation occurred during the following 24 hours. It appeared that a dark period was necessary to promote floret volatile emanation. Floret volatile emanation was drastically affected for at least 7 days following a photoperiod change. A photoperiod change caused 6-fold concentration oscillations every 2 hours. The results are interpreted on the basis of a very active floral metabolism controlled by photoperiodically induced rhythms.     

Characterization of a Small Sulphur-Rich Storage Albumin in Seeds of Alfalfa (Medicago sativa L.)

A 2S albumin fraction was characterized in seeds of alfalfa{^{Medicago sativa} L.). This low molecular weight (LMW) familyof disulphide-bonded proteins represents a major nitrogen andsulphur storage reserve for the alfalfa seed Characteristicof seed storage proteins, the 2S albumins are abundant in nitrogen-richglutarrune/glutamate/asparagine/aspartate (32%) In addition,this LMW fraction is high in cysteine (9%) and methionine (4%),amino acids which are under-represented in legume seed globulins.These 2S proteins start to accumulate during the early cotyledonstage of development, and are mobilized following germinationPulse-chase labelling experiments show that the 2S proteinsare synthesized as 'preproproteins', similar to 2S proteinsin other seeds. However, alfalfa 2S albumins are immunologicallyunrelated to these proteins. Key words: Seed development, sulphur-containing 2S storage protein, alfalfa (Medicago sativa)     

紫花苜蓿F-box蛋白基因MsFTL的克隆及功能分析

FTL(F-box Triple LRR protein)是F-box蛋白家族的成员,具有F-box保守结构域,在植物抵御逆境胁迫过程中起重要作用。本研究参考低温胁迫下紫花苜蓿转录组数据设计引物,通过RT-PCR克隆获得紫花苜蓿MsFTL基因,该基因的全长1422 bp,编码473个氨基酸。该蛋白含有1个F-box结构域及3个LRR重复。系统进化分析表明,MsFTL与蒺藜苜蓿XP_003626345.1 F-box/FBD/LRR-repeat protein亲缘关系最近。两者蛋白序列比对发现共有11个差异位点。在低温、盐、干旱以及外源ABA处理下,MsFTL基因受到诱导,表达量上调。构建植物过表达载体pCBM-MsFTL,通过农杆菌介导法转化烟草。对经过抗性筛选、PCR和Real-time PCR验证的转基因植株进行低温抗性鉴定。在-4℃低温胁迫下,野生型烟草叶片出现了明显的萎蔫失水现象,而转基因烟草萎蔫程度相对较轻。生理检测结果表明,4℃处理24 h之后,转基因烟草的可溶性蛋白含量、可溶性糖含量、SOD活性,CAT活性高于野生型,MDA含量低于野生型。本研究表明,MsFTL基因在提高植物对低温胁迫的抗性方面具有重要的作用。     

Isolation and Immunochemical Characterization of Plant Glutamine Synthetase in Alfalfa (Medicago sativa L.) Nodules

Host plant glutamine synthetase (GS) has been purified 100-fold from N₂-fixing alfalfa (Medicago sativa L.) nodules by a new procedure involving preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a final step. An SDS-polypeptide fraction corresponding to plant GS was identified and consisted of two major polypeptides of 40,000 to 45,000 molecular weight. Antibodies to the SDS-polypeptide fraction were raised in mice by intraperitoneal injection, and antisera were collected as ascitic fluid. Crude extracts of soluble protein from the plant fraction of nodules were resolved by SDS-PAGE and then subjected to electrophoresis in the second dimension into antibody-containing agarose gel. A single immunochemically active protein species was observed using this crossed immunoelectrophoresis method, even though both major GS SDS-polypeptides were apparently resolved in the first (SDS-PAGE) dimension. Plant GS protein in crude nodule extracts was quantitated immunochemically by comparison with immunoprecipitin arcs of similarly treated amounts of pure antigen. Using this technique, it was determined that plant GS was present at 150 micrograms per gram fresh weight or 1.2% of total plant soluble protein in N₂-fixing alfalfa nodules.

Results suggest that alfalfa nodule plant GS consists of two major subunit polypeptides, but only a single immunochemically active native protein was observed. The crossed immunoelectrophoresis procedure described here should be generally applicable for immunochemical detection of lower abundance components of crude plant extracts.

     

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.     

