Perennial Glycine Research Articles

Genomic relationships in the genusGlycine (Fabaceae: Phaseoleae): use of a monoclonal antibody to the soybean Bowman‐Blrk inhibitor as a genome marker

We investigated the use of a monoclonal antibody (MAb 238) to the soybean Bowman‐Birk inhibitor (BBI) to verify and understand the intergenomic relationships among the wild perennial Glycine species. Competitive enzyme linked immunosorbent assay and western blot screening studies revealed that the accessions of B‐genome (G. latifolia, G. microphylla, and G. tabacina, 2n = 40) and C‐genome (G. curvata and G. cyrtoloba) species did not contain the MAb 238 crossreactive proteins (BBI‐nulls). By contrast, all the A‐genome (G. argyrea, G. canescens, G. clandestina, and G. latrobeana), E‐genome (G. tomentella, 2n = 38), and F‐genome (G. falcata) species, G. arenaria (genome unknown), and the polyploid (2n = 78,80) G. tomentella accessions were BBI‐positive. The D‐genome G. tomentella (2n = 40) and tetraploid G. tabacina (2n = 80) contained both BBI‐null and BBI‐positive type accessions. Among the recently described species, G. hirticaulis (2n = 40), G. lactovirens, and G. pindanica contained the MAb 238 crossreactive proteins while G. albicans did not. Glycine hirticaulis, G. pindanica, and G. tomentella (2n = 38) displayed highly similar MAb 238 crossreactive isoelectric focusing banding patterns, indicating that they are genomically close to each other. Glycine hirticaulis was found to have both diploid (2n = 40) and tetraploid (2n = 80) cytotypes. We demonstrated that the MAb 238 was specific to the trypsin inhibitor domain of the BBI. The MAb 238 clearly reflected all the previously established relationships in the genus Glycine, validating its use as a genome marker.

Genomic Relationships in the Genus Glycine (Fabaceae: Phaseoleae): Use of a Monoclonal Antibody to the Soybean Bowman-Birk Inhibitor as a Genome Marker

We investigated the use of a monoclonal antibody (MAb 238) to the soybean Bowman-Birk inhibitor (BBI) to verify and understand the intergenomic relationships among the wild perennial Glycine species. Competitive enzyme linked immunosorbent assay and western blot screening studies revealed that the accessions of B-genome (G. latifolia, G. microphylla, and G. tabacina, 2n = 40) and C-genome (G. curvata and G. cyrtoloba) species did not contain the MAb 238 crossreactive proteins (BBI-nulls). By contrast, all the A-genome (G. argyrea, G. canescens, G. clandestina, and G. latrobeana), E-genome (G. tomentella, 2n = 38), and F-genome (G. falcata) species, G. arenaria (genome unknown), and the polyploid (2n = 78,80) G. tomentella accessions were BBI-positive. The D-genome G. tomentella (2n = 40) and tetraploid G. tabacina (2n = 80) contained both BBI-null and BBI-positive type accessions. Among the recently described species, G. hirticaulis (2n = 40), G. lactovirens, and G. pindanica contained the MAb 238 crossreactive proteins while G. albicans did not. Glycine hirticaulis, G. pindanica, and G. tomentella (2n = 38) displayed highly similar MAb 238 crossreactive isoelectric focusing banding patterns, indicating that they are genomically close to each other. Glycine hirticaulis was found to have both diploid (2n = 40) and tetraploid (2n = 80) cytotypes. We demonstrated that the MAb 238 was specific to the trypsin inhibitor domain of the BBI. The MAb 238 clearly reflected all the previously established relationships in the genus Glycine, validating its use as a genome marker.

