Agronomic evaluation of tissue-culture-derived soybean plants

Summary Genetic alterations of regenerated plants based on the tissue culture process (somaclonal variation) have become common for many plant species including soybean [Glycine max (L.) Merr.]. The objective of this study was to test for the presence of tissue-culture-derived genetic variation in eight agronomic traits in homozygous progeny regenerated by organogenesis using the commercially important cultivar Asgrow A3127. A total of 86 lines derived by repeated self-pollination of nine regenerated plants was grown in two locations for 2 years. When compared to the unregenerated parent, statistically significant variation (P<0.05) was found for maturity, lodging, height, seed protein and oil, but not for seed quality, seed weight, or seed yield. All of the variation noted was beneficial and did not involve decreased yield. Since the differences were not large, the results indicate that the tissue culture process is not necessarily detrimental to plant performance, which is an important consideration since tissue culture techniques are used in many genetic engineering methods.     

Inheritance of two independent isozyme variants in soybean plants derived from tissue culture

Summary Soybean [Glycine max (L.) Merr.] plants were regenerated via somatic embryogenesis from nine soybean cultivars. Our objective was to identify and characterize genetically novel mutations that would further our understanding of the soybean genome. Variant isozyme patterns were observed in two independent tissue culturederived lines. Genetic analyses were conducted on these two isozyme variants, and they were heritable. No variant isozyme patterns were evident in control (parental) soybean lines. In the cultivar BSR 101, a mutation of Aco2-b (aconitase) to a null allele was detected. The Aco2-bn mutant, Genetic Type T318, had not been previously observed in soybean. In the Chinese cultivar Jilin 3 (PI 427.099), a chlorophyll-deficient plant was identified that also lacked two mitochondrial malate-dehydrogenase (Mdh null) isozyme bands. These two mutant phenotypes, chlorophyll-deficient and Mdh null, were found to cosegregate. The Jilin 3 mutant, Mdh1-n (Ames 1) y20 (Ames 1) Genetic Type T317, was allelic to three chlorophyll-deficient, Mdh1 null mutants [Mdh1-n (Ames 2) y20 (Ames 2) (T323), Mdh1-n (Ames 3) y20 (Ames 3) (T324), and Mdh1-n (Ames 4) y20 (Ames 4) (T325)] previously identified from a transposon-containing soybean population, and to a chlorophyll-deficient, Mdh1 null mutant [Mdh1-n (Urbana) y20 (Urbana) k2, Genetic Type T253] which occurred spontaneously in soybean. The recovery of two isozyme variants from progeny of 185 soybean plants regenerated from somatic embryogenesis indicates the feasibility of selection for molecular variants.     

Proton efflux and transfer cell formation as responses to Fe deficiency of soybean in nutrient solution culture

Hydroponically grown Hawkeye soybeans with N supplied as NO₃^– did not show any measurable pH decrease of the nutrient solution during the first week of Fe deficiency as has been observed for other Fe-efficient dicotyledonous species. Only after prolonged Fe stress with no renewal of the nutrient solution could an unspecific pH reduction be measured as a consequence of a decrease in the NO₃^– content of the solution. On the other hand, Fe stress induced H⁺ efflux could be localized at the root tip region by day foru of-Fe treatment when intact plants were transferred from the nutrient solution to agar medium containing the pH indicator dye bromocresol purple. However, the activity of this H⁺ pump obviously was too weak to neutralize HCO₃-ions simultaneously excreted from older root parts and to acidify the bulk nutrient solution. Thus no remobilization of iron precipitated on older parts of the roots occurred and the plants remained chlorotic.Electron microscopy of the H⁺ extruding zone revealed hypodermal transfer cells with wall protuberances surrounded by cytoplasm especially rich in mitochondria. No transfer cells occurred in the rhizodermis as seen in other Fe-efficient dicots. Some cortical cells also showed transfer cell features with wall protuberances in the intercellular spaces. Often wall ingrowths were surrounded by a periplasmic space which reduced the potential surface amplification of the plasma membrane. It is concluded that the weak capacity of Hawkeye soybeans for Fe stress-induced H⁺ extrusion correlates with their less intense wall labyrinth formation as compared with other dicotyledonous species with higher Fe efficiency.     

