Ontogeny and ultrastructure of spontaneous nodules in alfalfa (Medicago sativa)

Summary The development of spontaneous nodules, formed in the absence ofRhizobium and combined nitrogen, on alfalfa (Medicago sativa L. cv. Vernal) was investigated at the light and electron microscopic level and compared to that ofRhizobium-induced normal nodules. Spontaneous nodules were initiated from cortical cell divisions in the inner cortex next to the endodermis, i.e., the site of normal nodule development. These nodules, on uninoculated roots, were white multilobed structures, histologically composed of nodule meristems, cortex, endodermis, central zone and vascular strands. Nodules were devoid of intercellular or intracellular bacteria confirming microbiological tests. Early development of spontaneous nodules was initiated by series of anticlinal followed by periclinal divisions of dedifferentiated cells in the inner cortex of the root. These cells formed the nodular meristem from which the nodule developed. The cells in the nodule meristems divided unequally and differentiated into two distinct cell types, one larger type being filled with numerous membrane-bound starch grains, and the other smaller type with very few starch grains. There were no infection threads or bacteria in the spontaneous nodules at any stage of development. This size differentiation is suggestive of the different cell sizes seen inRhizobium-induced nodules, where the larger cell type harbours the invading bacteria and the smaller type is essential in supportive metabolic roles. The ontogenic studies further support the claim that these structures are nodules rather than aberrant lateral roots, and that plant possess all the genetic information needed to develop a nodule with distinct cell types. Our results suggest that bacteria and therefore theirnod genes are not necessarily involved in the ontogeny and morphogenesis of spontaneous and normal nodules in alfalfa.Abbreviations EH smallest emergent root hair - EM electron microscope - enod2 early nodulin2 gene - RT root tip - RER rough endoplasmic reticulum - YEMG yeast extract-mannitol-gluconate     

Regulation of symbiotic nitrogen fixation in root nodules of alfalfa (Medicago sativa) infected with Rhizobium meliloti

Symbiotic nitrogen fixation of Rhizobium meliloti bacteroids in Medicago sativa root nodules was suppressed by several inorganic nitrogen sources. Amino acids like glutamine, glutamic acid and aspartic acid, which can serve as sole nitrogen sources for the unnodulated plant did not influence nitrogenase activity of effective nodules, even at high concentrations.Ammonia and nitrate suppressed symbiotic nitrogen fixation in vivo only at concentrations much higher than those needed for suppression of nitrogenase activity in free living nitrogen fixing bacteria. The kinetics of suppression were slow compared with that of free living nitrogen fixing bacteria. On the other hand, nitrite, which acts as a direct inhibitor of nitrogenase, suppressed very quickly and at low concentrations. Glutamic acid and glutamine enhanced the effect of ammonia dramatically, while the suppression by nitrate was enhanced only slightly.     

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.     

Colonization behaviour of Pseudomonas fluorescens and Sinorhizobium meliloti in the alfalfa (Medicago sativa) rhizosphere

The colonization ability of Pseudomonas fluorescens F113rif in alfalfa rhizosphere and its interactions with the alfalfa microsymbiont Sinorhizobium meliloti EFB1 has been analyzed. Both strains efficiently colonize the alfalfa rhizosphere in gnotobiotic systems and soil microcosms. Colonization dynamics of F113rif on alfalfa were similar to other plant systems previously studied but it is displaced by S. meliloti EFB1, lowering its population by one order of magnitude in co-inoculation experiments. GFP tagged strains used to study the colonization patterns by both strains indicated that P. fluorescens F113rif did not colonize root hairs while S. meliloti EFB1 extensively colonized this niche. Inoculation of F113rif had a deleterious effect on plants grown in gnotobiotic systems, possibly because of the production of HCN and the high populations reached in these systems. This effect was reversed by co-inoculation. Pseudomonas fluorescens F113 derivatives with biocontrol and bioremediation abilities have been developed in recent years. The results obtained support the possibility of using this bacterium in conjunction with alfalfa for biocontrol or rhizoremediation technologies.     