Backcross (BC2‐BC4)‐Derived Fertile Plants from Glycine max and G. tomentella Intersubgeneric Hybrids

Wild perennial relatives of the soybean [Glycine max (L.) Merr.] have not been utilized to broaden the genetic base of the crop. This study provides information on production, morphology, cytology, and breeding behavior of backcross (BC2 to BC4)‐derived fertile plants from soybean (2n = 40, genome GG) and G. tomentella Hayata (2n = 78, genome DDEE). The main hurdle was to obtain BC~ plants (G. max cv. Altona) × (G. tomentella, Pl 483218)] → F1, 2n = 59, genome GDE → colchicine treatment (CT) → 2n = 118, GGDDEE × soybean cv. Clark 63 → BC1, 2,n = 76 (expected 2n = 79). Three sterile BC2 plants (2n = 58, 2n = 56, 2n = 55) were produced. Procedures for immature seed‐rescue were utilized to obtain F, amphiploid, BC1 and BC2 plants. Soybean cv. Clark 63 was used as the recurrent parent in the backcrossing programs. The range of chromosome number among BC3 plants was 2n = 41 to 2n = 52, and among BC4 plants was 2n = 40 to 64. A hypertriploid (2n = 64) in BC4 plant originated after fertilization of an unreduced egg (n = 44) by a normal haploid (n = 20) male gamete. Phenotypes of BC3 and BC4 plants were dissimilar but generally resembled closely to the soybean cv. Clark 63. We expect to isolate a series of aneuploid lines from BC3 to BC6 generations that may open up the path to introgress desired traits from wild perennial Glycine species to the cultivated soybean.

Genotypic variation among Glycine spp. for ability to acclimate to saturated soil

The ability to acclimate to, and grow well under saturated soil culture conditions (SSC) is a ubiquitous trait in soybean (Glycine max) but little information has been reported about this novel adaptive trait in wild Glycine spp. The ability of 39 diverse accessions from one wild annual and eight wild perennial Glycine spp. to acclimate to, and grow in SSC was therefore evaluated, and responses compared with those of three cultivars of soybean. Plants were exposed to SSC 26 days after sowing, and measurements made, after a further month, of the number of nodules, leaf area, and dry matter accumulation of roots, stems and leaves. None of the wild perennial Glycine spp.(subgenus Glycine) acclimated to SSC. Necrotic tissue developed in some perennial accessions as soon as six days from the imposition of SSC, and all of the perennial accessions died during the experiment. In contrast, both of the annual species within subgenus Soja (soybean, and its presumed wild progenitor, G. soja) acclimated to SSC. The subsequent rate of recovery tended to be slower in G. soja than soybean. The responses are consistent with the hypothesis that the ability to acclimate to SSC is a wild-type trait, expression of which was amplified in soybean during domestication in rice-based agriculture.

Characterization of trypsin and chymotrypsin inhibitors in the wild perennial Glycine species

Seven wild perennial species were used for characterizing the seed protease inhibitors in the genus Glycine Willd. subgenus Glycine to determine the presence and examine the variability and expression patterns. Seeds of all the species contained trypsin and chymotrypsin inhibitors. There were highly significant variations among the wild perennial species in the electrophoretic profiles of trypsin and chymotrypsin inhibitors, migration patterns of anti-KTI and anti-BBI (mAB 238)immunocrossreactive proteins, and trypsin and chymotrypsin inhibitor activities of seeds

Sources of Resistance to Soybean Rust in PerennialGlycineSpecies

Accessions of 12 perennial Glycine species were evaluated for resistance to Phakopsora pachyrhizi, the causal agent of soybean rust. A total of 23% of the accessions were resistant, 18% were moderately resistant, and 58% were susceptible. In two experiments, 59 and 40% of the accessions of G. tabacina (2n=80) were resistant. Resistance to P. pachyrhizi was identified in accessions of G. argyrea, G. canescens, G. clandestina, G. latifolia, G. microphylla, and G. tomentella, but not in accessions of G. arenaria, G. cyrtoloba, G. curvata, and G. falcata

