Sonication-assisted Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merrill] embryogenic suspension culture tissue

Successful transformation of plant tissue using Agrobacterium relies on several factors including bacterial infection, host recognition, and transformation competency of the target tissue. Although soybean [Glycine max (L.) Merrill] embryogenic suspension cultures have been transformed via particle bombardment, Agrobacterium-mediated transformation of this tissue has not been demonstrated. We report here transformation of embryogenic suspension cultures of soybean using “Sonication-Assisted Agrobacterium-mediated Transformation” (SAAT). For SAAT of suspension culture tissue, 10–20 embryogenic clumps (2–4 mm in diameter) were inoculated with 1 ml of diluted (OD_600nm 0.1–0.5) log phase Agrobacterium and sonicated for 0–300 s. After 2 days of co-culture in a maintenance medium containing 100 μM acetosyringone, the medium was removed and replaced with fresh maintenance medium containing 400 mg/l Timentin^?. Two weeks after SAAT, the tissue was placed in maintenance medium containing 20 mg/l hygromycin and 400 mg/l Timentin^?, and the medium was replenished every week thereafter. Transgenic clones were observed and isolated 6–8 weeks following SAAT. When SAAT was not used, hygromycin-resistant clones were not obtained. Southern hybridization analyses of transformed embryogenic tissue confirmed T-DNA integration. Received: 22 August 1997 / Revision received: 22 October 1997 / Accepted: 11 November 1997     

Cytological evidence of a new male-sterile mutant in soybean (Glycine max (L.) Merr.)

Summary From one plant of soybean (Glycine max (L.) Merr.) with only two one-seeded pods, found in an F₄ population maintained by single-seed descent procedure, two fully fertile plants were obtained which, in turn, produced two progeny segregating for male sterility. Segregation ratios, observed on progeny from fertile plants in three successive generations, indicated that the male-sterility trait was under the control of a single recessive gene. Cytological observations made on malesterile, female-fertile plants showed the occurrence of a complete and properly timed cytokinesis with the formation of tetrad cells whose size was very variable, one of which sometimes had two nuclei. During pollen maturation binucleate microspores and grains with reduced size (micropollens) were frequently observed. Massive pollen degeneration occurred at a rather later stage. Structural evidence points to a normally functioning tapetum.On the basis of these cytological observations we conclude that the abnormalities observed in the mutant we studied have to be considered to be different from those caused by any other known ms allele. Tests of allelism with other sources of male sterility are in progress.     

Efficacy of some plant extracts in the management of cercospora leaf spot of groundnut in the Sudan savanna of Nigeria

Field trials were conducted to evaluate the efficacy of neem seed, garlic clove, onion bulb, ginger rhizome and pawpaw leaf extracts applied as foliar spray in controlling cercospora leaf spot of groundnut in the Sudan savanna of Nigeria during the 2002 and 2003 cropping seasons. Ridomil was used as a positive check while the control was left untreated. Three spray regimes (once, twice and thrice) were evaluated. A strip plot design with three replications was used in setting up the experiments. Ex-Dakar, a cercospora leaf spot susceptible Spanish Valencia groundnut variety, was used as planting material. All the plant extracts reduced the incidence and severity of cercospora leaf spot in both seasons compared to the untreated crops. However, neem seed and garlic clove extracts significantly reduced the incidence and severity of the disease compared to the other plant extracts. Ridomil was more effective in controlling the disease than the plant extracts. Three sprays with the plant extracts gave better control of the disease than one or two sprays in the season. The highest seed yield of 3115 kg/ha and 3311 kg/ha in 2002 and 2003, respectively, were obtained with neem seed extract treatment while the lowest seed yield of 2134 kg/ha and 2334 kg/ha in 2002 and 2003, respectively, were recorded from the untreated crops. Also the highest seed yield of 3124 kg/ha and 3418 kg/ha in 2002 and 2003, respectively were obtained following three sprays compared to 2523 kg/ha and 2641 kg/ha for one spray in 2002 and 2003, respectively and 2840 kg/ha and 3085 kg/ha for two sprays in 2002 and 2003, respectively. The highest haulm yield of 4121 kg/ha and 4089 kg/ha in 2002 and 2003, respectively were also obtained following treatment with neem seed extract. The lowest haulm yield of 2951 kg/ha and 3045 kg/ha in 2002 and 2003 respectively, were recorded from the control. Three sprays gave higher haulm yield (4265 kg/ha and 4279 kg/ha in 2002 and 2003, respectively) than one and two sprays in the season. Neem seed extract could therefore be used to control cercospora leaf spot and to improve groundnut production in the Sudan savanna of Nigeria.     