Responses of nitrogen-fixing and nitrate-supplied alfalfa (Medicago sativa L.) to iron chelates in an alkaline hydroponic medium

Ferric ethylenediamine di-(o-hydroxyphenylacetate) (FeEDDHA) and ferric hydroxyethylethylenediaminetriacetic acid (FeHEDTA) were evaluated as Fe sources for hydroponic growth of alfalfa (Medicago sativa L., cv. Mesilla), either dependent on N₂ fixation or supplied with NO₃. The hydroponic medium was maintained at pH 7.5 by addition of CaCO₃. Nitrogen-fixing cultures were inoculated with Rhizobium meliloti 102 F51 and grown in medium without added nitrogen. After five to seven weeks of growth under greenhouse conditions, plants were harvested. Nitrogen fixation was measured by the acetylene reduction method.When FeEDDHA was supplied, growth of alfalfa, whether dependent on N₂ fixation or supplied with NO₃, was severely limited at concentrations typically used in hydroponic medium (10 or 20 M). Maximum yield of NO₃-supplied alfalfa was obtained at 100 M while maximum yield of N₂-fixing alfalfa was obtained in the range of 33 to 200 M FeEDDHA. Nodule fresh weights and N₂ fixation rates increased with FeEDDHA concentration up to 33 M and remained essentially constant up to 200 M. With FeHEDTA, maximum yields of both NO₃-grown and N₂-fixing alfalfa were obtained at 10 M. Growth of NO₃-supplied plants was inhibited at 200 M FeHEDTA while growth of N₂-fixing plants was inhibited at 100 M FeHEDTA. The numbers of nodules per plant increased between 3.3 and 10 M FeHEDTA; however, inhibition of nodule formation occurred at a concentration of 33 M or higher. Nodule weights per plant and N₂ fixation rates were depressed at 3.3 M as well as at 100 M FeHEDTA. The results suggest that alfalfa dependent on N₂ fixation is more sensitive to limited Fe availability than alfalfa supplied with NO₃.     

Effect of plant genotype on the transformation of cultivated alfalfa (Medicago sativa) by Agrobacterium tumefaciens

The trait for somatic embryogenesis is being introduced sexually into alfalfa (Medicago sativa) breeding populations to facilitate genetic transformation of this crop. Cocultivation experiments were conducted with an agronomically-improved embryogenic clone from one such population as well as with two other embryogenic clones, one of which was the source of the embryogenic trait in the breeding populations. Transgenic plants were produced from the agronomically-improved clone whereas none were produced from the other two clones. Among the 16 transgenic plants analyzed there was a range in both copy number and number of integration sites for the NPT-II gene; those plants regenerated after a prolonged selection phase in vitro generally had the highest numbers in both respects. There was no evidence of sectoral chimerism of the transgene in a subsample of transgenic plants analyzed by PCR.     

Rhizobium meliloti distribution in the soil following alfalfa inoculation

Summary Alfalfa seeds, inoculated with an antibiotic-resistantRhizobium meliloti strain, were planted in three replicated field plots at Clayton, N.C. Core samples were taken three times in the next year at 0, 10, and 20 cm from the edge of each plot. Soil subsamples were taken from within each core sample at 0, 6, 12, and 18 cm depths. The numbers of the inoculum Rhizobium strain in each soil subsample were determined by inoculation of alfalfa plants with diluted soil samples. In general the distribution of rhizobia showed some movement outward and downward in the soil. Lower counts were obtained at the surface during summer. The Rhizobium persistence pattern in the soil differed in the three plots which is consistent with the variability in Rhizobium numbers often observed in established alfalfa stands. Cooperative investigation of the United States Department of Agriculture, Science and Education Administration, Agricultural Research and the North Carolina Agricultural Research Service, Raleigh, North Carolina. Paper No. 6818 of the Journal Series of the North Carolina Agricultural Research Service at Raleigh.     