Examination of 2,4-D tolerance in perennial Glycine species

High levels of tolerance to 2,4-D [(2,4-dichlorophenoxy)acetic acid] were expressed at the cell and whole plant level by several accessions of wild perennial Glycine species. To determine the mechanism(s) responsible for tolerance, heterotrophic cell suspension cultures of soybean [ Glycine max (L.) Merr.] and four perennial Glycine accessions were used to investigate[ 14C]2,4-D uptake and metabolism. Uptake of 2,4-D was similar between soybean and the perennials. However, soybean metabolized only 7% of the absorbed 2,4-D at 2 and 10 μ M concentrations. The four perennial accessions metabolized 2,4-D at least 79% at 10 μ M and 64% at 50 μ M 2,4-D. The primary metabolite produced by the tolerant perennial lines was the glycoside conjugate of 4-OH-2,5-D, as determined by high-performance liquid chromatography cochromatography and a gas chromatography-mass spectrometer. 2,4-D-sensitive soybean and the least tolerant perennial accession produced high quantities of amino acid conjugates of 2,4-D. Varying levels of 2,4-D tolerance among the perennial Glycine accessions may also be influenced by differential sensitivity of each accession to 2,4-D and/or metabolites.

Nutrient culture medium components affecting plant recovery from immature embryos of three Glycine genotypes and an interspecific hybrid grown in vitro

Immature embryos of Glycine max (L.) Merr. cv. ‘Bay’, G. tabacina (Labill.) Benth. and G. tomentella Hayata were cultured on 72 media combinations to identify a nutrient medium which would allow a greater percentage of interspecific plants to be recovered from cultured embryos of G. max × various perennial Glycine species. The highest mean plant recovery rate of 79% was from a medium containing ‘B5’ nutrient salts as reported by Gamborg et al. (1968), vitamin components according to Williams (1978) and 30 g/l sucrose. This is as compared with 25% from a medium used previously. In an additional test, 67% of hybrid embryos of G. max × G. tomentella were recovered from the same medium, from which G. tomentella was most effectively recovered in all testing.

Restriction fragment length polymorphism (RFLP) of wild perennial relatives of soybean

Total DNA from callus tissue of 28 accessions representing seven wild perennial Glycine species was compared using recombinant genomic probes derived from G. max, the soybean. Using two probes, we show that this molecular approach both confirms and extends the model for the taxonomic relationships between the species derived from morphological and cytogenetic data, and that it provides clear evidence that RFLP analysis of genomic sequences has the potential for revealing the derivation of the member species of the wild perennial Glycine taxon. Although, in this preliminary report, the sample size for each species is small, it is clear that the greatest between-accession variation occurs in G. tabacina (B2B2) and G. clandestine (A1A1), suggesting that these may be the taxa from which further speciation occurred in the subgenus.

Variation in Flowering Response to Photoperiod in Perennial Glycine Species

The wild perennial Glycine spp. offer a potential source of new germplasm for the improvement of soybean [Glycine max (L.) Merr.], but information is needed on useful characteristics in this material. The objective of this study was to determine the flowering response of the wild perennial Glycine species to different daylengths. Twenty‐five accessions of G. tomentella Hayata, one of G. arenaria Tind., and one of G. tabacina (Labill.) Benth. were grown under photoperiods of 8, 12, and 16 h at day/night temperature regimes of 28/24 and 24/20 °C in a phytotron. The accessions represent the latitudinal range within each of five diploid (D1–D5) and five tetraploid (T1–T5) isozymically distinct groups of G. tomentella, and include two previously allied accessions that now have been placed in separate species. The 12‐h daylength was most conducive to cleistogamous and chasmogamous flower development while the 16‐h daylength suppressed flowering. Plants generally took longer to reach comparable phenologicai stages of growth in the cooler temperature regime, but the initial flowers appeared at similar nodes in both warm and cool temperatures. Eight accessions flowered in all three daylengths. The accessions from North Queensland in the D4 (G1300, G1780) and T2 (G1769, G1146) isozyme groups were the least sensitive since they produced both flower types in all photoperiods tested and may be of value in soybean germplasm development.

