Synchronization of protoplasts from Glycine max (L.) Merr. and Brassica napus (L.)

Cells of Glycine max originating in a suspension culture and cells of Brassica napus prepared from hypocotyls were synchronized. Synchronization was achieved by preparing protoplasts in the usual way and subsequently letting the protoplasts regenerate into cells by removing the cell-wall-digesting enzymes. More than 70% of the cells had divided synchronously at the end of the first cycle as determined by the mitotic index. The high frequency of mitosis critically depended on the osmolality of the medium. The duration of the S-phase was estimated by measuring the activity of thymidylate kinase as well as incorporation of [³H]deoxythymidine into acid-insoluble material. The data indicate that synchronization is induced by resetting the cell cycle.Abbreviations dTMP deoxythymidine 5-monophosphate - TCA trichloroacetic acid     

Reproductive capacity of bacteroids in nodules of Trifolium repens L. and Glycine max (L.) Merr.

Reproductive growth of intracellular bacteria from isolated protoplasts in nodules of clover and soybean was directly investigated using a microchamber with visual and video recording. Differentiated bacteriods from clover nodules uniformly failed to reproduce. Such growth as occurred came from undifferentiated rhizobia from within the protoplast or extracellularly in the nodule. Plating investigation gave results in agreement with this conclusion. Osmoprotective media failed to secure the reproduction of differentiated clover bacteroids. Reproductive growth of bacteroids from protoplasts and crushed nodules of soybean was regularly observed in the microchamber and determined as proportionate colony-forming ability (CFA) on laboratory media. The CFA markedly increased with age of nodule and with the addition of nodule or root extract. The promoting effect of such extracts was reduced after heating for 60 min at 100°C, and lost completely after 20 min at 121°C. High osmolarity in the suspending and culture media was detrimental to bacteroid recovery.Abbreviations BMM Bergersen's modified medium - B^+m BMM with additional mannitol - CDB Chlamydomonas dilution buffer - PDB protoplast dilution buffer - PDB PDB without mannitol or sorbitol - RMM Rhizobium minimal medium - R^+m RMM with mannitol instead of sucrose - YMA, YMB yeast mannitol agar and broth, respectively. For details, see Materials and methods     

Intracellular redistribution of phytochrome in etiolated soybean (Glycine max L.) seedlings

The intracellular distribution of phytochrome in hypocotyl hooks of etiolated soybean (Glycine max L.) has been examined by immunofluorescence using a newly produced monoclonal antibody (Soy-1) directed to phytochrome purified from etiolated soybean shoots. Cortical cells in the hook region exhibit the strongest phytochrome-associated fluorescence, which is diffusely distributed throughout the cytosol in unirradiated, etiolated seedlings. A redistribution of immunocytochemically detectable hytochrome to discrete areas (sequestering) following irradiation with red light requires a few minutes at room temperature in soybean, whereas this redistribution is reversed rapidly following irradiation with far-red light. In contrast, sequestering in oat (Avena sativa L.) occurs within a few seconds (D. McCurdy and L. Pratt, 1986, Planta 167, 330–336) while its reversal by far-red light requires hours (J. M. Mackenzie Jr. et al., 1975, Proc. Natl. Acad. Sci. USA 72, 799–803). The time courses, however, of red-light-enhanced phytochrome pelletability and sequestering are similar for soybean as they are for oat. Thus, while these observations made with a dicotyledon are consistent with the previous conclusion derived from work with oat, namely that sequestering and enhanced pelletability are different manifestations of the same intracellular event, they are inconsistent with the hypothesis that either is a primary step in the mode of action of phytochrome.Abbreviations DIC differential interference contrast - FR far-red light - Ig immunoglobulin - Pfr, P far-red- and red-absorbing form of phytochrome, respectively - R red light This work was supported by National Science Foundation grant No. DCB-8703057.     