Cytological studies of triploids and their progeny from male-sterile (ms1) soybean

Summary Triploids (2n=3X=60) were obtained from genetic male-sterile (ms1 ms1) soybean [Glycine max (L.) Merr.] plants. Meiosis, pollen fertility, and chromosome number of their progeny were studied. Studies of meiosis in fertile and sterile triploids revealed no distinguishable differences in chromosome associations. Male-sterile plants formed coenocytic microspores characteristic of the ms1 mutant. Restitution of some dyad and tetrad nuclei were observed in male-sterile plants. Chromosomes of the triploids tended to occur in trivalents during diakinesis and metaphase I (MI), but multivalents, bivalents, and univalents also were observed. Average types and frequencies of chromosome associations per cell in diakinesis and MI from 542 pollen mother cells were 0.004 IX + 0.06 VI + 0.002 V + 0.005 IV + 16.99 III + 1.79 II + 5.03 I. Some secondary associations, nonhomologous pairing, and aberrant nucleolar distributions occasionally were observed. Such behavior support the hypothesis of duplicated genomes and the polyploid origin of soybean. Pollen fertility in male-fertile triploid plants (Ms1 ms1 ms1) varied from 57% to 82%, with an average of about 71%. Chromosome numbers of progenies obtained from these fertile triploids varied from 2n=40 to 2n=71, and exhibited a near-random distribution, with the majority (about 60%) being between 56 and 65. Progenies of the fertile triploids gave segregation ratios for the ms1 allele, which confirmed the Ms1 ms1 ms1 genotype.Joint contribution: Agricultural Research Service, U.S. Department of Agriculture, and Journal Paper No. J-11672 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011, USA, Project 2471     

Exclusion of inefficient Bradyrhizobium japonicum serogroups by soybean genotypes

Soybean [Glycine max (L.) Merr.] forms a symbiosis with serogroups of Bradyrhizobium japonicum that differ in their dinitrogen fixing abilities. The objectives of this study were to identify soybean genotypes that would restrict nodulation by relatively inefficient serogroups indigenous to a large portion of the southeastern USA, and then characterize the nodulation responses of selected genotypes with specific bradyrhizobial strains under controlled conditions. From field screening trials followed by controlled single and competitive inoculations of serogroups USDA 31, 76 and 110, twelve soybean genotypes out of 382 tested were identified with varying levels of exclusion abilities. Soybean nodule occupancies and nodulation characteristics were influenced by plant genotype, environment (i.e. field or greenhouse), bradyrhizobial serogroup, and location of nodules (i.e. tap or lateral root). The cultivar Centennial sustains high seed yields even though it nodulates to a high degree with the inefficient serogroup USDA 31. In contrast, data from the released cultivars Braxton, Centennial and Coker 368 indicate that they may have been selected to exclude the inefficient serogroup USDA 76 from their tap root nodules, possibly contributing to high seed yield.     

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.     

The frequency of polyembryonic seedlings and polyploids from ms1 soybean

Summary Seed from homozygous recessivems ₁ genetic male-sterile soybean (Glycine max (L.) Merr.) plants was studied for frequencies of polyembryonic seedlings and different levels of polyploidy among abnormal seedlings from six different source populations: Amesms ₁ (Ams), North Carolinams ₁ (NCms), Tonicams ₁ (Tms), Urbanams ₁ (Ums), and F₄ generation seed obtained from crosses ofms ₁ to two chromosome interchange lines (Ams x Clark T/T and Ums x KS-172-11-3). Frequencies of polyembryony observed in Tms, Ums, Ams, NCms, F₄ seed from Ams x Clark T/T, and F₄ seed from Ums x KS-172-11-3 were 3.6%, 2.4%, 3.1%, 2.5%, 2.2% and 0.1%, respectively. Frequencies of abnormal seedlings from these six sources varied from 1.7% (Ums X KS-172-11-3) to 16.8% (Ams X Clark T/T). Frequencies of polyploids among the abnormal seedlings ranged from 6.8% in Ums x Ks-172-11-3 to 66.7% in Tms. On average, the frequency of polyploid individuals from monoembryonic seedlings was 1.22%. Chromosome number of these seedlings varied from 20 to 200. Variation of the frequencies of polyembryonic seedlings and polyploid progeny among abnormal seedlings suggested that the mechanism(s) controlling the characters of polyembryony and formation of polyploids was associated with thems ₁ gene and was affected by other gene(s) or environmental factors.Joint contribution: Agricultural Research Service, US Department of Agriculture, and Journal Paper No. J-11255 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011, USA. Project 2471     

