<Emphasis Type="Italic">Soybean dwarf virus</Emphasis>-resistant transgenic soybeans with the sense coat protein gene

We transformed a construct containing the sense coat protein (CP) gene of Soybean dwarf virus (SbDV) into soybean somatic embryos via microprojectile bombardment to acquire SbDV-resistant soybean plants. Six independent T(0) plants were obtained. One of these transgenic lines was subjected to further extensive analysis. Three different insertion patterns of Southern blot hybridization analysis in T(1) plants suggested that these insertions introduced in T(0) plants were segregated from each other or co-inherited in T(1) progenies. These insertions were classified into two types, which overexpressed SbDV-CP mRNA and accumulated SbDV-CP-specific short interfering RNA (siRNA), or repressed accumulation of SbDV-CP mRNA and siRNA by RNA analysis prior to SbDV inoculation. After inoculation of SbDV by the aphids, most T(2) plants of this transgenic line remained symptomless, contained little SbDV-specific RNA by RNA dot-blot hybridization analysis and exhibited SbDV-CP-specific siRNA. We discuss here the possible mechanisms of the achieved resistance, including the RNA silencing.     

QTL analysis for resistance to Soybean dwarf virus in Indonesian soybean cultivar Wilis

Soybean dwarf virus (SbDV), a member of the Luteoviridae family, causes serious yield losses in soybean production in northern Japan. We previously found that an Indonesian soybean cv. Wilis had a high level of resistance to SbDV. Although Wilis is infected by SbDV, symptoms are always mild and develop considerably later compared with many susceptible cultivars. To identify the resistance gene(s) to SbDV in Wilis in a quantitative trait loci (QTL) analysis, we used 71 recombinant inbred lines derived from a cross between Wilis and the susceptible Japanese cv. Toyokomachi and a set of published simple sequence repeat (SSR) markers. The SbDV resistance in Wilis was mainly controlled by a single QTL located near the SSR marker Sat_271 on the linkage group A1. This QTL accounted for 79% of the phenotypic variance. A. Uchibori and J. Sasaki equally contributed to this work.     

Transformation of 12 different plasmids into soybean via particle bombardment

Particle bombardment offers a simple method for the introduction of DNA into plant cells. Multiple DNA fragments may be introduced on a single plasmid or on separate plasmids (co-transformation). To investigate some of the properties and limits of co-transformation, 12 different plasmids were introduced into embryogenic suspension culture tissue of soybean [Glycine max (L.) Merrill] via particle bombardment. The DNAs used for co-transformation included 10 plasmids containing KFLP markers for maize and 2 plasmids separately encoding hygromycin-resistance and ß-glucuronidase. Two weeks following bombardment with the 12 different plasmids, suspension culture tissue was placed under hygromycin selection. Hygromycin-resistant clones were isolated after an additional 5 to 6 weeks. Southern hybridization analysis of 26 hygromycin-resistant embryogenic clones verified the presence of introduced plasmid DNAs. All of the co-transforming plasmids were present in most of the transgenic soybean clones and there was no preferential uptake and integration of any of the plasmids. The copy number of individual plasmids was approximately equal within clones but highly variable between clones. While some clones contained as few as zero to three copies of each plasmid, others clones contained as many as 10 to 15 copies of each of the 12 different plasmids.     

Radiation as a tool to remove selective marker genes from transgenic soybean plants

The present study evaluated the use of γ-radiation to physically remove selective marker genes previously introduced into the soybean genome. Homozygous seeds from a transgenic soybean line carrying the gus and ahas transgenes were irradiated with γ-rays. Six plants presenting a deleted gus gene were analyzed by Southern blot to confirm removal of both ahas and gus genes. Line 1A presented an absence of the gus gene cassette and presence of the ahas gene cassette.     

Genetic characteristics of two genes for resistance to soybean mosaic virus in PI486355 soybean