Responses of photosynthesis to irradiance and temperature in soybean,Glycine max (L.) Merr

The response of photosynthesis to irradiance and temperature during growth was investigated in two soybean genotypes. Soybean is a species that can modify its structure and metabolism so as to adapt to differing light conditions; its responses to rapid changes in irradiance are characterized by their flexibility. However, the temperature during growth can change the response to irradiance: moreover, there may be a marked interaction with genotype.The response of photosynthesis to irradiance consists of changes in leaf thickness, which bring about variations in the mesophyll resistance to CO₂ transfer. The increase in net photosynthesis per unit of leaf area is due to the increase in the amount of assimilating material beneath unit of area, as corroborated by the stability of the net photosynthesis per unit volume. Moreover, the response of photosynthesis to temperature is due to the mesophyll diffusion constant which decreases with the growth temperature.     

Trifluralin-induced chlorosis in soybeans (Glycine max (L.) Merr.) grown on clayey,high pH soils

Summary The cause of leaf chlorosis, frequently observed on soybeans (Glycine max (L.) Merr.) grown on high pH soils of the Mississippi Blackland Prairie, is thought to be low Fe availability and restricted rooting. Three greenhouse experiments were conducted using two soils, Sumter, a Rendollic Eutrocrept and Okolona, a Typic Chromudert; nine soybean cultivars differing in Feefficiency; and trifluralin (α-α-α-trifluoro-2,6-dinitro-N, N-di-propyl-p-toludine). Trifluralin at rates greater than 0.56 kg/ha caused chlorosis which was more severe on the Sumter, a soil low in available Fe. Fe-efficient cultivars were more resistant to the chlorosis induced by trifluralin than the Fe-inefficient cultivars. It was concluded that the chlorosis is an Fe deficiency caused by reduced uptake. The herbicide-induced chlorosis can be avoided by proper dosage and placement of the herbicide.     

Carbohydrate oxidation by leucoplasts from suspension cultures of soybean (Glycine max L.)

The aim of this work was to discover how leucoplasts from suspension cultures of soybean (Glycine max L.) oxidize hexose monophosphates. Leucoplasts were isolated from protoplast lysates on a continuous gradient of Nycodenz with a yield of 28% and an intactness of 80%. Incubation of the leucoplasts with ¹⁴C-labelled substrates led to ¹⁴CO₂ production, that was dependent upon leucoplast intactness, from [U-¹⁴C]glucose 6-phosphate, [U-¹⁴C]glucose 1-phosphate, [U-¹⁴C] fructose 6-phosphate and [U-¹⁴C]glucose+ATP, but not from [U-¹⁴C]fructose-1,6-bisphosphate or [U-¹⁴C]triose phosphate. The yield from [U-¹⁴C]glucose 6-phosphate was at least four times greater than that from any of the other substrates. When [1-¹⁴C]-, [2-¹⁴C]-, [3,4-¹⁴C]-, and [6-¹⁴C]glucose 6-phosphate were supplied to leucoplasts significant ¹⁴CO₂ production that was dependent upon leucoplast intactness was found only for [1-¹⁴C]glucose 6-phosphate. It is argued that soybean cell leucoplasts oxidize glucose 6-phosphate via the oxidative pentose phosphate pathway with very little recycling, and that in these plastids glycolysis to acetyl CoA is negligible.S.A.C. thanks the Science and Engineering Research Council for a research studentship.     

The effect of silicon on the manganese nutrition of soybeans (Glycine max (L.) Merrill)

Summary Silicon during the early vegetative stage did not affect the oven dry weight of any of the various tissues of the soybean plant. Silicon did, however, decrease the Mn concentration in the youngest fully mature leaf at intermediate levels of Mn. This effect did not occur at the lowest or highest Mn levels. Deficiency and toxicity symptoms were moderated to a slight degree by Si except at the highest level of Mn.     

Manganese and iron interactions on their uptake and distribution in soybean (Glycine max (L.) Merr.)

