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.     

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     

低磷对大豆主根伸长生长的影响

文章采用卷纸培养和分层琼脂培养的方法,研究磷对大豆主根伸长影响的结果表明:低磷[0.2 μmol(KH2PO4)·L-1]显著促进大豆主根伸长,特别是延长大豆主根根尖至最新侧根间的距离;组织切片表明,低磷对主根伸长的促进主要是通过延迟主根伸长区的分化实现的,并且低磷对主根的促进作用不受亚磷酸盐的影响.琼脂分层培养的结果表明,在磷分布不均匀的条件下,低磷影响主根的仲长生长,上层或下层不施磷的大豆主根伸长均有增加.     

Phosphorus deficiency increases the acetylene-induced decline in nitrogenase activity in soybean (Glycine max (L.) Merr.)

Open-flow assays of acetylene reduction activity {ARA)and CO₂production in nodulated roots were performed in situ with soybean{Glycine max (L.) Merr.) cv. Kingsoy grown hydroponically withorthophosphate (Pi) nutrition either limiting (low-P) or non-limiting(control) for plant growth. Nodule growth was more limited thanshoot growth by P deficiency. During ARA assays, nitrogenaseactivity declined a few minutes after exposure of the nodulatedroots to C₂H₂, and this acetyleneinduced decline (C₂H₂-ID) wastwice as intense at low-P. Moreover, the minimum ARA after theC₂H₂-ID was reached about 10 min earlier at low-P. The intensityof the C₂H₂-ID was correlated negatively with nodule mass perplant and positively with the ratio of shoot/nodule mass. Afterinitial exposure to C₂H₂, the nodulated-root CO₂ productionwas transiently stimulated and, moreover, this increase was2-fold higher at low-P. Then, the nodulated-root CO₂ productiondecreased with nodule C₂H₄ production. During the C₂H₂-ID, thenodule nitrogenase-linked respiration, which was computed asthe variable component of the linear regression between CO₂and C₂H₄ production, was 2-fold higher at low-P. Furthermore,the microscopic observation of nodule sections revealed thatstarch deposits were decreased at low-P. However, nitrogenaseactivity, i.e. ARA before the C₂H₂-ID, was not affected by Pdeficiency. It is argued that P deficiency increased the C₂H₂-IDbecause it increased nodule permeability to O₂ diffusion. Key words: Acetylene reduction, nitrogen fixation, phosphorus, respiration, soybean, Glycine max (L.) Merr     

Genetics and cytology of a new genic male-sterile soybean [Glycine max (L.) Merr.]

 Genetic and cytological studies were conducted with a new male-sterile, female-fertile soybean [Glycine max (L.) Merr.] mutant. This mutant was completely male sterile and was inherited as a single-recessive gene. No differences in female or male gamete transmission of the recessive allele were observed between reciprocal cross-pollinations in the F₁ or F₂ generations. This mutant was not allelic to any previously identified soybean genic male-sterile mutants: ms1, ms2, ms3, ms4, ms5, or ms6. No linkage was detected between sterility and flower color (W1 locus), or between sterility and pubescence color (T1 locus). Light microscopic and cytological observations of microsporogenesis in fertile and sterile anthers were conducted. The structure of microspore mother cells (MMC) in male-sterile plants was identical to the MMCs in male-fertile plants. Enzyme extraction analyses showed that there was no callase activity in male-sterile anthers, and this suggests that sterility was caused by retention of the callose walls, which normally are degraded around tetrads at the late tetrad stage. The tapetum from male-sterile anthers also showed abnormalities at the tetrad stage and later stages, which were expressed by an unusual formation of vacuoles, and by accumulation of densely staining material. At maturity, anthers from sterile plants were devoid of pollen grains. Received: 13 May 1996 / Revision accepted: 19 August 1996     

Embryogenic response of multiple soybean [Glycine max (L.) merr.] cultivars across three locations