Soybean [Glycine max (L.) Merr.] PI486355 is resistant to all the identified strains of soybean mosaic virus (SMV) and possesses two independently inherited resistance genes. To characterize the two genes, PI486355 was crossed with the susceptible cultivars Lee 68 and Essex and with cultivars Ogden and Marshall, which are resistant to SMV-G1 but systemically necrotic to SMV-G7. The F₂ populations and F₂₃ progenies from these crosses were inoculated with SMV-G7 in the greenhouse. The two resistance genes were separated in two F₃₄ lines, LR1 and LR2, derived from Essex x PI486355. F₁ individuals from the crosses of LR1 and LR2 with Lee 68, Ogden, and York were tested with SMV-G7 in the greenhouse; the F₂ populations were tested with SMV-G1 and G7. The results revealed that expression of the gene in LR1 is gene-dosage dependent, with the homozygotes conferring resistance but the heterozygotes showing systemic necrosis to SMV-G7. This gene was shown to be an allele of the Rsv1 locus and was designated as Rsv1-s. It is the only allele identified so far at the Rsv1 locus which confers resistance to SMV-G7. Rsv1-s also confers resistance to SMV-G1 through G4, but results in systemic necrosis with SMV-G5 and G6. The gene in LR2 confers resistance to strains SMV-G1 through G7 and exhibits complete dominance. It appears to be epistatic to genes at the Rsv1 locus, inhibiting the expression of the systemic necrosis conditioned by the Rsv1 alleles. SMV-G7 induced a pin-point necrotic reaction on the inoculated primary leaves in LR1 but not in LR2. The unique genetic features of the two resistance genes from PI486355 will facilitate their proper use and identification in breeding and contribute to a better understanding of the interaction of SMV strains with soybean resistance genes.     

The use of the two T-DNA binary system to derive marker-free transgenic soybeans

Summary A binary vector, pPTN133, was assembled that harbored two separate T-DNAs. T-DNA one contained a bar cassette, while T-DNA two carried a GUS cassette. The plasmid was mobilized into the Agrobacterium tumefaciens strain EHA101. Mature soybean cotyledonary node explants were inoculated and regenerated on medium amended with glufosinate. Transgenic soybeans were grown to maturity in the greenhouse. Fifteen primary transformants (T₀) representing 10 independent events were characterized. Seven of the 10 independent T₀ events co-expressed GUS. Progeny analysis was conducted by sowing the T₁ seeds and monitoring the expression of the GUS gene after 21 d. Individual T₁ plants were subsequently scored for herbicide tolerance by leaf painting a unifoliate leaf with a 100 mgl⁻¹ solution of glufosinate and scoring the leaf 5 d post application. Herbicide-sensitive and GUS-positive individuals were observed in four of the 10 independent events. Southern blot analysis confirmed the absence of the bar gene in the GUS positive/herbicide-sensitive individuals. These results demonstrate that simultaneous integration of two T-DNAs followed by their independent segregation in progeny is a viable means to obtain soybeans that lack a selectable marker.     

Characterization of soybean vegetative storage proteins and genes

Summary Soybean vegetative storage proteins (VSPs) were purified and characterized. Anion exchange HPLC resolved partially purified VSPs into fractions containing 27-kD/27-kD and 29-kD/29-kD homodimers and 27-kD/29-kD heterodimers. Reversed-phase HPLC resolved partially purified VSPs into three fractions. One fraction contained only 27-kD VSP and the other two contained 29-kD VSP. The two 29-kD VSP fractions differed with respect to their cyanogen bromide cleavage patterns, an observation that indicated the 29-kD VSPs were heterogeneous. Genomic clones that contained 29-kD VSP genes were also isolated and characterized. One genomic clone contained a complete 29-kD VSP gene and was sequenced. The coding region in the clone contained two introns whose borders had regulatory sequences typical of other eukaryotic genes. Putative polyadenlyation signals were present in the 3-flanking region of the gene, while putative TATA, CAAT, and enhancer core sequences were found in the 5-flanking regions. A second genomic clone that was studied contained the 5 regions of two partial 29-kD VSP genes in an inverted linkage. Genomic DNA gel blots showed that the two genes were organized in the same arrangement in the soybean genome.Cooperative research between USDA/Agricultural Research Service and the Indiana Agricultural Experiment Station. Journal Paper No. 12,192 from the Indiana Agricultural Experiment Station     

Analysis of the genes encoding the largest subunit of RNA polymerase II in Arabidopsis and soybean

We have cloned and sequenced the gene encoding the largest subunit of RNA polymerase II (RPB1) from Arabidopsis thaliana and partially sequenced genes from soybean (Glycine max). We have also determined the nucleotide sequence for a number of cDNA clones which encode the carboxyl terminal domains (CTDs) of RNA polymerase II from both soybean and Arabidopsis. The Arabidopsis RPB1 gene encodes a polypeptide of approximately 205 kDa, consists of 12 exons, and encompasses more than 8 kb. Predicted amino acid sequence shows eight regions of similarity with the largest subunit of other prokaryotic and eukaryotic RNA polymerases, as well as a highly conserved CTD unique to RNA polymerase II.The CTDs in plants, like those in most other eukaryotes, consist of tandem heptapeptide repeats with the consensus amino acid sequence PTSPSYS. The portion of RPB1 which encodes the CTD in plants differs from that of RPB1 of animals and lower eukaryotes. All the plant genes examined contain 2–3 introns within the CTD encoding regions, and at least two plant genes contain an alternatively spliced intron in the 3 untranslated region. Several clustered amino acid substitutions in the CTD are conserved in the two plant species examined, but are not found in other eukaryotes. RPB1 is encoded by a multigene family in soybean, but a single gene encodes this subunit in Arabidopsis and most other eukaryotes.     