Seasonal and diurnal variations in nitrate reductase activity of soybean (Glycine max (L.) Merr.)

Summary The seasonal and diurnal variations in nitrate reductase (NR) activity of field grown Altona soybean, with and without applied nitrogen, were determined. The NR activity in the fortnightly collected leaf samples was, on the average, 20 percent higher throughout the season in N-treated plants, the highest being early in the season and declining gradually in the samples of subsequent dates. Diurnal variations were marked by increase in the NR activity from 7 a.m. to 7 p.m. and then declining gradually to a minimum at 7 a.m. the next morning.     

Development ofBradyrhizobium infections in supernodulating and non-nodulating mutants of soybean (Glycine max [L.] Merrill)

Summary The early events in the development of nodules induced byBradyrhizobium japonicum were studied in serial sections of a wild type (cv. Bragg), a supernodulating mutant (nts 382) and four non-nodulating mutants (nod49, nod139, nod772, andrj ₁) of soybean (Glycine max [L.] Merrill). Cultivar Bragg responded to inoculation in a similar manner to that described previously for cv. Williams; centres of sub-epidermal cell divisions were observed both with and without associated infection threads and most infection events were blocked before the formation of a nodule meristem. The non-nodulating mutants (nod49, nod772, andrj ₁) had, at most, a few centres of sub-epidermal cell divisions. In general, these were devoid of infection threads and did not develop beyond the very early stages of nodule ontogeny. Sub-epidermal cell divisions or infection threads were never observed on mutant nodl39. This mutant is not allelic to the other non-nodulating mutants and represents a defect in a separate complementation group or gene that is required for nodulation. The supernodulating mutant nts382, which is defective in autoregulation of nodulation, had a similar number of sub-epidermal cell divisions as the wild-type Bragg, but a much greater proportion of these developed to an advanced stage of nodule ontogeny. Mutant nts382, like Bragg, possessed other infection events that were arrested at an early stage of development. The results are discussed in the context of the progression of events in nodule formation and autoregulation of nodulation in soybean.Abbreviations nts nitrate tolerant symbiosis - RT root tip (i.e., position of the tap root tip at the time of inoculation) - SERH shortest emerging root hair (i.e., position of the shortest emerging root hair on the tap root at the time of inoculation) - SCD subepidermal cell divisions     

The Effects of Crop Rotation and Nitrogen Fertilization on Soil Chemical and Microbial Properties in a Guinea Savanna Alfisol of Nigeria

The impacts of crop rotation and inorganic nitrogen fertilization on soil microbial biomass C (SMBC) and N (SMBN) and water-soluble organic C (WSOC) were studied in a Guinea savanna Alfisol of Nigeria. In 2001, fields of grain legumes (soybean and cowpea), herbaceous legume (Centrosema pascuorum) and a natural fallow were established. In 2002, maize was planted with N fertilizer rates of 0, 20, 40 and 60 kg N ha⁻¹ in a split-plot arrangement fitted to a randomized complete block design with legumes and fallow as main plots and N fertilizer levels as subplots. Surface soil samples were taken at 4 weeks after planting and tasselling stage of the maize. Inorganic N fertilization had no significant (P>0.05) effect on SMBC, SMBN and WSOC, while crop rotation significantly (P<0.0001) affected both SMBC and WSOC. These results demonstrate that crop rotation do not necessarily influence the gross soil microbial biomass, but may affect physiologically distinct subcomponent of the microbial biomass. The soils under the various rotations had a predominance of fungi community as indicated by their wide biomass C/N ratio ranging from 9.2 to 20.9 suggesting fungi to be mainly responsible for decomposition in these soils. Soil microbial biomass and WSOC showed significant (P<0.05) correlation with both soil pH and organic carbon but no relationship with total N. Based on these results, it appears that the soil pH and organic carbon determined the flux of the soil microbial biomass and amount of WSOC in these soils.     