Summary Nine soybean [Glycine max (L.) Merr.] cultivars representing midwestern, mid-south, and southern US growing regions were evaluated at each of three locations (Athens, GA; Lexington, KY; and Wooster, OH) using uniform embryogenic induction and proliferation protocols in order to evaluate the portability of soybean somatic embryogenic protocols to different locations. The experimental design minimized variation between locations by having all cultivars present at all locations on all days. A quantitative weighted score for primary embryo induction was developed on average embryo number per explant and was used to describe non-embryogenic, poorly embryogenic, moderately embryogenic, and highly embryogenic responses. Ranking of cultivars remained similar across all locations, indicating a uniform transportability of the protocol, at least as far as embryo induction is concerned. Continued proliferation of embryogenic cultures was also measured using a repetitive growth measure but few meaningful conclusions could be made due to the high level of variability including inconsistent growth of cultures between each subculture. Overall, several cultivars were identified as being uniformly embryogenic or non-embryogenic at the primary induction phase at all locations, and we predict that those embryogenic cultivars could be used by any laboratory for high-efficiency induction of embryogenesis. The best of these cultivars, ‘Jack’, was uniformly responsive across all locations and should be selected as the genotype most likely to yield positive results when attempting to culture and genetically engineer soybeans via embryogenic protocols.     

Nitrate Reduction by Roots of Soybean (Glycine max [L.] Merr.) Seedlings

Studies were conducted with 9 to 12 day-old soybean (Glycine max [L.] Merr. cv. Williams) seedlings to determine the contribution of roots to whole plant NO(3) (-) reduction. Using an in vivo -NO(3) (-) nitrate reductase (NR) assay (no exogenous NO(3) (-) added to incubation medium) developed for roots, the roots accounted for approximately 30% of whole plant nitrate reductase activity (NRA) of plants grown on 15 mm NO(3) (-).Nitrogen analyses of xylem exudate showed that 53 to 66% of the total-N was as reduced-N, depending on the time of day of exudate collection. These observations supported enzyme data that suggested roots were contributing significantly to whole plant NO(3) (-) reduction. In short-term feeding studies using (15)N-NO(3) (-) significant and increasing atom percent (15)N excess was found in the reduced-N fraction of xylem exudate at 1.5 and 3 hours after feeding, respectively, which verified that roots were capable of reducing NO(3) (-).Estimated reduced-N accumulation by plants based on in vivo -NO(3) (-) NR assays of all plant parts substantially over-estimated actual reduced-N accumulation by the plants. Thus, the in vivo NR assay cannot be used to accurately estimate reduced-N accumulation but still serves as a useful assay for relative differences in treatment conditions.     

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.     

Morpho-physiological parameters affecting iron deficiency chlorosis in soybean (Glycine max L.)

Background and aims

Iron deficiency chlorosis (IDC) leads to severe leaf chlorosis, low photosynthetic rates, and yield reductions of several million metric tonnes each year. In order to devise breeding and genetic transformation programs that aim at generating high-yielding and IDC-tolerant soybean lines, it is necessary to better understand the mechanisms that enable tolerant plants to survive under Fe-limiting conditions.

Methods

An in silico analysis in the USDA soybean collection allowed the identification of a set of novel efficient and inefficient soybean cultivars which can be used in future studies concerning IDC response. Plants were grown in iron deficient and iron sufficient conditions using a bicarbonate system and several IDC-related aspects were studied.

Results

A new set of efficient and inefficient soybean lines were identified in silico, and their tolerance to IDC was confirmed under laboratorial conditions. New plant traits that are highly correlated to IDC scoring were identified: a negative correlation was found between SPAD values and stem weight, weight of the unifoliolates and iron concentration of the first unifoliolates was found; higher SPAD values were correlated with the amount of iron in the first trifoliate leaves. Our data also show that having higher concentrations of iron in the seeds provides increased resistance to IDC. No correlation was found between root iron reductase activity and chlorosis.

