Direct shoot formation in spontaneously occurring root pseudonodules of alfalfa (Medicago sativa L.)

Summary A simple and rapid procedure for direct organogenesis from root nodulelike structures of alfalfa (Medicago sativa L.) line SGg, spontaneously induced on growth regulator-free Gamborg (B₅) medium, was developed. Prolific adventitious shoot initiation was obtained using a combination of 1.0 mg/liter TIBA and 0.5 mg/liter 2iP. Transfer of shoots to a medium containing 0.5 mg/liter ABA and reduced concentration of TIBA (0.5 mg/liter) before rooting markedly stimulated shoot development. Regenerated shoots rooted easily and revealed the early appearance of nodulelike structures on basal medium (B₅) lacking growth regulators. Analysis of endogenous growth regulator levels of SGg roots maintained on growth regulators free media, showed that spontaneous shoot appearances was correlated with high cytokinin-to-auxin ratios.     

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     

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.     

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.     

Arylsulphatase activity of corn (Zea mays L.) seedling roots

The crude lysosomal fraction of corn seedling root tips contains an arylsulphatase (E.C. 3.1.6.1) which hydrolysed p-nitrophenyl sulphate at pH 8.0 but had no activity towards p-nitrocatechol sulphate. The Km value for p-nitrophenyl sulphate was 1.24 mM. The hydrolysis of p-nitrophenyl sulphate was linear up to 2 h and the rate was proportional to the amount of enzyme added. The enzyme was strongly inhibited by cyanide, fluoride and phosphate ions and did not resemble the arylsulphatases of bacterial and animal origin.     

Biochemical and molecular characterization of corn (Zea mays L.) root elongases

Root surfaces are protected against the soil environment by the deposition of lignin and suberin. In order to obtain more insight into the regulation of root suberin biosynthesis, elongases from primary roots of corn (Zea mays L.) seedlings were characterized. Elongase activities (acyl-CoA and ATP-dependent) were located in the microsomal fraction of the root cells. C(20), C(22) and C(24) fatty acids were detected as primary products of elongases. Preferred substrates of the acyl-CoA elongases were C(18:0)-CoA and C(20:0)-CoA. Applying a molecular approach, using PCR and degenerate primers derived from the sequences of known leaf and seed 3-ketoacyl-CoA synthases (KCSs), catalysing the first step of very-long-chain fatty acid synthesis, the cDNA of a putative root KCS was obtained showing high homology to known leaf and seed KCSs at the DNA and amino acid levels. Thus, our approach provides the first direct evidence for the presence and the activity of root elongases in Z. mays. Ongoing research is focusing on the molecular analysis and the regulation of KCS expression in roots in reaction to different environmental stimuli.     

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.     

Effect of nodules on dehydration response in alfalfa (Medicago sativa L.)

Drought is a key abiotic stress that negatively affects growth and development as well as symbiotic nitrogen fixation in alfalfa (Medicago sativa L.). To understand whether nodulation would affect drought stress response in alfalfa, we analyzed the lipid peroxidation, activities of antioxidant enzymes including superoxide dismutase (SOD), and catalase (CAT), contents of superoxide anion radical, non-enzymatic antioxidants including reduced glutathione (GSH) and proline, total protein, and soluble sugar in dehydration-stressed alfalfa. Three-month-old alfalfa plants without nodule, with active nodules, or with inactive nodules were dehydrated for 0, 1, 2, 4, 6, 8, and 10 h. We found that roots and leaves from plants with nodules, especially with active nodules, showed less lipid peroxidation which was associated with higher CAT activities and higher levels of GSH. Roots and leaves with active nodules also accumulated less free proline and soluble sugar compared to plants without nodules, suggesting that proline and soluble sugar may have a limited role in osmotic adjustment in these plants. The results suggested that active nodules may have a positive effect on drought stress tolerance in alfalfa.     

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 effect of soil aggregate size on early shoot and root growth of maize (Zea mays L.)

