The genomic relationships among six wild perennial species of the genus Glycine subgenus Glycine Willd.

Summary Based on meiotic chromosome behavior in intra- and interspecific hybrids, genome symbols were assigned to the following diploid (2n=40) Glycine species: G. canescens = AA; G. clandestina- Intermediate pod (Ip)=A ₁ A ₁; G. clandestina-Short pod (Sp)=BB; G. latifolia = B ₁ B ₁; G. tabacina = B ₂ B ₂; G. cyrtoloba = CC; and G. tomentella = DD. Genome symbol GG was reserved for the soybean, G. max. At metaphaseI, loose chromosome associations were observed in completely sterile interspecific hybrids whose parents differed in their genomes, suggesting some chromosome homologies among species. Although G. clandestina-Sp, G. latifolia and G. tabacina are morphologically distinct species, they differ only by a paracentric inversion. Similar observations were recorded for G. canescens and G. clandestina-Ip. Evidence is presented that demonstrates that G. tabacina (2n=80) and G. tomentella (2n=78, 80) are allotetraploid species complexes. Hybrid weakness, sterility, seedling lethality and seed inviability were found in intra- and interspecific hybrids.This research was supported in part by the Illinois Agricultural Experiment Station and the U.S. Department of Agriculture (Special grant 82-CRSR-2-2007). Travel grants to collect Glycine germplasm were received from the Rockefeller Foundation, the Illinois Soybean Program Operating Board, the National Science Foundation (INT76-14753) and the International Board for Plant Genetic Resources     

Wide hybridization between Brazilian soybean cultivars and wild perennial relatives

Employing a different culture strategy, we obtained a greatly improved frequency of embryo rescue in intersubgeneric soybean hybrids. Successful crosses were obtained in 31 different genotype combinations between nine Brazilian soybean lines as the female parents and 12 accessions from Glycine canescens, G. microphylla, G. tabacina and G. tomentella. The hybrid pod retention rate dropped to about 10% during the first 8 days after pollination and stayed largely unchanged up to the 20th day. Immature harvested seeds fell into three size groups: Group 1, smaller than 1.3 mm (mostly empty seed coats); Group 2, 1.9–5.0 mm; Group 3, larger than 5 mm (from selfing). A total of 90 putative hybrid embryos were rescued using a highly enriched B5 medium to nourish the newly dissected embryos. The growing embryos were then placed in a high osmotic, modified B5 medium to induce maturation and dormancy. Schenk and Hildebrandt medium was used to germinate the dormant, partially dehydrated, physiologically mature embryos. Approximately 37% of the rescued embryos developed into plantlets in vitro, and approximately 8% grew into mature plants in the greenhouse. Morphological, cytological and isoenzyme patterns confirmed the hybrid status of all seven mature plants, all of which were generated using G. tomentella G 9943 as the paternal parent. It was observed that all soybean lines crossed with G 9943 were capable of producing mature hybrid plants. There was no correlation between the initial size of Group 2 seeds and plant survival rate. The hybrids were cloned by grafting and treated with colchicine. One of the treated plants displayed chromosome doubling.     

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     

Genomic relationships among diploid wild perennial species of the genus Glycine Willd. subgenus Glycine revealed by crossability,meiotic chromosome pairing and seed protein electrophoresis

Summary The nomenclature of species beased on classical taxonomy can be verified from cytogenetic, biochemical and molecular studies. The objective of the study presented here was to provide further information on genomic affinities among species of the genus Glycine Willd. based on crossability, meiotic chromosome pairing of F₁ hybrids and seed-protein profiles. Meiotic chromosome pairing data revealed no genomic similarity between G. microphylla (BB) and G. falcata (FF), nor between G. tomentella (2n = 38; EE) and G. microphylla (BB). Despite morphological similarity between G. cyrtoloba (CC) and G. curvata no F₁ hybrid was obtained, although 748 flowers were pollinated. The seed-protein banding patterns showed G. latrobeana to be closer to the A-genome species than to others. Based on these results we assign genome symbol A₃A₃ to G. latrobeana. Likewise, G. curvata was allotted the designation C₁C₁ because the seed-protein banding patterns of G. curvata and G. cyrtoloba are similar. The genome designations of Glycine species based on cytogenetic investigations may be further extended by results obtained from biochemical and molecular approaches.Research supported in part by the Illinois Agricultural Experiment Station and US Department of Agriculture Competitive Research Grant 88-37231-4100     