Conclusions

Soybean differential chlorosis susceptibility between different accessions is linked to specific morpho-physiological parameters such as unifoliolate leaf size, stem weigh, concentration of iron in the seeds, and tissue iron partitioning.     

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.     

Soluble carbohydrate content of soybean [Glycine max (L.) merr.] somatic and zygotic embryos during development

Summary Somatic and zygotic embryos of soybean cv. Jack were analyzed for soluble carbohydrate, total lipids, and protein during development. Zygotic embryos accumulated trace amounts of fructose, galactose, and galactinol., whereas somatic embryos contained only trace amounts of galactose. Somatic embryos accumulated much higher glucose levels than zygotic embryos. Both somatic and zygotic embryos contain low levels of sucrose, myoinositol, and pinitol. Raffinose and stachyose accumulated in the late developmental stages of zygotic embryos, but only stachyose was found to accumulate in the late stage somatic embryos. Zygotic embryos contained low total lipid levels up to 50 d after flowering (DAF) and then the levels increased to 16% by 55 DAF and 21% at 65 DAF. Somatic embryos had low levels of total lipids throughout development with the maximum of only 4.7%. Soybean zygotic embryos contained about 40% protein throughout development, while the protein concentration of somatic embryos decreased from 44% to 25% as maturation approached. These studies demonstrate that the composition of Jack zygotic embryos is similar to that described for other cultivars during development while the somatic embryo composition and size is markedly different. The low somatic embryo germination often noted might be due to the abnormal development as shown by a composition different from that of mature zygotic embryos. The low concentration of the raffinose series sugars might be especially important factors.     

Genistein accumulation in soybean (Glycine max [L.] Merr.) root systems under suboptimal root zone temperatures

Genistein, as a plant-to-bacteria signal, plays an importantrole in the establishment of the soybean (Glycine max [L.] Merr.)-Bradyrhizobiumjaponicum nitrogen-fixing symbiosis. It is essential to thedevelopment of effective root nodules and responsible for inducingthe nod genes of B. japonicum. Because sub-optimal root zonetemperature (RZT) delays infection and early nodule development,and decreases plant nodule number, and genistein addition overcomessome of this, it is reasonable to hypothesize that suboptimalRZT disrupts the inter-organismal signal exchange by inhibitinggenistein synthesis. Four experiments were conducted to testthese hypotheses. The results of these studies indicated that:(1) when soybean plants were germinated and maintained at RZTsranging from 13 to 17C, root genistein concentration and contentper plant were lower than those of plants with roots maintainedat RZTs above 17C; (2) when plants were germinated at an optimalRZT (25 C) then transferred to RZTs below 17C, and acclimatedfor a few days, root genistein concentration and content perplant were higher than those of plants with roots maintainedeither at optimal RZT, or transferred to RZT above 17 C, althoughby the end of the experiment, the genistein concentration ofroot systems at below 17C RZT appeared to be declining to valuesbelow those of plants with above 17 C RZT; (3) the root genisteinconcentration increased before the onset of nitrogen fixationand decreased thereafter; and (4) part of the effect of RZTson genistein content per plant root system was from reductionsin genistein concentration at lower RZT5, and part was due todecreased plant root growth. Key words: Genistein, Glycine max, suboptimal temperature     

Boron deficiency in soybean [Glycine max (L.) Merr.] peanut (Arachis hypogaea L.) and black gram [Vigna mungo (L.) Hepper]: Symptoms in seeds and differences among soybean cultivars in susceptibility to boron deficiency