Effects of the relationship between area under disease progress curve of angular leaf spot and yield of seed cotton in northeastern Nigeria

Bacterial blight induced by Xanthomonas campestris pv. malvacearum causes substantial yield losses of cotton in Nigeria annually. A field study was carried out for two seasons to investigate the reaction of a selection of 10 different cotton genotypes to bacterial blight and to examine the relationship between the area under disease progress curve (AUDPC) and the yield of seed cotton in the northeastern cotton region of Nigeria. All 10 genotypes reacted positively to the disease with SAMCOT-13 having the highest incidence particularly at Dowaya and Kem locations during the two seasons. This investigation also revealed that SAMCOT-13 and Ex-Benin had the highest AUDPC while TX-CDP37HH-1-83, TAMCOT CAMD-E and TAMCOT SP-21S had the lowest AUDPC indicating relative susceptibility and resistance, respectively. However, it was observed that TX-CDP37HH-1-83, SAMCOT-11 and Ex-Benin had the highest mean yields of 1441.5 kg/ha and 1494.7 kg/ha at Dowaya, Kem and Ngurore, respectively. The results also show a highly significant (P = 0.01) negative correlation between AUDPC and yield in the three locations. These findings suggest that TX-CDP37HH-1-83, TAMCOT CAMD-E and TAMCOT SP-21S are moderately resistant to the disease in view of their lower AUDPC and are considered promising.     

Nigerian Man and Biosphere research plots in Guinea savanna: floristics and structure of the vegetation

A study was made of the vegetation of three plots used in the Nigerian Man and Biosphere Research Programme for Savanna Studies. The aim is to provide detailed information on the vegetation of the plots which may possibly contribute to an understanding of factors influencing savanna structure and relative abundances of forbs, grasses and woody species. Results show that there are differences in the species composition of the plots. There are more forb species than grass species in each plot. The woody basal areas and crown areas of the plots do not depend entirely on the density but also on the size of the woody species. There are differences in herbage yield in the plots that could be attributed to differences in soil properties, species composition and level of human and animal activities between the plots.     

Molecular characterization and genetic diversity analysis of soybean (Glycine max (L.) Merr.) germplasm accessions in India

Molecular characterization and genetic diversity among 82 soybean accessions was carried out by using 44 simple sequence repeat (SSR) markers. Of the 44 SSR markers used, 40 markers were found polymorphic among 82 soybean accessions. These 40 polymorphic markers produced a total of 119 alleles, of which five were unique alleles and four alleles were rare. The allele number for each SSR locus varied between two to four with an average of 2.97 alleles per marker. Polymorphic information content values of SSRs ranged from 0.101 to 0.742 with an average of 0.477. Jaccard’s similarity coefficient was employed to study the molecular diversity of 82 soybean accessions. The pairwise genetic similarity among 82 soybean accessions varied from 0.28 to 0.90. The dendrogram constructed based on genetic similarities among 82 soybean accessions identified three major clusters. The majority of genotypes including four improved cultivars were grouped in a single subcluster IIIa of cluster III, indicating high genetic resemblance among soybean germplasm collection in India.

Electronic supplementary material

The online version of this article (doi:10.1007/s12298-014-0266-y) contains supplementary material, which is available to authorized users.     