Genetic transformation of commercial breeding lines of alfalfa (Medicago sativa)

Bio-engineering technologies are now routinely used for the genetic improvement of many agricultural crops. However, breeding lines of Medicago sativa are not easily amenable to genetic transformation and therefore cannot benefit from the molecular tools that have been developed for genetic manipulations. This paper describes a strategy that has been developed to transfer DNA into commercially important breeding lines of winter-hardy alfalfa via Agrobacterium infection. Three highly regenerative genotypes have been selected from ca 1000 genotypes within 11 breeding lines. They have been used as basic material for an extensive genetic transformation trial. Combinations of genotypes (11.9, 8.8, 1.5) expression vectors (pGA482, pGA643, pBibKan) and bacterial strains (C58, A281, LBA4404) were tested for their ability to produce stable transgenic material. Putative transgenic plantlets were further screened by nptII-specific PCR amplification, Southern hybridization and recallusing assays. One genotype (1.5) gave only one transformant out of 432 individual trials. With the two other genotypes, efficiency of transformation (kanamycin-resistant calluses obtained/explant tested) ranged from 0 to 0.92 depending on the strain/vector combination used. Statistical interactions underline the possibility of obtaining good genotype-strain-vector combinations for alfalfa transformation. Predicted transformation probability indicates that with strain LBA4404 containing the vector pGA482 and genotype 11.9, transformation efficiency is above 60% and 10% or more of the calluses retain embryogenic potential. PCR amplification and Southern hybridization of randomly chosen regenerated plantlets demonstrated that all embryos developing on 50 g ml^-1 kanamycin had a stable genomic insertion of nptII. Sexual crosses with untransformed genotypes showed that segregation of the transgenic trait followed Mendelian heredity.     

Hairy root transformation in alfalfa (Medicago sativa L.)

Summary The widely cultivated forage legume alfalfa (Medicago sativa L.) was transformed with the agropine type Agrobacterium rhizogenes NCPPB 1855. Sterile root and callus cultures were derived from tumorous hairy roots which were easily obtained independent of the plant variety or genotype. Plant regeneration, via somatic embryogenesis, was achieved only when a selected alfalfa line, characterized by high regenerative capability, was utilized. Genetic transformation was confirmed by the presence of agropine and T-DNA. Phenotypic alterations, mainly affecting the root system, were observed in transformed plants. The possibility that T-DNA-induced variations could be useful in the improvement of M. sativa is discussed.Research work was partially supported by Progetto Strategico Agrobiotecnologia C.N.R., Italy     

Reducing the tetraploid non-nodulating alfalfa (Medicago sativa) MnNC-1008(NN) germ plasm to the diploid level

MnNC-1008(NN) (referred to as MN-1008) is a tetraploid alfalfa mutant with two recessive genes (nn ₁ and nn ₂ )conditioning the non-nodulating trait. The tetraploid level (2n=4x=32) of this Medicago sativa germ plasm was reduced to the diploid (2n=2x=16) level using the 4x-2x genetic cross originally described as a workable method for the induction of haploidy in alfalfa by T. E. Bingham. In our experiments more than 7000 emasculated flowers of a single non-nodulating MN-1008 mutant alfalfa plant with purple petals were cross-pollinated with pollen from a single, diploid, yellow-flowered alfalfa plant. Mature seeds from these crosses were collected and germinated, after which the plants were subjected to morphological and cytogenetic analyses as well as to DNA fingerprinting. Out of 26 viable progeny, 6 were hybrid plants, 19 proved to be self-mated derivatives of MN-1008, while one descendant turned out to be a diploid (2n=2x=16), purple flowered, non-nodulating plant denoted as M. sativa DN-1008. This diploid, non-nodulating alfalfa plant can serve as starting material to facilitate the comprehensive morphological, physiological and genetic analysis (gene mapping and cloning) of nodulation in order to learn more about the biology of the symbiotic root nodule development. To produce diploid, nodulating hybrid F₁ plants, DN-1008 was crossed with a diploid, yellow-flowered M. sativa ssp. quasifalcata plant. An F₂ population segregating the nn ₁ and nn ₂ genes in a diploid manner, in which the genetic analysis is more simple than in a tetraploid population, can be established by self-mating of the F₁ plants.     