Alfalfa (Medicago sativa L.) seed yield in relation to phosphorus fertilization and honeybee pollination

This investigation was conducted at the Agricultural and Veterinary Training and Research Station, King Faisal University, Al-Ahsa, Saudi Arabia, during the alfalfa growing season in 2014. The study aimed to evaluate the impact of phosphorus fertilization and honeybee pollination on alfalfa seed production. The experiment was divided into 9 treatments of open pollination, honeybee pollination, and non-pollination with three different levels (0, 300 or 600 kg P₂O₅/ha/year) of triple super phosphate. All vegetative growth attributes of Hassawi alfalfa were significantly higher in the non-insect pollination plots, while the yield and yield component traits were significantly higher with either open pollination or honeybee pollination in parallel with the increasing level of phosphorus fertilizer up to 600 kg P₂O₅/ha/year in light salt-affected loamy sand soils. There was no seed yield in Hassawi alfalfa without insect pollination. Therefore, placing honeybee colonies near the fields of Hassawi alfalfa and adding 600 kg P₂O₅/ha/year can increase seed production.     

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     

A Comparison of Screening Techniques for Resistance to Verticillium Wilt in Alfalfa (Medicago sativa L.)

Three screening tests for resistance to Verticillium wilt in alfalfa using stem cuttings were compared including: stem infusion with a fungal culture filtrate (SIF), leaf injection with a spore inoculum (LII) and stem infusion with a spore inoculum (SII). The disease severity indice (DSI) determined by the three tests were very similar on average and were significantly correlated for a population of 142 plants regenerated from tissue culture. The DSIs determined by the LII test were also significantly correlated with the DSIs determined by the North American Standard test in a population of 20 Vertus (c.v.) plants. The results suggest that the assays using stem cuttings are effective for determining resistance to Verticillium wilt in alfalfa. Because they are nondestructive and quicker than the North American Standard test, these methods should find application in alfalfa breeding.     

Effects of Cold Hardening on the Regulation of Polyamine Levels in Wheat (Triticum aestivum L.) and Alfalfa (Medicago sativa L.)

When leaves of wheat (Triticum aestivum L.) are exposed to a cold hardening temperature, a major accumulation of putrescine (6-9 times) takes place. Spermidine accumulates to a lesser extent and, conversely, spermine decreases slightly. These variations are completely reversible when plants are returned to initial growing conditions. A similar response is obtained with crowns. During cold hardening, arginine decarboxylase activity remains near its initial level while a considerable loss of activity is observed in control plants. Ornithine decarboxylase and diamine oxidase activity levels are not substantially modified by the treatment. Alfalfa (Medicago sativa L.) also accumulates putrescine under low temperature stress, indicating that this phenomenon is not typical of cereals. The physiological significance of this accumulation of putrescine is still unexplained but the results obtained suggest the involvement of polyamines in the biochemical processes of cold hardening.     

Freezing Tolerance and Alteration of Translatable mRNAs in Alfalfa (Medicago sativa L.) Hardened at Subzero Temperatures

We analyzed changes in populations of translatable mRNAs occurringin crowns of the cold-tolerant alfalfa (Medicago sativa L.)cv. Apica (CT) and the cold-sensitive cv. CUF-101 (CS) aftertheir acclimation at low nonfreezing temperatures and at subzerotemperatures. Both cultivars showed very similar translationprofiles under all treatments. Low temperatures induced significantchanges in the populations of translatable mRNAs. We observeda relationship between the accumulation of cold-regulated (COR)translation products and freezing tolerance within cultivars.Moreover, at least three COR translation products were specificto the CT and might be related to hardiness potential in alfalfa.Whereas extension of the cold acclimation period at 2C reducedcold tolerance, incubation at subzero temperatures increasedor maintained freezing tolerance. This increased hardiness wasassociated with enhanced translation of COR polypeptides andalso with the appearance of new translatable mRNAs. This is,to our knowledge, the first report of altered gene expressionin plants incubated at subzero temperatures. Marked changesin populations of translatable mRNAs at temperatures below freezingmight be related to previous reports that alfalfa achieves maximumhardiness under snow cover when the soil has frozen. Translationin the presence of [³H]glycine showed that a large proportionof the COR genes encode for glycine-rich proteins (GRPs) andthat some of the GRPs are specific to the CT. (Received May 29, 1992; Accepted October 13, 1992)     

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.