Cytokinin inhibition of respiration by cells and mitochondria of soybean,Glycine max (L.) Merrill

Cells of a soybean tissue strain, suspended in an aerated liquid medium, caused disappearance of p-coumaric acid from the medium and oxidation of guaiacol, benzidine, pyrogallol, L-dihydroxyphenylalanine and L-epinephrine. Both the disappearance and the oxidations were inhibited by 6-benzylaminopurine (BAP) at a concentration of 0.5 mM. BAP at other concentrations either promoted or inhibited oxidation of epinephrine in precisely the pattern reported earlier for the disappearance of coumarate; therefore, the disappearance of coumarate probably involves its oxidation. The effectiveness of other cytokinins in inhibiting the oxidation was studied.At 0.5 mM, and perhaps even at 0.5 M, some of the several cytokinins tested inhibited oxygen consumption by the soybean cells. This inhibition, which did not require any of the above metabolizable compounds, was especially marked in the presence of cyanide, azide or Antimycin A, and was detectable in 10 min or less. Either Antimycin A or salicylhydroxamic acid alone promoted O₂ consumption but together they were quite inhibitory. The soybean cells apparently have an alternate respiratory pathway and cytokinins may influence its operation.Several cytokinins at 0.5 mM, and perhaps at 0.5 M, also inhibited oxygen consumption by mitochondrial preparations from the soybean cells, the inhibition being evident in about 20 s. The consumption required a substrate such as malate, succinate or NADH. Cytokinins and related compounds varied in effectiveness as follows: BAP and 6-isopentenyla-minopurine 9-tetrahydropyranyl-BAP > kinetin, ribosyl-isopentenylaminopurine, 9-methyl-BAP and 9-methoxymethyl-BAP > 6,6-dimethylaminopurine and zeatin (slight activity) > 6-methylaminopurine, nicotinamide and adenine (ineffective). To a great extent this order parallels the order of effectiveness of the compounds in causing cell division. Mitochondria, therefore, may contain a site for an important cytokinin action.Abbreviations BAP 6-benzylaminopurine - IPA 6-(²-isopentenyl)aminopurine     

The distribution,abundance, and the effects of fire on mound building termites (Trinervitermes and Cubitermes spp., Isoptera: Termitidae) in northern guinea savanna West Africa

Summary Termite mound densities in typical guinea savanna, Detarium, and grassland (boval) habitats in northern guinea savanna were determined by random quadratting of 2–3 sites in each habitat (100, 10x10 m quadrats per habitat). Dominant species in guinea savanna were T. geminatus (46 mounds ha^-1) and T. oeconomus (21 mounds ha^-1), in Detarium T. geminatus (59 mounds ha^-1) and C. curtatus (45 mounds ha^-1) and in boval C. curtatus (72 mounds ha^-1) and T. geminatus (22 mounds ha^-1). Only C. curtatus densities and total densities differed significantly between sites within habitats, but all species differed significantly in abundance between habitats. The composition of each community was related to general environment but no particular environmental variable was shown to be a major determinant of termite distribution. Evidence for the limitation of termite populations was obtained from indirect evidence of competition between colonies in Detarium, and by experimental manipulation of fire regimes in the typical guinea savanna habitat. Harvester termites increased four-five fold over two years in fire-protected plots as a result of increased food supplies. Total termite densities in the fire-protected community equilibrated to the new population density (100 mounds ha^-1) after only two-three years.     

Manganese and iron interactions on their uptake and distribution in soybean (Glycine max (L.) Merr.)

Summary The uptake and distribution of iron and manganese were studied in a manganese-sensitive soybean cultivar (‘Bragg’) grown over a range of supply levels of these nutrients in solution culture. At high (90 and 275 μM) manganese levels, increasing the iron concentration in solution from 2 to 100 μM partially overcame the effects of manganese toxicity. Interactions between manganese and iron occurred for dry matter yields, rate of Mn absorption by the roots, and the proportions of manganese and iron transported to the tops. No interaction was observed for the rate of root absorption of iron. The percentage distribution of manganese in the plant top increased with increasing iron, despite a reduced rate of Mn uptake. On the other hand, iron uptake was independent of solution Mn concentration and increased with increasing solution Fe. Also more iron was retained in the roots at high Mn and/or Fe levels in solution. Concentrations of manganese and iron in roots, stems and individual leaves were affected independently by the manganese and iron supplyi.e. without any interaction occurring between the two elements. In general, the concentration in a plant part was related directly to the solution concentration. Symptoms resembling iron deficiency correlated poorly with leaf Fe concentrations whereas high levels of manganese were found in leaves displaying Mn toxicity symptoms.     