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.     

Cotransformation frequencies of foreign genes in soybean cell cultures

Summary Through the use of electroporation and a soybean (Glycine max L.) protoplast system, we generated stably transformed cell lines expressing a number of foreign genes (neomycin phosphotransferase,-glucuronidase, chloramphenicol acetyl transferase, and phosphinothricin acetyl transferase). Selected and unselected marker genes were cointroduced either linked on a single plasmid or as separate plasmids. Calli expressing multiple genes were recovered, and Cotransformation frequencies were established for both cases. Our results show a 50% cotransformation frequency in the case of linked genes. In situations in which two genes are introduced on independent plasmids, cotransformation frequencies are 18%–27%. Similar rates of cotransformation were observed among various marker pairs.     

5S ribosomal gene variation in the soybean and its progenitor

Summary The soybean, Glycine max and its wild progenitor, Glycine soja, have been surveyed for repeat length variation for the nuclearly encoded 5S ribosomal RNA genes. There is little variation among the 33 accessions assayed, with a common repeat length of 345 bases being typical of both taxa. A 334 base size variant was encountered in individuals from two populations of G. soja from China. The low level of variability is in marked contrast to the variation observed within and between the species of the perennial subgenus Glycine.     

Macronutrient concentrations of soybean infected with soybean cyst nematode

Soybean cultivars (Glycine max(L.) Merr.) infected with soybean cyst nematode (SCN; Heterodera glycinesIchinohe) often show symptoms similar to K deficiency. The objectives of this experiment were to determine if SCN infection affected macronutrient concentrations in soybean seedling vegetative tissues, determine whether increased K fertility can overcome these possible effects, and to determine if these possible effects are localized at the site of infection or expressed systemically throughout the root system. Soybean plants were grown with root systems split into two halves. This allowed differential K (0.2, 2.4 and 6.0 mM K nutrient solutions) and SCN (0 and 15 000 eggs/plant) treatments to be applied to opposite root-halves of the same plant. Thirty days after plants were inoculated with SCN, macronutrient concentrations of shoot and root tissues were determined. Potassium concentration in leaf blades was not affected; but K concentrations in leaf-petiole and stem tissues were increased with SCN infection. Roots infected with SCN contained lower K concentrations than uninfected roots, but only for the 2.4 mM K treatment. Thus, at the medium level of K fertility, SCN reduced K concentration in soybean roots, and increasing K fertility to the high level overcame the effect. Because K concentrations in the shoot tissues were not reduced by SCN infection, above ground portions of the plant may be able to overcome limitations that occur in roots during the first 30 days of infection. Increasing K fertility level in soybean fields may not benefit vegetative growth of soybean infected with SCN.     

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.     

Restriction fragment length polymorphism diversity in soybean

Summary Fifty-eight soybean accessions from the genus Glycine, subgenus Soja, were surveyed with 17 restriction fragment length polymorphism (RFLP) genetic markers to assess the level of molecular diversity and to evaluate the usefulness of previously identified RFLP markers. In general, only low levels of molecular diversity were observed: 2 of the 17 markers exhibited three alleles per locus, whereas all others had only two alleles. Thirty-five percent of the markers had rare alleles present in only 1 or 2 of the 58 accessions. Molecular diversity was least among cultivated soybeans and greatest between accessions of different soybean species such as Glycine max (L.) Merr. and G. soja Sieb. and Zucc. Principal component analysis was useful in reducing the multidimensional genotype data set and identifying genetic relationships.     