A novel plasma membrane-bound thioredoxin from soybean

Two thioredoxin cDNAs from soybean were isolated by screening an expression library using an anti-(plasma membrane) serum. The nucleotide sequences of the two cDNAs were found to be 89% identical. The polypeptides encoded by the two cDNAs, designated TRX1 and TRX2, contain a disulfide active site, as found in other thioredoxins. TRX1 was expressed as a fusion protein in Escherichia coli and shown to possess thiol-disufide interchange activity. Unlike other eukaryotic thioredoxins, these two soybean thioredoxins contain a putative transmembrane domain in their N-terminal regions. To determine subcellular location, the TRX1 was fused with a reporter epitope at its C-terminus and expressed in transgenic tobacco plants. The fusion protein was co-purified with plasma membrane markers 1,3-glucan synthase and vanadate-sensitive ATPase, indicating the plasma membrane location of TRX1. When the reporter epitope was inserted between the start codon and the transmembrane domain in the N-terminus, the fusion protein was found in the soluble fraction, possibly due to disruption of the transmembrane domain by the highly hydrophilic epitope sequence. Taken together, our results demonstrate that soybean TRX1 is a plasma membrane-bound thioredoxin, which is most likely anchored to the membrane through the N-terminal transmembrane domain. It is known that plant plasma membranes contain various proteins with thiol-disulfide interchange activity. The soybean thioredoxins reported here are the first group of such proteins to be characterized at the molecular level. However, the biological function of the plasma membrane-bound thioredoxin remains to be determined.     

Induction and development of somatic embryos from cell suspension cultures of soybean

Glycine max (L.) Merr. (soybean) andGlycine soja Sieb. and Zucc. cell suspension cultures were grown and used as inoculum sources for growing callus on agar-solidified nutrient media. Concentrations and chemical forms of the growth regulators in liquid and solidified media were altered in an attempt to achieve in vitro plant regeneration. Numerous embryoids, particularly ofG. soja, were produced on basal nutrient media supplemented with 100 ppm casein hydrolysate, 0.1 μM abscisic acid, 2.25 μM 2,4-dichlorophenoxyacetic acid, and 15 μM adenine or 0.46 μM kinetin. Often the roots of the embryoids elongated. This was enhanced in the presence of an inhibitor of gibberellin synthesis (1 to 20 μM Amo 1618). Callus recovered from aG. soja suspension culture produced one shoot structure when grown on a solid medium containing 0.2 μM Amo 1618 and 80 μM glutathione. The shoot structure consisted of two distinct buds, one producing two leaves. The shoot did not develop into a plant. Although regeneration of soybean plants was not achieved, these observations suggest that it may be achievable. The investigations reported in this paper (no. 81-3-100) were performed in connection with a project of the Kentucky Agriculture Experimental Station and the paper is published with the approval of the Director.     

Differential responses of soybean genotypes to excess manganese in an acid soil

To determine the tolerance of soybean genotypes to Mn toxicity, a green house study was conducted. Hayesville sandy loam (clayey, oxidic, mesic, Typic Hapludult), high in manganese, was used for the experiment. The experimental design was split-plot with three replications. Forty-one different soybean genotypes were planted in pots at two different pH levels: 5.2 (original soil pH) and 6.4 (amended with lime). Soybean genotypes were allowed to grow to the dry pod stage.Soil pH levels affected the soybean genotypes yields significantly (p < 0.01). Tolerant genotypes showed a higher or similar seed yield at pH 5.2 compared to pH 6.4. Sensitive genotype yields were lower at pH 5.2 than at pH 6.4. In general, Mn in leaves was higher at pH 5.2 than at pH 6.4. Some of the sensitive genotypes at pH 5.2. showed severe chlorosis and crinkle leaf symptoms as a result of Mn toxicity. Excess available Mn at pH 5.2. induced Ca deficiency. Soybean genotypes PI423758, PI417440, Aoda, Kingston, Rokusum and some others were tolerant to Mn toxicity, whereas PI417288, Verde, Wilson 5, Sango, Funk Delicious and some others were sensitive to Mn toxicity. The genotypes found to be tolerant can be recommended to plant breeders for development of Mn-tolerant cultivars.     

Effect of soybean plant growth on spore production byGlomus mosseae

Plants of two soybean cultivars infected withGlomus mosseae were physiologically stressed by top removal and were harvested at seven bi-weekly intervals. Removing tops stopped root growth, stimulated branching, delayed plant growth stages by approximately two weeks, but did not affect spore production. Spore numbers were significantly related only to time of harvest. Pot variation in spore number was not significantly correlated with infection percentage or root dry weight. Harvest, cultivar, and cutting effects were highly significant for root and shoot weights and root/shoot ratios.Approved by the Director as a contribution from the Missouri Agricultural Experiment Station (Journal Series No. 8123).     