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

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

Intergeneric hybridization between Diplotaxis siifolia,a wild species and crop brassicas

Summary Attempts were made to obtain intergeneric hybrids between Diplotaxis siifolia, a wild species, and cultivars of Brassica (B. campestris, B. juncea, and B. napus). The crosses showed unilateral incompatibility. When the wild species was used as female parent, pollen germination and pollen tube growth were normal, but hybrid seeds aborted due to post-fertilization barriers. Reciprocal crosses (cultivars as female parent) showed strong pre-fertilization barriers; although pollen grains showed germination, pollen tubes failed to enter the stigma. Hybrids were realized in two of the crosses, D. siifolia x B. juncea and D. siifolia x B. napus, through ovary culture. The hybrids were multiplied in vitro by multiplication of axillary shoots, or somatic embryogenesis. Detailed studies were carried out on the hybrid D. siifolia x B. juncea. F₁ hybrids had shrivelled anthers and were pollen sterile. Amphiploids of this hybrid showed 60% pollen fertility and produced seeds upon self-pollination as well as backcross pollination with the pollen of B. juncea.     

Plant regeneration from seedling explants of perennial Glycine species

More than 5000 cultures, from 30 accessions of six Glycine species, were established to assess the rôle of plant genotype in the response to an agar-solidified culture medium containing B5 salts and vitamins, 3% w/v sucrose, 1.1 mg 1^–1 BAP and 0.005 mg 1^–1 IBA, already known to induce shoot regeneration in callus of G. clandestina. Shoot initiation was obtained in a variety of explants from G. canescens, G. falcata, G. latrobeana and G. tomentella. With the exception of G. latrobeana, development of buds into shoots followed transfer to B5-based medium with 0.2 mg^–1 BAP and 0.005 mg 1^–1 IBA. Shoots readily produced roots in hormone-free half-strength B5 medium. In G. latrobeana, both extension and rooting occurred on this medium. Shoot regeneration was obtained in 12 of 30 accessions evaluated, but one accession of G. canescens, G1171, produced shoots and plantlets at a consistently higher frequency than other accessions, with plantlet recovery in more than 70% of the cultures. Bud formation in callus of G. canescens G1171 also occurred if BAP was replaced by 1.0 mg 1^–1 kinetin, 2i-p or zeatin, albeit at a lower frequency.     

Chloroplast DNA evidence for non-random selection of females in an outcrossed population of soybeans [Glycine max (L.)]

Summary Restriction fragment length polymorphisms (RFLPs) were used to assess chloroplast DNA (cpDNA) variation in a population of soybeans subjected to continuous cycles of forced outcrossing. This population was derived by crossing 39 female lines with four male-sterile (Ms₂ms₂) maintainer lines and advancing each generation by selecting only outcrossed seed borne on male-sterile (ms₂ms₂) plants. Analysis of the original 39 female lines revealed three groups based on cpDNA RFLPs. These three groups had been previously documented in soybeans, and the distribution of these groups among the female parents of this population was similar to that observed in germ plasm surveys of soybean. Thirty-four of the female parents had group I cpDNA, 3 had group II, and 2 had group III. Plants collected from this population after seven cycles of outcrossing were scored for four morphological traits (flower color, pubescence color, seed color, and pubescence type) known to be controlled by alleles at single nuclear loci. The frequencies of the phenotypes observed in this study indicated that the population underwent random mating with respect to flower and pubescence color, but deviated from random mating at the other two loci. Analysis of 158 of these same plants collected from the population after seven cycles of outcrossing revealed no individuals with group II or group III cpDNAs. The fixation of the group I cpDNA marker in this outcrossing population was judged to result primarily from selection against individuals in the population with the rare cpDNAs.Published as Paper no. 9640, Journal Series, Nebraska Agric Res Div Project no. 12-187. This research was supported in part by the University of Nebraska-Lincoln Research Council and NIH Biomedical Research Support Grant # RR-07055     