Summary The uptake and distribution of iron and manganese were studied in a manganese-sensitive soybean cultivar (‘Bragg’) grown over a range of supply levels of these nutrients in solution culture. At high (90 and 275 μM) manganese levels, increasing the iron concentration in solution from 2 to 100 μM partially overcame the effects of manganese toxicity. Interactions between manganese and iron occurred for dry matter yields, rate of Mn absorption by the roots, and the proportions of manganese and iron transported to the tops. No interaction was observed for the rate of root absorption of iron. The percentage distribution of manganese in the plant top increased with increasing iron, despite a reduced rate of Mn uptake. On the other hand, iron uptake was independent of solution Mn concentration and increased with increasing solution Fe. Also more iron was retained in the roots at high Mn and/or Fe levels in solution. Concentrations of manganese and iron in roots, stems and individual leaves were affected independently by the manganese and iron supplyi.e. without any interaction occurring between the two elements. In general, the concentration in a plant part was related directly to the solution concentration. Symptoms resembling iron deficiency correlated poorly with leaf Fe concentrations whereas high levels of manganese were found in leaves displaying Mn toxicity symptoms.     

Development of a highly efficient, repetitive system of organogenesis in soybean (Glycine max (L.) Merr).

A highly efficient, repetitive system of organogenesis was developed in soybean. Seeds of soybean cv. White hilum pretreated with TDZ formed multiple bud tissue(s) (MBT) at the cotyledonary nodes. MBT initiation occurred only if the axillary buds were not removed from the cotyledonary node. The best MBT formation was achieved by pretreating the seeds for 1 week on medium supplemented with 0.1 mg/l TDZ, followed by culture of the cotyledonary node on medium supplemented with 0.5 mg/l BA for 4 weeks. Culture of the MBT on medium supplemented with 0.1 mg/l TDZ resulted in the proliferation of MBT. MBT was maintained in this way for 12 months. Three hundred thirty six shoots were obtained when 1 g of MBT was subcultured on medium supplemented with 0.5 mg/l BA. Plants were rooted on medium without growth regulators. The regenerated plants grew normally in the greenhouse. Unfortunately, they did not set seeds because of the long-day conditions during growth. This system was successfully applied in three other genotypes.     

Metabolism of 2,4-dichlorophenoxyacetic acid in cell suspension cultures of soybean (Glycine max L.) and wheat (Triticum aestivum L.)

Cell suspension cultures of soybean (Glycine max L.) and wheat (Triticum aestivum L.) incorporated 2,4-dichlorophenoxyacetic acid (2,4-D) into a metabolite fraction which was insoluble in ethanol, water, and hot sodium dodecylsulphate. Further treatment with hot dimethylformamide solubilized a material which by the following criteria appeared to consist of 2,4-D derivatives covalently bound to lignin: i) co-chromatography of radioactivity and of UV-absorbing material upon gel permeation chromatography; ii) spectral similarity with authentic lignins (IR- and UV-spectra, phloroglucinol reaction), 2,4-D appeared to be incorporated as the intact molecule, as shown by comparison of ring- and sidechain-labeled 2,4-D and by detection of monohydroxylated and intact 2,4-D as the major radioactive products of acid hydrolysis. The same compounds were released from the metabolite material which could not be solubilized in dimethylformamide. The incorporation of xenobiotics or their metabolites into lignin, followed by deposition in the cell wall, is suggested as a general pathway for local excretion and detoxification by plant cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 4-OH-2,5-D 4-hydroxy-2,5-dichlorophenoxyacetic acid - SDS sodium dodecylsulphate - DMF dimethylformamide     

Maturation and germination of somatic embryos as affected by sucrose and plant growth regulators in soybeans Glycine gracilis Skvortz and Glycine max (L.) Merr.