Differences in plant growth arising from differences in aggregate size in the seedbed are normally atributed to limitations in nutrient or water supply during the early growth period. This study was initiated to determine if these were the only mechanisms by which aggregate size influences plant response. Four different aggregate size fractions (less than 1.6 mm, 1.6 to 3.2 mm, 3.2 to 6.4 mm and 6.4 to 12.8 mm diameter) were sieved from a silt loam soil. Nutrients were added to the soil and maize was grown in the aggregates for eighteen days after seedling emergence. Soil matric potential was maintained between — 3 and −20 kPa. Shoot dry weight declined by 18% as aggregate size increased from less than 1.6 mm to 1.6–3.2 mm. There was little further decline as aggregate size increased to 6.4–12.8 mm. Final leaf area showed a similar decline. The availability of nutrients or water were not limiting. Total root length in the coarsest aggregate system was less than 60% of that in the finest system. Main axes of seminal and nodal roots were longer in the coarser aggregate systems, the length of primary laterals was not affected, and length of secondary laterals was lower in the coarser systems. A greater proportion of the roots penetrated the larger aggregates than the smaller aggregates; however, the larger aggregates offered greater resistance to penetration by a rigid micropenetrometer (150 μ diameter probe). Diameter of the main axes roots were greatest in the largest two aggregate fractions. it is speculated that a combination of increased endogenous ethylene in roots in the finest aggregate system due to entrapment by water and increased mechanical resistance in the coarsest aggregate system accounts for the observed effects on root norphology.     

In-vitro morphogenesis of corn (Zea mays L.)

The objective of this research was to study the in-vitro morphogenetic pattern of corn (Zea mays L.) shoot tips excised from aseptically-grown seedlings, and of expiants of axillary shoot buds, immature tassels and ears (staminate and pistillate inflorescences) obtained from greenhouse-grown corn plants. The seedling shoot tips and immature ears first regenerated clumps of multiple shoots within four weeks of culture on Murashige and Skoog (MS) basal medium supplemented with 500 mg/L casein hydrolysate (CH) and 9.0 M N⁶-benzyladenine (BA). Multiple shoot clumps were also differentiated from spikelets of immature tassels cultured on MS medium containing 500 mg/L CH, 4.5 M BA and 0.45 M 2,4-dichlorophenoxy acetic acid (2,4-D). All these multiple shoot clumps in turn differentiated clusters of ears after further four subcultures at four-week intervals under light on MS medium supplemented with 500 mg/L CH and 2.25, 4.5, 9.0 or 18 M BA. Axillary shoot buds readily differentiated clusters of ears within four weeks of the initial culture on these media. Secondary and tertiary ear clusters were initiated following subculture of primary ears on MS medium containing 500 mg/L CH and 4.5 or 9.0 M BA. Most of the ear primordia developed into ears with well-developed ovaries and styles on subculture on MS medium containing 500 mg/L CH and 1.0 M BA. Corn kernels were obtained after pollination of in-vitro-formed ears with pollens collected from greenhouse-grown corn. These kernels germinated in vitro and developed into mature corn plants in the greenhouse. Clusters of tassels were also differentiated in darkness from the multiple shoot clumps after six months successive subcultures but the spikelet primordia of tassels failed to develop fully under the in-vitro conditions tested. Somatic embryos arose directly from spikelet primordia of young tassels or ears on MS medium containing 500 mg/L CH and 4.5 M 2,4-D, or indirectly from calli derived from spikelets of young tassels and ears on MS medium containing 500 mg/L CH and 9.0 M 2.4-D.Abbreviations BA N⁶-benzyladenine - CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (basal medium) Heng Zhong is a Rockefeller Foundation Fellow on leave from the Institute of Botany, Academia Sinica, Beijing, P.R. China. This work was supported by a grant from the MidWest Plant Biotechnology Consortium and U.S.-A.I.D. grant No. DAN-4197-A-00-1126-00 to M.B. Sticklen. Thanks are due to Illinois Foundation Seeds, Champaign, USA for the supply of Honey N Pearl sweetcorn seeds and the Services of Center for Electron Optics, Michigan State University, for the electromicroscopic work as related to this publication.     