Ferulic acid uptake by soybean root in nutrient culture

Ferulic acid uptake by soybean root in nutrient culture was investigated by the depletion method at different concentrations, temperatures and pH. Results showed that soybean roots absorbed this compound at greater rates in the concentrations between 0.05-mM and 1.0-mM and it was concentration dependent. Ferulic acid uptake was unaffected at pH 4.5 or 6.0 but reduced at pH 7.0. At pH 6.0, uptake rates decreased significantly with increasing temperature of nutrient solution.     

Interactions between iron-deficiency chlorosis and soybean cyst nematode in Minnesota soybean fields

Experiments were conducted in four commercial fields differing in severity of iron-deficiency chlorosis (IDC), and soybean cyst nematode (SCN) in Waseca and Lamberton, Minnesota to determine the interaction between the IDC and SCN. Each experiment was a randomized complete block with a factorial treatment design including 23 cultivars with or without traits of resistance to SCN, and IDC. The study illustrated the interactive effects of the two defensive traits on the diseases and soybean yields. IDC rating was higher in SCN-susceptible than SCN-resistant soybean, suggesting SCN infection increased IDC. Resistance to IDC apparently increased SCN reproduction due to better soybean plant growth. Yield response to the defensive traits depended on the disease pressures in a field. When both IDC and SCN were present in a field, deploying SCN-resistance was the best solution to the problems. However, SCN-resistance suppressed soybean yields when used in fields without the disease problems. IDC-resistance increased yield of SCN-susceptible cultivars, but it did not result in detectable yield benefit of SCN-resistant cultivars in SCN-infested sites. Effective use of the defensive traits for management of IDC and SCN requires specific knowledge of the disease problems present in a field. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U. S. Department of Agriculture and the University of Minnesota.     

Effects of phosphorus and iron fertilizers on the growth of two soybean varieties at two soil temperatures

The influence of FeEDDHA (0, 0.2 and 2 μg Fe g⁻¹ soil) and NaH₂PO₄·H₂O (0 and 120 μg Pg⁻¹ soil) on the growth of two Fe-ineffective soybean (Glycine max L. Merr.) varieties (anoka and T203) on a calcareous soil at two soil temperatures (16 and 24°C) was compared under greenhouse conditions. The two soybean varieties differed in the following respects: (a) T203 accumulated smaller concentrations of Fe in washed tops than Anoka under comparable conditions; (b) T203 was more susceptible to Fe deficiency and its accentuation by high levels of fertilizer P than Anoka; (c) T203 accumulated lower quantities of Mn in tops than Anoka under comparable conditions; (d) T203, but not Anoka, developed Mn deficiency symptoms when treated with P and 2 μg Fe g⁻¹ at 16°C. Fe deficiency was more severe in both varieties at the higher soil temperature due apparently to: (a) greater plant concentration of P in tops at 24°C; and/or (b) an increased rate of plant growth and greater dilution of Fe in young tissue at 24°C. Foliar P concentration was increased much more than foliar Fe concentration by an increase in soil temperature. Severely Fe deficient T203 plants grown without FeEDDHA at 24°C accumulated less foliar Mn than their FeEDDHA counterparts. Comparisons of Fe effectiveness of various soybean cultivars based on relative responses to FeEDDHA can be influenced by differential effects on Mn nutrition.     

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.     

The influence of soybean planting density on dinitrogen fixation and yield

We investigated the effect of planting density on soybean (Glycine max (L.) Merr.) yield in glasshouse and field experiments. Because net canopy photosynthesis increases with increasing plant density, we hypothesized that increasing planting density would result in increasing rates of dinitrogen fixation in soybeans and higher yields per unit land area.In glasshouse studies, Wayne variety soybeans were planted in 10-cm diameter pots, 1 plant pot^-1 in matrices of 10-, 15-, 20-, 25-, or 30-cm equidistant intervals. Bradyrhizobium japonicum inoculum was added to half of the plants in each treatment. Replicate measurements of total stem height, internode lengths, leaf mass, stem mass, root mass, nodule number, nodule mass, and nitrogenase activity were obtained at 3, 6, and 9 weeks post-emergence. Fruits were harvested and counted at week 14. As planting density increased there were (1) altered morphology and growth rates, (2) increased apparent specific nodule activity (SNA), (3) decreased nodule number and mass, and (4) nearly constant fruit and seed production/plant. Expressed on a unit area basis, nitrogen influx and yield increased geometrically as planting density increased, with maximum values observed for 10-cm plantings.Field studies of Wayne, Stein, Williams, and Gold Harvest soybean varieties were made in 1985. Plots were established containing 100 plants spaced at 10-, 20-, and 30-cm distances. Measurements made during the growing season and at harvest established the same relative trends identified from the glasshouse studies. Increasing plant densities resulted in higher yields per unit land. Varietal differences were almost significant.     