The effects of sucrose on maturation and of plant growth regulators on germination of soybean somatic embryos were investigated for the purpose of developing an efficient culture method for plant recovery. Somatic embryos produced on medium with a low sucrose concentration (5 gl^-1), less than 1 mm in length, 0.6 mg in fresh weight, and green in color, were grown for 2 weeks on MS medium containing 5 gl^-1 or 30 gl^-1 sucrose and then for another 5 weeks on MS medium containing 5–90 gl^-1 sucrose. The highest increase in fresh weight of somatic embryos was obtained in the treatment of transferring from 30 gl^-1 sucrose (2 weeks) to 60 gl^-1 (5 weeks). With the increase in fresh weight, the somatic embryos gradually changed color from green to yellow, and finally to white, when they stopped growth. Soybean seed storage proteins (-conglycinin and glycinin) were accumulated in somatic embryos under tissue specific and stage specific control analogous to that in zygotic embryos. Exogenous gibberellic acid was effective in promoting precocious germination of premature soybean somatic embryos, but was not necessary for the germination of mature somatic embryos. The efficiency of somatic embryo germination was as high as 77% from semi-wild soybean and 60–64% from cultivated soybeans, showing that the plant regeneration system developed in this study was efficient and practical.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BR brassinolide - GA₃ gibberellic acid - IBA indolebutyric acid - NAA -naphthaleneacetic acid - PAGE polyacrylamide gel electrophoresis - SDS Sodium Lauryl Sulfate     

Cytokinin inhibition of respiration by cells and mitochondria of soybean,Glycine max (L.) Merrill

Cells of a soybean tissue strain, suspended in an aerated liquid medium, caused disappearance of p-coumaric acid from the medium and oxidation of guaiacol, benzidine, pyrogallol, L-dihydroxyphenylalanine and L-epinephrine. Both the disappearance and the oxidations were inhibited by 6-benzylaminopurine (BAP) at a concentration of 0.5 mM. BAP at other concentrations either promoted or inhibited oxidation of epinephrine in precisely the pattern reported earlier for the disappearance of coumarate; therefore, the disappearance of coumarate probably involves its oxidation. The effectiveness of other cytokinins in inhibiting the oxidation was studied.At 0.5 mM, and perhaps even at 0.5 M, some of the several cytokinins tested inhibited oxygen consumption by the soybean cells. This inhibition, which did not require any of the above metabolizable compounds, was especially marked in the presence of cyanide, azide or Antimycin A, and was detectable in 10 min or less. Either Antimycin A or salicylhydroxamic acid alone promoted O₂ consumption but together they were quite inhibitory. The soybean cells apparently have an alternate respiratory pathway and cytokinins may influence its operation.Several cytokinins at 0.5 mM, and perhaps at 0.5 M, also inhibited oxygen consumption by mitochondrial preparations from the soybean cells, the inhibition being evident in about 20 s. The consumption required a substrate such as malate, succinate or NADH. Cytokinins and related compounds varied in effectiveness as follows: BAP and 6-isopentenyla-minopurine 9-tetrahydropyranyl-BAP > kinetin, ribosyl-isopentenylaminopurine, 9-methyl-BAP and 9-methoxymethyl-BAP > 6,6-dimethylaminopurine and zeatin (slight activity) > 6-methylaminopurine, nicotinamide and adenine (ineffective). To a great extent this order parallels the order of effectiveness of the compounds in causing cell division. Mitochondria, therefore, may contain a site for an important cytokinin action.Abbreviations BAP 6-benzylaminopurine - IPA 6-(²-isopentenyl)aminopurine     

Fatty-acid composition and biosynthesis in cell suspension cultures of Glycine max (L.) Merr., Catharanthus roseus G. Don and Nicotiana tabacum L.

The fatty-acid composition of C. roseus and N. tabacum cell suspension cultures was unaffected by subculture on Wood and Braun, Murashige and Skoog, or Gamborg B5C media. However, placing the cultures — which were normally grown at 25° C — at 15° C reduced growth but resulted in enhanced formation of oleic and linolenic acids in C. roseus cultures and increased levels of linoleic and linolenic acids in cultures of G. max and N. tabacum, respectively. The incorporation of [¹⁴C]acetate into [¹⁴C]linoleic acid was more rapid in N. tabacum cells than in G. max cells, but was very poor in C. roseus where the [¹⁴C] label was distributed mainly between palmitic and oleic acids.     

