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     

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     

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.     

Sowing seasons and drying methods during post-harvest influence the seed vigour of soybean (Glycine max (L.) Merr.)

Experiments were conducted to study the influence of sowing seasons and drying methods on the seed vigour of two spring soybean (Glycine max (L.) Merr.) cultivars. Two cultivars, ‘Huachun18’ and ‘Huachun 14’, were sown in three seasons viz., spring, summer and autumn and the harvested seeds were dried using three different methods. The results showed that soybean sown in spring had a higher number of branches per plant, pods per branch and seed weight, and consequently resulted in higher seed yields than that of soybean sown in autumn or summer seasons. Seeds sown in the autumn season had the lowest values of electrical conductivity during seed imbibitions, higher peroxidase (POD) activity in germinated seedlings and lower contamination by the seed-borne fungi on the MS medium, which indirectly improved the seed vigour, which was followed by summer sown seeds. Seeds sown during the spring season resulted in poor seed vigour. In addition, the effect of drying methods on the seed vigour was also clarified. Seeds that hung for four days before threshing and then air-dried had the poorest seed vigour which was determined by germination, electrical conductivity, POD activity and seed borne fungal growth. There was no difference in seed vigour between other methods, i.e. seeds threshed directly at harvest and then air-dried on a bamboo sifter or concrete floor. These results indicated that autumn sowing soybean and the drying method in which seeds were threshed directly at harvest and then air-dried on a bamboo sifter resulted in higher seed vigour.     

Early seedling and seasonal N2-fixing symbiotic activity of two soybean [Glycine max (L.) Merr.] cultivars inoculated with Bradyrhizobium strains of diverse origin

In areas with a short growing season the poor adaptability of soybean [Glycine max Meer. (L.)] to cool soil conditions is considered the primary yield limiting factor. Soybean requires temperatures in the 25 to 30°C range for optimum N₂-fixation and yield. Field studies were conducted in 1990 and 1991 at Montreal, Quebec to determine whether adaptability to cool soil conditions, with respect to earlier symbiosis establishment and function, existed among either Bradyrhizobium strains or soybean genotypes. An early maturing isoline of the soybean cultivar Evans and the cultivar Maple Arrow were inoculated with one of four strains isolated from the cold soils of Hakkaido, northern Japan, or the commercially used strains 532C or USDA110, at two planting dates. Plot biomass and nodulation were assessed at seedling (V2), and flowering(R2) growth stages and harvest maturity. Soybean genotypes did not differ for pre-flowering nodulation or N₂-fixation in the cool spring conditions of the first year. Seasonal N₂-fixation rates were also determined at the final harvest by the N-balance and ¹⁵N-isotope dilution methods. Significantly higher symbiotic activity was found for two of the four Hakkaido strains and was reflected in higher final soybean seed yield and total N₂-fixation for the growing season, as compared to the two commercial strains. Planting 14 days earlier resulted in greater early vegetative and total seasonal N₂ fixation and yield in the second year when soil temperatures were warmer, emphasizing the need for the development of soybean-Bradyrhizobium combinations superior in nodule development and function under cool soil conditions.     

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.     

Fatty acid composition of soybean (Glycine max (L.) merr.) somatic embryos

Summary Somatic embryos from four soybean cultivars were matured for 30 and 45 d. Success of embryo germination was evaluated for each length of maturation. The percentage of somatic embryos undergoing successful germination, as defined by rooting and shoot emergence, was greater for embryos matured 45 d than for embryos matured 30 d. Therefore, embryos matured for 45 d are probably physiologically more mature than embryos matured for 30 d. Relative percentages of fatty acids comprising oils and lipids of somatic embryos were determined for each length of maturation and for each cultivar. Variation in relative percentages of palmitic acid, oleic acid, and linoleic acid was affected by length of maturation. However, these changes were genotype dependent. A significant interaction between the cultivars Clark and Maple Arrow and stage of maturation was observed for levels of oleic acid. No other interactions were observed. These data suggest that if changes in relative percentages of certain fatty acids are associated with soybean somatic embryo maturation the changes are genotype dependent. This is journal paper No. J-12870 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 2763. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that also may be suitable. This research was supported in part by grants from the American Soybean Association Development Foundation and the Iowa Soybean Promotion Board.     

Development of an embryogenic suspension culture of soybean (Glycine max Merrill.)