Transformation of soybean via particle bombardment of embryogenic suspension culture tissue

Summary Embryogenic suspension culture tissue of soybean (Glycine max Merrill.) was bombarded with particles coated with plasmid DNAs encoding hygromycin resistance andβ-glucuronidase (GUS). One to two weeks after bombardment, embryogenic tissue was placed in a liquid proliferation medium containing hygromycin. Four to six weeks after bombardment, lobes of yellow-green, hygromycin-resistant tissue, which began as outgrowths on brown clumps of hygromycin-sensitive tissue, were isolated and cultured to give rise to clones of transgenic embryogenic material. In vivo GUS assays of hygromycin-resistant clones showed that the early outgrowths could be negative, sectored, or positive for GUS activity. Transgenic, fertile plants could be routinely produced from the proliferating transgenic embryogenic clones. Southern hybridization analyses confirmed stable transformation and indicated that both copy number and integration pattern of the introduced DNA varied among independently transformed clones. Hybridization analysis of DNA from progeny plants showed genetic linkage of multiple copies of introduced DNA. An average of three transgenic clones were obtained per bombardment making this procedure very suitable for transformation of soybean.     

Yield and composition of soybean seed as a function of potassium supply

Summary A reduction in K supply to soybean plants to deficiency levels during both vegetative and reproductive development resulted in reductions not only in yield, but also in oil and K concentrations in the seed and a concomittant increase in seed protein concentration. Correlations between mean fruit yield and oil, protein and K concentrations, over a wide range of K regimes, were 0.97, −0.94 and 0.98, respectively. When K supply was increased well above the level necessary to produce maximum yields,i.e. luxury consumption, there was no significant change in K concentration in the seed, indicating a high degree of control in the movement of K to the developing seed under high K regimes. When the K supply to the plant was limiting, the rate of accumulation of oil and carbohydrate fractions, but not of seed protein, declined during the latter part of podfilling. This resulted in a fall in the C/N ratio in the non-structural seed components during this part of seed development. Depriving plants of K only during seed development had no effect on seed composition or yield, whereas resupplying K to deficient plants after anthesis resulted in almost the same seed composition and yield as that which occurred with control plants. Possible mechanisms whereby K deficiency influences soybean seed composition and yield are discussed in terms of movement of carbohydrate and nitrogen to the seed. We suggest that potassium-deficient soils are likely to produce crops with low yields and low seed oil levels; the crop may respond to K fertilizers as late as anthesis.     

Nitrogen accumulation and redistribution in soybean genotypes with variation in seed protein concentration

Breeding for high seed protein concentration in soybean [Glycine max (L.) Merrill] often results in lower yield, but the basis for this negative relationship is not well understood. To address this question, we evaluated the N acquisition characteristics of three high protein and three normal soybean genotypes in the field for 3 years. Plants were grown in 0.76 m rows following conventional cultural practices and water stress was minimized with sprinkler irrigation. We determined the mass and N concentration of leaves, petioles and stems at the beginning of seed filling (growth stage R5) and of stems at maturity. The N concentration of abscised leaves and petioles was also determined. There was significant variation among genotypes in total seed N (g m⁻²) at maturity (range from 14.7 to 24.4 g N m⁻²) as a result of variation in seed N concentration and yield. There was no evidence that the larger amounts of mature seed N were associated with a larger vegetative N reservoir at growth stage R5 as determined by vegetative mass at R5 or the concentration of N in vegetative tissues. Increasing seed N at maturity did not lower the N concentration in abscised leaves and petioles, or in the stems at maturity. The rate and timing of leaf senescence (loss of chlorophyll) was essentially the same for all genotypes. With no increase in the contribution from redistributed N, increases in N uptake or fixation during seed filling must have been responsible for the higher levels of seed N at maturity in high-protein genotypes. These data suggest that increasing total seed N at maturity by selecting for higher seed protein concentration or higher yield in soybean does not require, as some models suggest, a larger vegetative N reservoir at the beginning of seed filling or more rapid senescence.