Fertile progeny of a hybridization between soybean [Glycine max (L.) Merr.] and G. tomentella Hayata

Summary A colchicine-doubled F₁ hybrid (2n=118) of a cross between PI 360841 (Glycine max) (2n=40) x PI 378708 (G. tomentella) (2n=78), propagated by shoot cuttings since January 1984, produced approximately 100 F₂ seed during October 1988. One-fourth of the F₂ plants or their F₃ progeny have been analyzed for chromosome number, pollen viability, pubescence tip morphology, seed coat color, and isoenzyme variation. Without exception, all plants evaluated possessed the chromosome number of the G. max parent (2n=40). Most F₂ plants demonstrated a high level of fertility, although 2 of 24 plants had low pollen viability and had large numbers of fleshy pods. One F₂ plant possessed sharp pubescence tip morphology, whereas all others were blunt-tipped. All evaluated F₂ and F₃ plants expressed the malate dehydrogenase and diaphorase isoenzyme patterns of the G. max parent and the endopeptidase isoenzyme pattern of the G. tomentella parent. Mobility variants were observed among progeny for the isoenzymes phosphoglucomutase, aconitase, and phosphoglucoisomerase. This study suggests that the G. Tomentella chromosome complement has been eliminated after genetic exchange and/or modification has taken place between the genomes.Journal Paper No. J-13776 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA, USA, Project 2763     

Cryopreservation of the SB-M photosynthetic soybean [Glycine max (L.) Merr.] suspension culture

Summary The photosynthetic cell suspension culture of soybean [Glycine max (L.) Merr. cv. Corsoy] (SB-M) was successfully cryopreserved in liquid nitrogen using a preculture and controlled freezing to −40° C (two-step) freezing method. The effective method included a preculture treatment with gradually increasing levels of sorbitol added to the 3% sucrose already present in the medium. The cells were then placed in a cryoprotectant solution [10% DMSO (dimethylsulfoxide) and 9.1% sorbitol, or 10% DMSO and 8% sucrose], incubated for 30 min at 0° C, cooled at a rate of 1° C/min to −40° C, held at −40° C for 1 h, and then immersed directly into liquid nitrogen. The cells were thawed at 40° C and then immediately placed in liquid culture medium. The cell viabilities immediately after thawing were 75% or higher in all cases where cell growth resumed. The original growth rate and chlorophyll level of the cells was recovered within 40 to 47 d. If the sorbitol level was not high enough or the preculture period too short, growing cultures could not be recovered. Likewise, survival was not attained with cryoprotectant mixtures consisting of 15% DMSO, 15% glycerol, and 9.1% sucrose or 15% glycerol and 8% sucrose. The successful method was reproducible, thus allowing long-term storage of this and certain other unique photosynthetic suspension cultures in liquid nitrogen.     

In vitro root induction by 24-epibrassinolide on hypocotyl segments of soybean [Glycine max (L.) Merr.]

Soybean hypocotyl segments were treated in the dark with 24-epibrassinolide (BR) at a range of concentrations for different durations. The maximum effect on adventitious root induction, both in terms of number and length was obtained at very low concentration (0.0001 ppm) of BR applied for 8 h. Higher concentrations were supraoptimal unless applied for a shorter period (4 h). BR was ineffective when applied at low concentration in continuous light.     

RFLP mapping using near-isogenic lines in the soybean [Glycine max (L.) Merr.]