The susceptibility of soybean cultivars (cvs) to boron (B) deficiency was examined in three experiments on a Typic Tropaqualf in Northern Thailand: one experiment also included peanut cv Tainan 9 and black gram cv Regur.Without added B (B0), B deficiency depressed seed yield by 60% in soybean cv NW1 compared with 30% in cv SJ5, 40% in cv 7016, 45% in peanut, and 93% in black gram. B deficiency also induced a localised depression on the internal surface of one or both cotyledons of some soybean seeds resembling the symptom of hollow heart in peanut seeds. It induced 50% hollow heart in peanut, 17% in soybean cv NW1, 5% in SJ5, and 1% in 7016: black gram seeds had no symptoms. Addition of B decreased or eliminated the symptoms.In a comparison of 19 soybean cvs, the incidence of hollow heart symptoms in seeds at B0 varied widely from none in two cvs to 75% in cv Buchanan: cv NW1 showed intermediate susceptibility to B deficiency with 36% hollow heart in its seed while SJ5 was insensitive with 1%. Application of B eliminated hollow heart except from one seed in one cultivar. The results suggest that susceptibility to B deficiency is sufficiently important and variable among soybean genotypes to warrant its inclusion as a selection criterion when breeding cultivars for areas with low soil B.     

Effects of sethoxydim on the metabolism of isolated leaf cells of soybean [Glycine max (L.) Merr.]

The effects of the herbicide sethoxydim {2-[1-ethoxyimino)-butyl]-5-[2-(ethylthio)-propyl]-3-hydroxy-2-cyclohexen-1-one} on selected metabolic processes of enzymatically isolated leaf cells from soybeans [Glycine max (L.) Merr., cv. Essex] were studied. Photosynthesis, protein, ribonucleic acid (RNA), and lipid synthesis were measured by the incorporation of NaH¹⁴CO₃, [¹⁴C]leucine, [¹⁴C]uracil, and [¹⁴C]acetic acid into the isolated soybean cells, respectively. Time-course and concentration studies included incubation times of 30, 60, and 120 min and concentrations of 0.1, 1, 10, and 100 M of sethoxydim. Lipid synthesis was the most sensitive and first metabolic process inhibited by the lowest concentration of sethoxydim. Photosynthesis was not affected significantly by sethoxydim and did not appear to be a target site involved in its herbicidal action. RNA and protein syntheses were inhibited significantly but only by the high concentrations of sethoxydim. It is suggested that sethoxydim exhibits its phytotoxic action by altering or modifying the lipid composition of plant membranes.Abbreviations MES [2-(N-morpholino)-ethanesulfonic acid] - HEPES [N-2-hydroxyethyl piperazine-N-2-ethane sulfonic acid]     

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     

Perception of Bradyrhizobium japonicum Nod factor by soybean [Glycine max (L.) Merr.] root hairs under abiotic stress conditions

Suboptimal growth conditions, such as low rhizosphere temperature, high salinity, and low pH can negatively affect the rhizobia-legume symbioses, resulting in poor nodulation and lower amounts of nitrogen fixed. Early stages of the Bradyrhizobium japonicum-soybean [Glycine max (L.) Merr.] symbiosis, such as excretion of genistein (the plant-to-bacteria signal) and infection initiation can be inhibited by abiotic stresses; however, the effect on early events modulated by Nod factors (bacteria-to-plant signalling), particularly root hair deformations is unknown. Thus, the objective of this study was to evaluate the perception of Nod factor by soybean root hairs under three stress conditions: low temperature, low pH, and high salinity. Three experiments were conducted using a 1:1 ratio of Nod Bj-V (C(18:1), MeFuc) and Nod Bj-V (Ac, C(16:0), MeFuc). Nod factor induced four types of root hair deformation (HAD), wiggling, bulging, curling, and branching. Under optimal experimental conditions root hair response to the three levels of Nod factor tested (10(-6), 10(-8), and 10(-10) M) was dose-dependent. The highest frequency of root hair deformations was elicited by the 10(-6) M level. Root hair deformation decreased with temperature (25, 17, and 15 degrees C), low pH, and high salinity. Nod factor concentration did not interact with either low temperature or pH. However, salinity strongly inhibited HAD responses to increases in Nod factor concentration. Thus, the addition of higher levels of Nod factor is able to overcome the effects of low pH and temperature stress, but not salinity.