A rapidly growing, maintainable, embryogenic suspension culture of Glycine max L. Merrill. has been generated. The culture consisted almost entirely of clumps of proliferating globular embryos with very little nonembryogenic tissues. The number and size of somatic embryo clumps were used to quantify growth of embryogenic tissues under various conditions. Initiation and proliferation of this embryogenic suspension culture were dependent on the inoculum, method of subculture, and composition of the subculture medium. Twenty to 50 mg of highly embryogenic, early-staged soybean tissue were inoculated into 35 ml of liquid culture medium containing 5 mg 1^–1 2,4-D and either 15 mM glutamine or preferably 5 mM asparagine. Suspension cultures were subcultured at the same inoculum density every 4 weeks. The embryos matured and germinated following placement on solid media, resulting in consistent plant regeneration.     

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.     

Emergence and growth of two non-nodulated soybean genotypes (Glycine max (L.) Merr.) in response to soil acidity

Toxic levels of extractable soil Al limit production of important crops in many areas of the world. The nature of the limitation in soybeans is not completely understood. Our objectives were to investigate the cause of acid-soil-induced delays in seedling emergence, the effect of acidity on productivity in non-nodulated soybeans and further test the Al tolerance of PI 416,937 compared to a sensitive control, Essex. Growth characteristics of the two genotypes through the flowering stage were measured on a Corozal clay (Aquic Tropudult) in Puerto Rico which had been differentially limed to provide a wide range of soil Al. Early growth was also studied in the laboratory using soil from the field experiment. Highly acidic soil conditions, coupled with high Al levels, reduced growth in both Essex and PI 416,937. The principal factor responsible for delayed emergence in the high Al soil was not delayed radicle initiation, but delayed initiation of hypocotyl elongation. Hypocotyl initiation was highly associated with rate of tap root growth, with the former possibly determined by the latter, because a minimum tap root length of 60 mm was required in both high and low Al soils before hypocotyl initiation commenced. In seedlings, the high acidity reduced root more than shoot growth. By 44 days after planting (DAP), however, soil acidity had reduced shoot growth greatly. Although the soybean plants were not nodulated, foliar N levels and shoot growth were decreased by high Al levels, indicating that interference with N fixation may not be the sole mechanism by which nitrogen accumulation and plant growth is reduced in the field.Joint contribution from the USDA, ARS, Tropical Agriculture Research Station, Mayaguez, PR; USDA, ARS, Soybean and Nitrogen Fixation Research Unit, Raleigh, NC, and the Agricultural Experiment Station-University of Puerto Rico (AES-UPR), Rio Piedras, PR.Joint contribution from the USDA, ARS, Tropical Agriculture Research Station, Mayaguez, PR; USDA, ARS, Soybean and Nitrogen Fixation Research Unit, Raleigh, NC, and the Agricultural Experiment Station-University of Puerto Rico (AES-UPR), Rio Piedras, PR.     

Low concentrations of ammonium inhibit specific nodulation (nodule number g-1 root DW) in soybean (Glycine max [L.] Merr.)

