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     

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.     

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.     

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.     

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.     

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.     

Iron-deficiency chlorosis evaluation of soybean with tissue culture

Summary The objectives of this study were: (i) to develop a tissue culture technique for the evaluation of Fe efficiency in soybean, and (ii) to compare the laboratory technique with field Fe chlorosis scores. Nineteen genotypes that had low and high levels of Fe efficiency were evaluated in the laboratory and at five field locations. Friable callus was induced from epicotyl sections, weighed, and placed on two different modified Murashige and Skoog media; one low in -naphthaleneacetic acid and the other low in Fe. Callus growth was rated as lack of growth compared to respective controls. As an example, Fe-inefficient cultivars (Asgrow A3205 and Pride B216) had significantly reduced growth compared to Fe-efficient germ plasm lines (All and A14). Correlation between the laboratory and field chlorosis rating was highest for the low auxin medium (r ² = 0.78), although correlation for the low Fe medium was also significant (r ² = 0.72). These results show that in vitro evaluation for Fe efficiency can be a useful tool for plant breeders.     

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).     

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.     

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.     

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     

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.     

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.     

Physiological comparisons of two soybean cultivars differing in canopy photosynthesis. II. Variation in specific leaf weight,nitrogen, and protein components

Cultivar differences in canopy apparent photosynthesis (CAP) have been observed in soybean (Glycine max (L.) Merr.) but little is known about the physiological mechanisms which are responsible for such differences. This study was initiated to determine if variation in ribulose 1,5-bisphosphate carboxylase (RuBPCase) and soluble protein exists among cultivars which differ in CAP during reproductive growth. In addition, the relationship between specific leaf weight (SLW) and leaf protein was examined. Two Maturity Group VI cultivars, Tracy (high CAP) and Davis (low CAP), were grown in the field during 1979, 1980, and 1981 and in a greenhouse experiment. Leaves located at two canopy positions (topmost, fully expanded leaf and eighth node from the top) in 1979 and three canopy positions (those mentioned, plus the fourth node from the top) in 1980 and 1981 were sampled. Leaves at the two upper canopy positions exhibited greater SLW, RuBPCase m^–2, and soluble protein m^–2 than found at the eighth node down. Photosynthetic capacity of leaves at inner canopy regions was therefore affected by both light penetration into the canopy and leaf protein status. Over the three year period, the SLW was 23 percent and the soluble protein m^–2 leaf 21 percent greater in Tracy than in Davis. Although the trend in RuBPCase m^–2 leaf was not significant, it was consistently greater in Tracy in the field and greenhouse. No cultivar differences were observed when the proteins were expressed on a unit of leaf dry weight. The quantity of RuBPCase per unit leaf area was positively correlated with SLW with significant partial correlation coefficients of 0.62, 0.67, 0.35, and 0.82 for 1979, 1980, 1981, and the greenhouse study, respectively. Since these cultivars have similar leaf area indices during September, the greater SLW of Tracy is translated into more photosynthetic proteins per unit ground area and higher CAP rate.Abbreviations AP Leaf Apparent Photosynthesis - CAP Canopy Apparent Photosynthesis - DAP Days After Planting - DTT Dithothreitol - HEPES N-2-hydroethylpiperazine N-2 ethanesulfonic acid - LAI Leaf Area Index - LSD Least Significant Difference - PPFD Photosynthetic Photon Flux Density - PVP-40 Polyvinylpolypyrroledone (molecular weight, 4000) - RuBPCase Ribulose 1,5-bisphosphate Carboxylase - SLW Specific Leaf Weight     

The ms1 mutation in soybean: involvement of gametes in crosses with tetraploid soybean

Summary Previous studies indicated that ms1ms1 malesterile female-fertile soybean (Glycine max [L.] Merr.) plants can produce seeds with different ploidy levels. The codominant chlorophyll-deficient mutant y11 was used in attempts to understand the embryo-endosperm relationship in seed production in ms1ms1 plants and to determine the mechanism of gamete formation in the ms1 mutation. Crosses were conducted between yellow-green male-sterile plants (ms1ms1Y11y11) and green fertile tetraploid cultivars (Ms1Ms1Ms1Ms1Y11Y11Y11Y11) in the greenhouse in the summers of 1987 and 1988. A total of 2,007 cross-pollinations were made. Thirty hybrid seeds were obtained, and plants were analyzed for chromosome number, fertility, and color. All the hybrid seedlings were tetraploid and fertile. No triploids were found. Among the 30 F₁ plants, 7 were green (Y11Y11Y11Y11), 17 were green-yellow (Y11Y11Y11y11), and 6 were yellow-green (Y11Y11y11y11). The segregation ratio was close to the expected 1 green: 2 green-yellow: 1 yellow-green (X² = 0.38; 0.90>p>0.75). From the results of this experiment, we conclude that: (1) triploids were not produced by crossing diploid ms1ms1 soybean plants with tetraploid plants; (2) tetraploid progeny can be produced from these crosses by the fusion of 2n ms1 eggs, or fusion of other 2n gametophyte cells in the embryo sac with a 2x sperm from tetraploid plants; (3) the megaspore mother cell of male-sterile plants undergoes meiotic division without cytokinesis after telophase II and forms more than the normal number of gametes, which can fuse with each other to generate tetraploid gametophyte cells.Joint contribution: U.S. Department of Agriculture, Agricultural Research Service, Cereal and Soybean Research Improvement Unit, Midwest Area, and Journal Paper No.-13838 of the Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa     

Separately and simultaneously induced dark chilling and drought stress effects on photosynthesis, proline accumulation and antioxidant metabolism in soybean

The effects of separately or simultaneously induced dark chilling and drought stress were evaluated in two Glycine max (L.) Merrill cultivars. For the separately induced dark chilling treatment (C), plants were incubated at 8 °C during 9 consecutive dark periods. During the days, plants were kept at normal growth temperatures. For the separately induced drought treatment (D), plants were maintained at normal growth temperatures without irrigation. For the simultaneously induced dark chilling and drought stress treatment (CD), plants were dark chilled without irrigation. All treatments caused similar decreases in pre-dawn leaf water potential, but resulted in distinct physiological and biochemical effects on photosynthesis. In Maple Arrow, where C had the smallest effect on photosynthesis, prolonged CD caused less inhibition of photosynthesis compared to D. Compared to Fiskeby V, the photosynthetic apparatus of Maple Arrow appears to possess superior dark chilling tolerance, a property which probably also conveyed enhanced protection against CD. Proline accumulation was prevented by CD at the ψ_PD where D already resulted in considerable accumulation. The superior capacity for proline accumulation in Maple Arrow would seem to be an important factor in its stress tolerance. Antioxidant activity evoked by CD and D was higher than for C alone. In Fiskeby V, the small increase in ascorbate peroxidase (EC 1.11.1.7) activity, which was in most cases not accompanied by increased gluthatione reductase (EC 1.6.4.2) activity, could impact negatively on its stress tolerance. These results demonstrate large genotypic differences in response to chilling and drought stress, even between soybean cultivars regarded as chilling tolerant.