紫花苜蓿内生和非内生根瘤菌多样性及共生差异

【背景】对根瘤菌多样性的研究有助于推进根瘤菌种质资源的利用。【目的】研究紫花苜蓿内生和非内生根瘤菌的表型和遗传多样性,比较菌株在5个苜蓿品种上的共生效应,验证根瘤菌群体共生效应由苜蓿品种决定的假设。【方法】从甘肃省白银会宁旱作区、兰州安宁灌区、武威凉州灌区3个栽培区域的陇中、清水、WL168HQ、甘农3号、甘农9号等紫花苜蓿品种中分离内生(植株种子、花、叶、茎、根表皮、根中柱和根瘤)和非内生(根际土壤和田间土壤)根瘤菌菌株,通过表型数值分类、 16SrRNA基因限制性片段长度多态性(Restrictionfragmentlengthpolymorphism,RFLP)、16S rRNA基因测序、持家基因多位点序列分型(Multilocus sequence typing,MLST),以及结瘤基因nodC和固氮基因nifH片段序列测定,研究紫花苜蓿根瘤菌的表型和遗传多样性,并采用主成分分析研究根瘤菌菌株在5个紫花苜蓿品种上的共生效应差异。【结果】共分离得到43株内生根瘤菌和10株非内生根瘤菌,叶片和花中没有分离到根瘤菌菌株。53株根瘤菌以及对照菌株R.GN5和S.12531表型特征数值分类聚为8个群,菌株表型多样性丰富。经16SrRNA-RFLP分析共形成22种RFLP分型组合,基因型Ⅰ分布最广泛(24),其次为基因型Ⅻ(5)、ⅩⅤ(5)和ⅩⅨ(3),其余16株菌各代表1种基因型,菌株遗传多样性丰富。16S rRNA基因测序和MLST分析将所有菌株划分为Rhizobiumradiobacter、R.rosettiformans和Ensifermeliloti。仅从7株E.meliloti代表菌株和对照菌株S.12531中扩增到nodC和nifH基因,说明E.meliloti菌株均能结瘤固氮。E.meliloti菌株G3L3接种甘农3号,LP3、LL1和LL2接种陇中,QL2接种清水,LL1、LL2和WLP2接种WL169HQ苜蓿均能显著促进植株的单株结瘤数、地上干重和粗蛋白含量。E.meliloti菌株接种甘农3号、甘农9号和清水苜蓿品种后所有参数值在PC1轴上分别聚在-1-1之间,在PC3轴上聚在-1.5-1.5之间;接种陇中和WL168HQ苜蓿的参数值较分散,PC1轴上分散在-1.5-4之间,PC3轴上分散在-3-4之间。【结论】紫花苜蓿内生和非内生根瘤菌菌株多样性丰富,表型和遗传多样性与其来源没有直接关系。菌株G3L3与甘农3号,LP3、LL1、LL2与陇中,QL2与清水、LL1、LL2、WLP2与WL169HQ苜蓿品种共生匹配和适应能力强。在甘农3号、甘农9号和清水紫花苜蓿品种上群体共生效应相似,在陇中和WL168HQ紫花苜蓿上共生效应差异明显。本研究内生和非内生根瘤菌菌株的群体共生效应根据苜蓿品种而定,根瘤菌菌株与苜蓿品种间的信号识别程度存在差异。     

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     

The occurrence of calyx nodules inPsychotria spp. (Rubiaceae)

Summary The occurrence and structure of calyx nodules in the flowers of two leaf nodulated rubiaceous speciesPsychotria punctata Vatke andPsychotria kirkii Hiern. has been described for the first time at the ultrastructural level. Bacteria, resident in colleter-secreted mucilage in the space between calyx and corolla, invade stomatal pores which develop on the calyx protoderm. The bacteria proliferate in the substomatal cavity and then invade the calyx mesophyll. This invasion is most pronounced inP. punctata where the bacteria even penetrate and enter the cells of the vascular tissue. Although no sheath forms around the calyx nodules, the calyx mesophyll cells surrounded by the bacteria become identical in shape, size and secretory function to the invasive mesophyll cells of leaf nodules. The functional and evolutionary significance of calyx nodulation is discussed.     