Experiments on peas (Gulden and Vessey, 1997) have indicated that NH ₄ ⁺ stimulates both whole plant (nodules plant^-1) and specific nodulation (nodules g^-1 root DW). The effect of low concentrations of NH ₄ ⁺ on the soybean/Bradyrhizobium symbiosis is unknown. The objectives of the current study were to determine the immediate and residual effects of NH ₄ ⁺ on nodulation and N₂ fixation in soybean (Glycine max [L.] Merr.) in sand culture. Soybean (cv. Maple Ridge) were exposed to 0.0, 0.5, 1.0 and 2.0 mM of ¹⁵N-labelled (NH₄)₂SO₄ for 28 days after inoculation (DAI). From 29 to 56 DAI the plants were grown on NH ₄ ⁺ -free nutrient solution. Plants were harvested at 7, 14, 21, 28 and 56 DAI for root, shoot and nodule dry weight (DW), total N content, nodule counts and ¹⁵N enrichment of plant composites. Nitrogenase activity was measured by gas exchange at 28 DAI. The plants in the control (0.0 mM NH ₄ ⁺ ) treatment had consistently lower relative growth rates than the plants in the NH ₄ ⁺ treatments during the first 28 DAI. Plant growth was also less at 2.0 mM NH ₄ ⁺ compared to growth at 0.5 and 1.0 mM NH ₄ ⁺ . At 28 DAI, plants exposed to 0.5 and 1.0 mM NH ₄ ⁺ had significantly more nodules per plant and larger individual nodules than either the NH ₄ ⁺ -free controls or the 2.0 mM NH ₄ ⁺ -supplied plants. However, specific nodulation (nodule number g^-1 root DW) and specific nitrogenase activity (nitrogenase activity g^-1 nodule DW) were on average approximately 286% and 60% higher in the control plants, respectively, than for plants in the NH ₄ ⁺ treatments at 28 DAI. Also at 28 DAI, specific nodule DW (nodule DW g^-1 root DW) were 17, 44 and 53% higher in control plants than plants that had been exposed to 0.5, 1.0 and 2.0 mM NH ₄ ⁺ . At 56 DAI, after an additional 4 weeks of NH ₄ ⁺ -free nutrition, the plants which had previously received 0.5 and 1.0 mM NH ₄ ⁺ still maintained the highest plant DW and N contents, however, specific nodule DW had become similar at 600 mg nodule DW g^-1 root DW among all treatments. It is concluded that NH ₄ ⁺ has a negative effect on the nodulation process in the soybean/Bradyrhizobium symbiosis (as best indicated by the negative effect of NH ₄ ⁺ on specific nodulation). Despite this negative effect on specific nodulation, 0.5 and 1.0 mM NH ₄ ⁺ resulted in higher whole plant nodulation and N₂ fixation due to a compensating, positive effect on overall plant growth (i.e. fewer nodules g^-1 root DW, but much larger roots). Once NH ₄ ⁺ was removed from all treatments, the soybean plants appeared to move toward a consistent level of nodule DW relative to root DW.     

Effects of norflurazon (San 9789) on light-increased extractable nitrate reductase activity in soybean [Glycine max (L.) Merr.]seedlings

Abstract. The effects of norflurazon (San 9789) on light-increased extractable NADH nitrate reductase activity (NRA) in soybean seedlings were studied. Continuous white light (W) increased NRA steadily in root and cotyledonary tissues over a 5 d period. Morflurazon, a pyridazinone herbicide which causes chlorophyll bleaching in W, reduced the initial NRA induction rate in roots and cotyledons. However, in cotyledons of norfiurazon-treated plants NRA increased at a more rapid rate than in the control after 24 h of W, with activity levels reaching three times those of control seedlings after 5 d. NRA induced by W in control and norflurazon-treated cotyledons was fluence-rate dependent. Continuous FR induced equal amounts of NRA in control and norflurazontreated tissues, suggesting that the superinduceable NRA of norflurazon-treated plants under W is not phytochrome induced. The FR-induced NRA of control and norflurazon-treated cotyledons had pH optima of 6.6, but during development under W the pH optimum of control cotyledons changed from 6.3 to between 6.6 and 7.1. The pH optimum of the norflurazon-induced NRA of the cotyledon under W was about 7.5. The NADH/NADPH NRA ratio after 4 d of W was 1.3 in control and 2.5 in norflurazontreated cotyledons. These data indicate that photosynthelic pigments are involved only secondarily in light-induction of NRA in this system.     

Mapping QTLs for tissue culture response in soybean (Glycine max (L.) Merr.)

Hempseed is rich in polyunsaturated fatty acids (PUFAs), which have potential as therapeutic compounds for the treatment of neurodegenerative and cardiovascular disease. However, the effect of hempseed meal (HSM) intake on the animal models of these diseases has yet to be elucidated. In this study, we assessed the effects of the intake of HSM and PUFAs on oxidative stress, cytotoxicity and neurological phenotypes, and cholesterol uptake, using Drosophila models. HSM intake was shown to reduce H₂O₂ toxicity markedly, indicating that HSM exerts a profound antioxidant effect. Meanwhile, intake of HSM, as well as linoleic or linolenic acids (major PUFA components of HSM) was shown to ameliorate Aβ42-induced eye degeneration, thus suggesting that these compounds exert a protective effect against Aβ42 cytotoxicity. On the contrary, locomotion and longevity in the Parkinson’s disease model and eye degeneration in the Huntington’s disease model were unaffected by HSM feeding. Additionally, intake of HSM or linoleic acid was shown to reduce cholesterol uptake significantly. Moreover, linoleic acid intake has been shown to delay pupariation, and cholesterol feeding rescued the linoleic acid-induced larval growth delay, thereby indicating that linoleic acid acts antagonistically with cholesterol during larval growth. In conclusion, our results indicate that HSM and linoleic acid exert inhibitory effects on both Aβ42 cytotoxicity and cholesterol uptake, and are potential candidates for the treatment of Alzheimer’s disease and cardiovascular disease.     