Conditional lethality involving nuclear and cytoplasmic chlorophyll mutants in soybeans

Summary A conditionally lethal phenotype occurred when a nuclear chlorophyll mutant (y ₂₀-k ₂) was present with a cytoplasmic chlorophyll mutant (cyt-Y ₂) in soybean (Glycine max [L.] Merr.). Nuclear mutant y ₂₀-k ₂, Genetic Type Collection Number T253, has yellow foliage, tan-saddle-pattern seed and is viable. The y ₂₀-k ₂ mutant cannot be separated by classical genetic tests into two separate components, y ₂₀ (yellow foliage) and k ₂ (tan-saddle-pattern seed). Mutant cyt-Y ₂, T275, is inherited cytoplasmically, has yellow foliage, and is viable. The genotype cyt-Y ₂ y ₂₀-k ₂/ y ₂₀-k ₂ is a conditional lethal; the genotype is lethal under field conditions, but plants survive under greenhouse conditions. This interaction is unique to y ₂₀-k ₂. This conditionally lethal genotype may be useful in molecular studies on the interaction between nuclear and plastid genomes.This is a joint contribution of North Central Region, USDA ARS, and Journal Paper No. J-11429 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, and the Agriculture Experiment Station, Univ. of Puerto Rico, Mayaguez Campus, Mayaguez, Puerto Rico 00708. Projects 2471 and 2475. The research was supported in part by the Iowa Soybean Promotion Board.     

Generation of transgenic potato plants highly resistant to potato virus Y (PVY) through RNA silencing

In this study we applied RNA silencing to engineer potato plants that are resistant to potato virus Y (PVY). We expressed double-stranded (ds) RNA derived from the 3 terminal part of the coat protein gene of PVY, which is highly conserved in sequence amongst different PVY isolates, in transgenic potatoes of the commercial variety Spunta. Transgenic plants were analyzed for generation of transgene-derived short interfering RNAs (siRNAs) prior to virus inoculation. Twelve of fifteen transgenic lines produced siRNAs and were highly resistant to three strains of PVY, each belonging to three different subtypes of the virus (PVY^N, PVY^O and PVY^NTN). Infection of transgenic plants with Potato virus X (PVX) simultaneously or prior to the challenge with PVY did not interfere with PVY-resistance.Anastasia Missiou: M.A. and K.K. have contributed equally to this workKriton Kalantidis: M.A. and K.K. have contributed equally to this work     

Genetic improvement of the somatic embryogenesis and regeneration in soybean and transformation of the improved breeding lines

Somatic embryos of soybean [Glycine max (L.) Merrill] have been used to generate transgenic plants by particle bombardment. The induction and proliferation of somatic embryos from immature cotyledons are dependent on the genotype of the cultivar. Whereas somatic embryogenesis and plant regeneration are inefficient in most cultivars, they are efficient in the cultivar Jack. We previously established a breeding line, QF2, by the integration of null mutations of each subunit of the major seed storage proteins glycinin and β-conglycinin, but the embryogenic response of this line is insufficient to allow efficient transformation. We have now backcrossed QF2 to cultivar Jack in order to combine the null traits with competence for somatic embryogenesis. The backcrossed breeding lines selected on the basis of the absence of the major storage proteins exhibited an improved capacity for the induction and proliferation of somatic embryos compared with that of QF2. The induced somatic embryogenic tissue of these breeding lines was successfully used for the production of transgenic plants by particle bombardment. These results also indicate that somatic embryogenesis in soybean is genetically controlled and inherited in a manner independent of the null traits of the major seed storage proteins.     

Direct effects of soybean varietal selection and Aphis glycines-resistant soybeans on natural enemies

The direct effects of three soybean parentages, each represented by an Aphis glycines-resistant and susceptible isoline, on the fitness and performance of two key predators (Orius insidiosus and Harmonia axyridis) were evaluated in the laboratory. Predators were reared from hatch through adulthood in Petri dishes with cut trifoliolate leaves of the designated soybean variety, using eggs of Ephestia kuehniella as surrogate prey to eliminate prey-mediated effects of the host plant. Preimaginal survival and development, sex ratio, adult longevity, fecundity, and size were compared among treatments and a no-plant control. An additional experiment compared life-history parameters of predators caged with soybean versus Ipomoea hederacea (ivyleaf morning glory). Aphid resistance reduced the adult longevity of H. axyridis, but O. insidiosus was unaffected by resistance traits. However, adult O. insidiosus lived longer on soybeans with Group C base genetics than the other soybean varieties. Other parameters were not affected by soybean base genetics or resistance, but both predators generally performed worse on soybean than on I. hederacea or no-plant controls. The results suggest that soybean varietal selection, particularly with respect to A. glycines-resistance, may directly affect biological control agents. Also, implications of the generally poor suitability of soybean for natural enemies are discussed within the context of current crop production practices. Handling editor: Michael Smith     

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.