Nodulation of lucerne (Medicago sativa L.) in an acid soil: Effects of inoculum size and lime-pelleting

The effects of inoculum level and lime-pelleting were studied in an acid soil with respect to the nodulation and growth of lucerne (Medicago sativa cv Resis) and the population dynamics of Rhizobium meliloti. In small root-boxes (rhizotrons), the in-situ survival of inoculated rhizobia was studied in the micro-environment around the seed for a period of 12 days after sowing. During the initial 24 hours, a strong increase in rhizobial numbers was measured, concomitantly with the development of roots. As a result of lime-pelleting, rhizobial numbers were higher only at 3 days after sowing (P<0.05). Later, this difference diminished steadily. Addition of lime did not increase the adhesion of the rhizobia to the seedling tap root. Plant responses to inoculation were studied in pots. To obtain optimal nodulation, the soil had to be neutralized around the seed with lime and at least 10⁵ cells of R. meliloti were required. With more than 10⁵ rhizobia per seed, lime-pelleting increased the number of crown-nodulated seedlings from 24% to 77%. Higher numbers of rhizobia could not compensate the effect of lime. A strong correlation was found between crown nodulation, nitrogen content and dry weight of the shoots.     

Paternal inheritance of plastids in Medicago sativa

Summary Plastids are plant cellular organelles that are generally inherited from the maternal parent in the angiosperms. Many species exhibit biparental inheritance of plastids, but usually with a predominantly maternal influence. In contrast to this, we report strong paternal inheritance of plastids in reciprocal crosses of alfalfa, Medicago sativa, by following restriction fragment length polymorphisms for plastid DNA in two normal green plastids. Mitochondrial inheritance remained exclusively maternal.     

Nitrogen fixation of lucerne (Medicago sativa) in an acid soil: The use of rhizotrons as a model system to simulate field conditions

The effect of pelleting seeds of lucerne with lime was studied in an acid sandy soil. In pot experiments, the fraction of seedlings with crown nodules, i.e. nodules on the upper 10 mm of the taproot, increased from 26% to 71%. In rhizotrons, the application of CaCO₃ resulted in an even stronger response.An agar-contact method was used to study pH changes in the rhizosphere during a period up to 12 days. Application of 1.0 µmol of CaCO₃, in drops of 12 µL volume, resulted in an initial soil pH of 6.1 and yielded 75% crown nodulation. In the absence of CaCO₃, roots induced a pH increase from 5.1 (day 0) to 5.7 (day 12). However, this did not increase nodulation (5%). Obviously, this type of alkalinization does not overcome the acid-sensitive step of the nodulation process.     

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.     

Dissolution of ferric phosphate by alfalfa (Medicago sativa L.) root exudates

Alfalfa (Medicago sativa L.) was grown in hydroponic culture to investigate adaptation to Fe-deficiency. Root exudates released into the nutrient solution from Fe-deficient plants were trapped and condensed on an amberlite XAD-4 resin column. The diethyl ether fraction of these exudates dissolved ferric phosphate remarkably. The dissolving capability was about 62 times higher than that of root exudates obtained from Fe-sufficient plants in complete nutrient solution. The Fe-dissolving compound was separated and identified. It was a new natural compound with molecular formula C₁₄H₁₀O₅ and was identified as 2-(3,5-dihydroxyphenyl)-5,6-dihydroxybenzofuran by means of mass spectrometry and ¹H-nuclear magnetic resonance. This new compound worked as a phytoalexin and inhibited completely the fungal growth of Fusarium oxysporum f. sp. phaseoli.