Formation and function of secondary aerenchyma in hypocotyl, roots and nodules of soybean (Glycine max) under flooded conditions

Flooding is a major problem in many areas of the world and soybean is susceptible to the stress. Understanding the morphological mechanisms of flooding tolerance is important for developing flood-tolerant genotypes. We investigated secondary aerenchyma formation and function in soybean (Glycine max) seedlings grown under flooded conditions. Secondary aerenchyma, a white and spongy tissue, was formed in the hypocotyl, tap root, adventitious roots and root nodules after 3 weeks of flooding. Under irrigated conditions aerenchyma development was either absent or rare and phellem was formed in the hypocotyl, tap root, adventitious roots and root nodules. Secondary meristem partially appeared at the outer parts of the interfascicular cambium and girdled the stele, and then cells differentiated to construct secondary aerenchyma in the flooded hypocotyl. These morphological changes proceeded for 4 days after the initiation of the flooding. After 14 days of treatment, porosity exceeded 30% in flooded hypocotyl with well-developed secondary aerenchyma, while it was below 10% in hypocotyl of irrigated plants that had no aerenchyma. When Vaseline was applied to the hypocotyl of plants from a flooded treatment to prevent the entry of atmospheric oxygen into secondary aerenchyma, plant growth, especially that of roots, was sharply inhibited. Thus secondary aerenchyma might be an adaptive response to flooding.     

In situ expression of the GmNMH7 gene is photoperiod-dependent in a unique soybean (Glycine max [L.] Merr.) flowering reversion system

Soybean is a short-day plant and its flowering process can be reversed when switching from short-day to long-day conditions. Flowering reversion provides a useful system to study the flowering process in both forward and backward directions. In this study, we optimized a soybean flowering reversion system using a photoperiod-sensitive cultivar Zigongdongdou. Three types of terminal structures were found during flowering reversion: reversed terminal raceme (RTR), short terminal raceme (STR), and vegetative terminal (VT). The relative frequency of these terminal structures during flowering reversion under long day was dependent on the duration of the prior short day (SD) pretreatment. This process is phytochrome dependent and young plants were more susceptible to flowering reversion. Leaf removal increased the minimal SD period needed for the induction of STR. To demonstrate the application of this system, we studied the patterns of in situ expression of the GmNMH7 gene during flowering development and reversion. NMH7 family members encode MADS-box proteins and are unique in legume families since their expression can be detected in both developing flowers and nodules. In situ hybridization experiments using plants grown under different photoperiod cycles provided several lines of evidence supporting a close relationship between GmNMH7 gene expression and floral development in soybean. Furthermore, it seems that GmNMH7 may participate in flower development at different stages. Interestingly, the expression pattern of GmNMH7 in root nodules was also found to be regulated by photoperiod. These results support the notion that the photoperiod sensitive GmNMH7 gene may play multiple roles in growth and development in soybean.C. Wu and Q. Ma contributed equally to this work.     

Isolation and culture of soybean hypocotyl protoplasts

Summary Protoplasts were isolated seedling hypocotyls of soybean (Glycine max), and cultured in both liquid and agarose-solidified, modified K8P medium. Nuclear staining revealed that only 2% of protoplasts lacked a nucleus, 93% contained a single nucleus, and 5% contained more than one. Maximum protoplast yields and subsequent division frequencies, in liquid medium, were obtained from 5 days-old seedlings. Maximum division frequencies (54%) were obtained from hypocotyl protoplasts plated at a density of 5×10⁴ ml⁻¹. Using different osmolality reduction régimes for liquid cultures, hypocotyl protoplasts developed into green, nodular callus, similar to that which has previously given rise to shoot buds in perennialGlycine species. This tissue, however, did not produce shoot buds in soybean. N. H. was supported by a SERC CASE studentship and a postdoctoral fellowship from Shell Research Ltd., Sittingbourne, Kent, UK.