Biochemical characterization of elongase activity in corn (Zea mays L.) roots

Chemical analysis of 4-day-old corn (Zea mays L.) root cell walls revealed that the lipophilic biopolymer suberin forms an important constituent of rhizodermal and hypodermal cell walls. Identified aliphatic monomers had chain lengths ranging from C16 to C26 and they belonged to 5 substance classes (omega-hydroxycarboxylic acids, 1,omega-dicarboxylic acids, 2-hydroxycarboxylic acids, carboxylic acids and alcohols) by which suberin is characterized. Biochemical experiments proved the occurrence of elongase activities in corn roots. Highest enzymatic activities were found in corn root microsomes, and major products synthesized by root elongases were elongated fatty acids with chain lengths ranging from C20 to C24. Preferred substrates of root elongases were acyl-CoAs of the chain length C18 and C20, whereas monounsaturated acyl-CoAs (C16:1 and C18:1) and acyl-CoAs of lower (C12-C16) and higher chain lengths (C22-C24) were rarely elongated. Elongase activities significantly decreased over the length (40 cm) of 10-day-old corn roots going from the young tip to the older base of the root. Thus, results presented here show the presence and activity of elongases in roots of plants.     

Biological activity and chemical isolation of root saponins of six cultivars of alfalfa (Medicago sativa L.)

Wheat, cheat and Trichoderma viride bioassays were used to establish the relationship between the content of biologically active saponins in the roots and the degree of winter dormancy and/or time of six cultivars of alfalfa over the period January to August, 1988. Wheat and cheat bioassay results indicated no significant difforences among cultivars, whereas T. viride was inhibited most by extracts of roots collected during months with high rainfall and rapid growth. Cheast seedling roots were inhibited 8–10% more than those of wheat seedling roots indicating that alfalfa root saponins were more effective as allelopathic compounds in preventing growth of cheat than that of wheat alone. An average of 14 different saponins per cultivar were separated by thin-layer chromatograms. Saponins, and the aglycones produced by acid hydrolysis of the May samples, were separated by thin-layer chromatography. The conclusion is that the amount, structure, and type of saponins present in alfalfa roots vary with time.     

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.     

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.     

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.     

Effect of different levels of humic acids on nutrient content and growth of corn (Zea mays L.)

Summary The effect of humic acids on nutrient uptake and growth of corn plants (Zea mays L.) was investigated by growing corn in plastic growth pouches containing a Hoagland nutrient solution to which were added 0, 320, 640, 1,600 or 3,200 ppm HA, pH 7.0. The experiments were carried out in three replicates for a growing period of 16 days after germination. Humic acid was in general beneficial to shoot and root growth of corn plants. Dry matter yield in corn shoots was stimulated by HA, especially by treatments with 640 ppm HA. Nutrient uptake showed a number of differences as a result of treatments with HA. Moderate applications with HA resulted in a significant increase in N content of corn shoots, while large amounts of HA had a tendency to reduce the N concentration in corn shoots. As a result of the HA treatments, P concentrations in corn shoots were decreased, but differences in K contents were statistically nonsignificant and Mn contents in shoots were also nonsignificantly different among the treatments. However, Zn content showed a tendency to increase with increasing applications of HA.Contribution of the University of Georgia, College Agri. Exp. Stn., College Stn., Athens, GA 30602. Current address of junior author: c/o Soil and Fertilizer Section, Field Crop Division, Department of Agriculture, Bangkok, Thailand.     

A re-examination of corn (Zea mays L.) ear volatiles

Helicoverpa zea (Boddie) is a major insect pest of corn and other agricultural crops. An improved understanding of semiochemcials that control adult behavior is needed to develop alternative control measures. In this study, overnight SPME collection of volatiles from corn ears enclosed in Teflon bags in the field at two stages of development were made. C₈–C₁₀ aldehydes, a C₈-alcohol, C₆–C₉ alcohol acetates, and numerous monoterpenes, sesquiterpenes, sequiterpene alcohols, and geosmin were identified after thermal desorption and GC/MS. Structural assignments of the alcohol acetates, Z-3-hexenyl acetate, 2-heptyl acetate, 2-nonyl acetate, and 4-nonyl acetate, the monoterpenes, α- and β-ocimene, and geosmin were made by analysis of standards that were purchased or prepared in the laboratory. All other assignments were based on published Kovat’s retention time indices (KI) and mass spectra. Pair-wise comparison of the relative amounts of each component between two groups of corn ears defined by silk weight did not identify significant differences, thus it is unknown whether or not silk weight impacted volatile emission composition and rate. To our knowledge three compounds detected in SPME collections, 2-heptyl acetate, 2-nonyl acetate, and 4-nonyl acetate have not been previously reported in corn ear or silk volatiles. Their impact on the flight response of gravid earworm females was evaluated in a flight chamber. No significant response to the individual compounds or a blend of all three was observed. Thus, their impact on moth behavior remains uncertain.