Drought tolerance processes in soybeans

Fifteen soybean cultivars were evaluated in two water supply conditions, inducing or not a drought stress. Main canopy traits were measured several times during the reproductive period and, at maturity date, the yield components were estimated. Using principal components analysis, the main physiological functions involved in soybean drought tolerance are described: leaf cells enlargement and assimilates transport. These processes could be a good basis on which to define new selection criteria for soybean drought tolerance.     

Genetic structure of Glycine canescens,a perennial relative of soybean

Summary Allozyme variation as detected by starch gel electrophoresis was used to assess the extent and spatial organization of genetic variation across the entire range of Glycine canescens sensu lato. Eleven enzyme systems were assayed in 116 accessions of this taxon and 102 alleles were detected at a total of 31 loci. Eighty-one percent of loci were polymorphic. Most of this variation occurred between and very little within accessions. Three major groupings were detected. These groupings (groups 1, 2, and 3) also differed with respect to mean seed size and their geographic distribution. A further ten accessions stood out from these distinct groups. These accessions were most closely related to group 3 but were variable among themselves. In general, they were collected from highly dissected terrain, often in the remote interior of the continent. A final group of 18 problematic accessions (group X), originally tentatively identified as G. canescens on morphological grounds, was shown to be isozymically distinct from this species and was reclassified as one form of the polytypic species G. clandestina.     

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.     

MiniMax,a new diminutive <Emphasis Type="Italic">Glycine max</Emphasis> genotype with a rapid life cycle,embryogenic potential and transformation capabilities

We developed Glycine max cv MiniMax (PI643148) that has a rapid life cycle, short stature and characteristic simple sequence repeat (SSR) markers that could make it useful for mutant screening, functional genomics, genetic mapping and other studies involving soybeans. We demonstrate that MiniMax is able to make somatic embryos (SEs) that rapidly develop into plantlets. Thus, the rapid cycling habit carries over into aspects of plant regeneration. Chimaeras (having transformed roots with untransformed aerial stocks) have been produced rapidly under non-axenic conditions using Agrobacterium rhizogenes-mediated transformation. Part of these experiments involved the engineering an enhanced green fluorescent protein (eGFP) reporter cassette outside the multi-cloning site of a plant expression vector, permitting non-invasive visual screening of the transformed roots. The rapid cycling growth habit of MiniMax, its ability to efficiently generate SEs and ability to be transformed should prove useful for basic aspects of G. max molecular and genetic research.     

Fluorescentin situ hybridization to soybean metaphase chromosomes

Repetitive DNA sequences were detected directly on somatic metaphase chromosome spreads from soybean root tips using fluorescentin situ hybridization. Methods to spread the forty small metaphase chromosomes substantially free of cellular material were developed using protoplasts. The specific DNA probe was a 1.05 kb internal fragment of a soybean gene encoding the 18S ribosomal RNA subunit. Two methods of incorporating biotin residues into the probe were compared and detection was accomplished with fluorescein-labeled avidin. The rDNA probe exhibits distinct yellow fluorescent signals on only two of the forty metaphase chromosomes that have been counterstained with propidium iodide. This result agrees with our previous analyses of soybean pachytene chromosome [27] showing that only chromosome 13 is closely associated with the nucleolus organizer region. Fluorescentin situ hybridization with the rDNA probe was detected on three of the forty-one metaphase chromosomes in plants that are trisomic for chromosome 13.     