Interspecific hybrids between the soybean and wild perennial relatives

Successful crosses were obtained in 390 different genotype combinations between the soybean, Glycine max (L.) Merr., and wild perennial Glycine species. A total of 1534 Immature embryos were excised prior to abortion and cultured, and 1.4 percent of these germinated to produce shoots representing 18 genotype combinations. Sixteen F1 Interspecific hybrids encompassing 12 different combinations were subsequently grown to maturity: 7 G. max (2n = 40) × G. tomentella Hayata (2n = 78), 3 G. tomentella × G. max, and 2 G. canescens F. J. Herm. (2n = 40) × G. max. The F1 hybrids differed morphologically depending on the genotype combination, and showed variable degrees of anther development. Hybrids with wild Glycine as the female parent gave the most normal anther development. Expected chromosome numbers of 2n = 59 and 2n = 40 were confirmed for G. tomentella and G. canescens hybrids, respectively. The F1 hybrids exhibited low chromosome pairing and perturbed melosis in pollen mother cells and were sterile. Eight hybrid combinations were doubled with colchicine to give 2n = 118 and 2n = 80 chromosomes for G. tomentella and G. canescens hybrids, respectively. Doubled plants exhibited high levels of chromosome pairing as expected, but remained highly sterile because of abnormal microspore formation. Fertility was partially restored in one G. max × G. tomentella (2n = 118), which set mostly one-seeded, cleistogamous pods at a low rate. Chromosome numbers of selfed progeny were similar to the doubled parent. Variation in viability and fertility shown by different hybrid combinations within one interspecific cross indicate that genotype interactions play a large part in determining the ease with which hybrids can be made between wild perennials and the soybean, and will most likely influence which Glycine collections represent potential candidates for gene exchange with the soybean.

Intersubgeneric Crossability in the Genus Glycine Willd.

AbstractIn intersubgeneric crosses between wild perennial Glycine species of submenus Glycine and soybeans, Glycine max (L.) Merr., of subgenus Soja, all the putative hybrid pods aborted from 9 to 30 days after pollination (DAP). Spraying of 70 ppm gibberellic acid on hybridized gynoecia facilitated pod retention. Fluorescence microscopy revealed normal pollen germination and pollen tube growth in all the intersubgeneric crosses except in the G. cyrtoloba × G. max cross.

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 role 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.

An intersubgeneric hybrid between Glycine tomentella Hayata and the soybean, G. max (L.) Merr.

The objective of the present paper is to provide information on the morphology and cytology of an intersub-generic hybrid (2n=59) between Glycine tomentellaHayata (2n=78) and G. max (L.) Merr. (2n=40) obtained through in vitro immature seed culture. The hybrid plant was slow in vegetative growth and twinning like the female parent but morphologically was intermediate between both parents for several traits. At metaphase I, the average chromosome associations and ranges for 25 cells were 44.0 I (37–51)+7.5 II (3–11). The plant was completely pollen and seed sterile. The present investigation suggests that wild perennial Glycine species can be exploited as either the male or female parent in wide hybridization programs with the soybean, G. max.

Interspecific Hybridization in Glycine Willd. Subgenus Glycine (Leguminosae)

The meiotic configurations of 12 new hybrid combinations among eight perennial Glycine species were analysed. These data together with those of Putievsky and Broué and of Newell and Hymowitz showed close genomic relationships among the diploid species G. canescens, G. clandestina and G. Latrobeana and the euploid tetraploid G. tomentella. Distant from these species was a second evolutionary branch, which included the diploids G. latifolia and an undescribed species, G. 'reducta'. The diploid G. tomentella (2n = 38, 40) appeared to be intermediate between these two lines. Interspecies hybrids using either the diploid species G. falcata or the tetraploid G. tabacina have so far been achieved with members of the G. canescens-related group, from which both species are very distant.

Interspecific hybridisation of soybeans and perennial Glycine species indigenous to Australia via embryo culture

Five sterile hybrids (2n=59) between a synthetic amphiploid of Glycine tomentella (2n=38) and G. caneseens (2n=40) as female and soybean cultivars Lincoln and Hark as males have been produced by embryo or ovule culture using transplanted endosperm. The hybrid plants are twining perennials like the female parent but possess a number of morphological characters which reflect the presence of the soybean genome. Indophenol oxidase isozymes from leaf extracts also provide good evidence of the hybrid nature of the cultured plants. These hybrids open the way for the exploitation of the diverse germplasm resources of the perennial Glycine species in soybean breeding.