Summary A molecular marker analysis of a near-isogenic line (NIL), its donor parent (DP), and its recurrent parent (RP) can provide information about linkages between molecular markers and a conventional marker introgressed into the NIL. If the DP and RP possess different alleles for a given molecular marker, and if the NIL possesses the same allele as the DP, then it is reasonable to presume a linkage between that molecular marker and the introgressed marker. In this study, we examined the utility of RFLPs as molecular markers for the NIL genemapping approach. The allelic status of fifteen RFLP loci was determined in 116 soybean RP/NIL/DP line sets; 66 of the Clark RP type and 50 of the Harosoy RP type. Of the 1740 possible allelic comparisons (116 NILs x 15 RFLP loci), 1638 were tested and 462 (33.9%) of those were informative (i.e., the RP and DP had different RFLP alleles). In 15 (3.2%) of these 462 cases the NIL possessed the DP-derived RFLP allele, leading to a presumption of linkage between the RFLP locus and the introgressed conventional marker locus. Two presumptive linkages, pK-3 — and pK-472 — Lf _i, were subsequently confirmed by cosegregation linkage analysis. Although not yet confirmed, two other associations, pk-7 ab and pK-229 — y ₉ seemed to be plausible linkages, primarily because the pk-7 — ab association was detected in two independently derived NILs and both markers of the pK-229 — y ₉ association were known to be linked to Pb. The data obtained in this investigation indicated that RFLP loci were useful molecular markers for the NIL gene-mapping technique.Published as Paper no. 9101, Journal Series, Nebraska Agric. Res. Div. Project no. 12-091. Research partially funded by a grant from the Nebraska Soybean Development, Utilization, and Marketing Board     

Factors underlying genotypic differences in the induction of photosynthesis in soybean [Glycine max (L.) Merr.]

Crop leaves are subject to continually changing light levels in the field. Photosynthetic efficiency of a crop canopy and productivity will depend significantly on how quickly a leaf can acclimate to a change. One measure of speed of response is the rate of photosynthesis increase toward its steady state on transition from low to high light. This rate was measured for seven genotypes of soybean [Glycine max (L.) Merr.]. After 10 min of illumination, cultivar ‘UA4805’ (UA) had achieved a leaf photosynthetic rate (P_n) of 23.2 μmol · m^?2 · s^?1, close to its steady‐state rate, while the slowest cultivar ‘Tachinagaha’ (Tc) had only reached 13.0 μmol · m^?2 · s^?1 and was still many minutes from obtaining steady state. This difference was further investigated by examining induction at a range of carbon dioxide concentrations. Applying a biochemical model of limitations to photosynthesis to the responses of P_n to intercellular CO₂ concentration (C_i), it was found that the speed of apparent in vivo activation of ribulose‐1:5‐bisphosphate carboxylase/oxygenase (Rubisco) was responsible for this difference. Sequence analysis of the Rubisco activase gene revealed single nucleotide polymorphisms that could relate to this difference. The results show a potential route for selection of cultivars with increased photosynthetic efficiency in fluctuating light.     

Identification of an UDP-glucose: Flavonol 3-O-glucosyl-transferase from cell suspension cultures of soybean (Glycine max L.)

A glucosyltransferase, which catalyses the glucosylation of flavonols, using uridine diphosphate-D-glucose as glucose donor, has been isolated and purified about 5–10 fold from cell suspension cultures of soybean (Glycine max L., var. Mandarin). The pH optimum for this reaction was ca. 8.5 in glycine-NaOH buffer, and no additional cofactors were required. The enzyme glucosylated the following flavonols predominantly at the 3-position: quercetin (Km 126 M), kaempferol (Km 172 M), isorhamnetin (Km 200 M) and fisetin (Km 270 M). With quercetin as substrate, the apparent Km value for uridine diphosphate-D-glucose was 0.3 M. Glucosylation of flavonols and flavones by this preparation occurred weakly also at the 7-position. No activity was found with dihydroquercetin, naringenin, 4,2,4-trihydroxychalcone, daidzein or texasin. The enzyme was specific for flavonoid compounds, since no activity was observed towards cinnamic acids or simple phenols. However, the preparation was contaminated by a vanillic acid glucosyltransferase, from which it could be partially separated by ionexchange chromatography. The specific activity of the flavonol 3-O-glucosyltransferase increased with age of the culture, reaching a maximum late in the growth cycle of the culture.Abbreviations SAM S-adenosyl-L-methionine - CMT, SAM caffeate 3-O-methyltransferase - FMT, SAM flavonoid O-methyltransferase - UDP-glucose uridine diphosphate-D-glucose - PAL phenylalanine ammonia-lyase