Effect of soil pH and sewage sludge on VA mycorrhizal infection of soybeans

Summary Small plots were amended in 1976 or 1978 with four kinds of sewage sludge. The sludges represented samples considered to be relatively free of heavy metals as well as sludges highly contaminated with heavy metals. Sludges were added to a silt loam soil at rates of 224 or 448 Mgha⁻¹. The soils were maintained at a high or low pH regime. In 1984, soybeans (Glycine max L. Merril. var. ‘Clark’) were planted and grown to the R4 stage. After harvest, roots were removed from the soil, washed, and examined for VA mycorrhizal infection. It was found that the heavy metal content of the sludge alone was generally not related to determining the extent of mycorrhizal infection. A heat treated sludge, high in heavy metals, exhibited the highest degree of mycorrhizal infection when the soil was maintained at a pH of 6.2. With this treatment, 52% of the root segments examined were infected by mycorrhiza. When the same sludge was added to a soil with a slightly lower pH (5.7) none of the roots examined were infected by mycorrhiza. When soybean roots were examined from soils that received no sludge and were maintained at either a low (5.6) or high (6.2) pH, there was no significant difference in mycorrhizal infection between the pH regimes. These results therefore indicate that sewage sludge may inhibit mycorrhizal infection if the sludge contains a high concentration of heavy metals and the sludge is applied to the soil with a low pH. Scientific Article No. A-4093 and Contribution No 7078 of the Maryland Agric. Exp. Stn., Dept. of Agronomy, University of Maryland, College Park, MD 20742.     

Interspecific hybridization of perennial Medicago species using ovule-embryo culture

Summary New interspecific hybrids between alfalfa (Medicago sativa L.) and several perennial Medicago species were obtained by embryo rescue techniques. The methodology, designated ovule-embryo culture, involved preculturing the fertilized ovule (10 to 20 days post-pollination) for a period of six to 12 days followed by excision and direct culture of the embryo. Placement of the hybrid embryo directly onto culture medium without the interim ovule culture was unsuccessful. Ovule culture to germination without removing the embryo also was unsuccessful. Ovule-embryo culture was essential for recovering interspecific hybrids between diploid alfalfa (2n=2x=16) and the following diploid (2n=2x=16) species: M. hybrida Traut., M. marina L., M. papillosa Boiss., M. rhodopea Velen. and M. rupestris M.B. In addition, trispecies hybrids between M. sativa x M. dzhawakhetica Bordz. F₁ hybrids (2n=3x=24) and either M. cancellata M.B. (2n=6x=48) or M. saxatilis M.B. (2n=6x=48) were obtained from ovuleembryo culture. Media manipulations using M. sativa x M. rupestris F₁ and first backcross generation embryos demonstrated the optimum concentration of 12.5 mM NH₄ ⁺ for successful embryo rescue; ammonium salt formulation (whether chloride, nitrate or sulfate) was not critical. From a few thousand crosses, hybrids between M. sativa and either M. rhodopea or M. rupestris were recovered relatively efficiently with 157 and 66 hybrids, respectively. However, only 13 hybrids between M. sativa and M. papillosa were obtained from more than 2,000 crosses, and just two hybrids each have been recovered from the combinations M. sativa x M. hybrida and M. sativa x M. marina from 2,000 to 3,000 crosses. The predominant chromosome number between diploid alfalfa and the other diploid perennial species was 2n=2x=16. Morphology of the hybrids was generally intermediate. Electrophoretic analysis of the F₁ hybrids and parental clones on uniform or gradient polyacrylamide gels demonstrated that peroxidase phenotypes could be used to confirm hybridity. For all interspecific combinations there was at least one peroxidase isozyme unique to the wild species that was present in the F₁ interspecific hybrid.     

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.     

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.     

Effect of vesicular-arbuscular mycorrhizae on the growth and yield of rice-stubble cultured soybeans

Summary Inoculation with vesicular-arbuscular (VA) mycorrhizal fungiGlomus fasciculatus, G. mosseae, G. etunicatus orAcaulospora scrobiculatus, increased plant dry weight and seed yields of pot-grown soybean plants in sterilized soil. Inoculation with a mixture ofG. fasciculatus, G. mosseae andG. etunicatus, orG. fasciculatus alone, increased seed yields and other agronomic traits of soybean plants grown in a no-tillage, rice-stubble field.