Somatic embryogenesis from cultured epicotyls and primary leaves of soybean [Glycine max (L.) Merrill]

Summary Regeneration of several varieties of soybean [Glycine max (L.) Merrill] by somatic embryogenesis from cultured epicotyls and primary leaves has been demonstrated. Somatic embryogenesis was induced from epicotyls and primary leaves when cotyledon halves with the intact zygotic embryo axes were cultured on Murashige and Skoog (MS) medium supplemented with 10 mg 1⁻¹ (45.2 μM) 2,4-D. Stable, continuously proliferating globular embryo cultures (GEC) were established from small groups of somatic embryos on MS medium supplemented with 20 mg 1⁻¹ (90.5 μM) 2,4-dichlorophenoxyacetic acid (2,4-D). Rapid multiplication of shoot tips from germinating somatic embryos was achieved on Cheng’s basal medium (CBO) containing 2.5 mg 1⁻¹ (11.3 μM) 6-benzyladenine. Fertile plants were obtained from individual somatic embryos and in vitro propagated adventitious shoot bud cultures.     

Genetics and cytology of a new genic male-sterile soybean [Glycine max (L.) Merr.]

 Genetic and cytological studies were conducted with a new male-sterile, female-fertile soybean [Glycine max (L.) Merr.] mutant. This mutant was completely male sterile and was inherited as a single-recessive gene. No differences in female or male gamete transmission of the recessive allele were observed between reciprocal cross-pollinations in the F₁ or F₂ generations. This mutant was not allelic to any previously identified soybean genic male-sterile mutants: ms1, ms2, ms3, ms4, ms5, or ms6. No linkage was detected between sterility and flower color (W1 locus), or between sterility and pubescence color (T1 locus). Light microscopic and cytological observations of microsporogenesis in fertile and sterile anthers were conducted. The structure of microspore mother cells (MMC) in male-sterile plants was identical to the MMCs in male-fertile plants. Enzyme extraction analyses showed that there was no callase activity in male-sterile anthers, and this suggests that sterility was caused by retention of the callose walls, which normally are degraded around tetrads at the late tetrad stage. The tapetum from male-sterile anthers also showed abnormalities at the tetrad stage and later stages, which were expressed by an unusual formation of vacuoles, and by accumulation of densely staining material. At maturity, anthers from sterile plants were devoid of pollen grains. Received: 13 May 1996 / Revision accepted: 19 August 1996     

An original mutation in soybean (Glycine max (L.) Merrill) involving degeneration of the generative cell and causing male sterility.

A spontaneous mutation causing male sterility has been detected in line BR97-17739 from the soybean breeding program conducted by Embrapa-National Soybean Research Center. Meiotic division and male gametophyte development were analyzed in 10 male-sterile, female-fertile plants. Meiotic process had few irregularities related to chromosome segregation and affected about 2% of tetrads. Despite the high frequency of normal microspores, pollen sterility was total. After callose dissolution, microspores were released into the anther loculle and interphase nucleus was displaced from the center to one side of the cell. Displacement continued throughout normal microspore mitosis (PMI). After telophase, the hemispherical phragmoplast marked the place of cytokinesis. A typical generative cell, adjacent to the plasma membrane, and the vegetative one, containing most of the cytoplasm, were formed. In spite of the well-formed generative cell, pollen mitosis (PMII) failed to occur. The generative cell degenerated and was completely destroyed. The 3:1 segregation for male sterility in this line and its progenies indicate that a single recessive gene controls mutation.     

Quantitative detection of transgenes in soybean [Glycine max (L.) Merrill] and peanut (Arachis hypogaea L.) by real-time polymerase chain reaction

Quantitative real-time polymerase chain reaction (PCR) assays were designed that enabled the zygosity of transgenes in soybean [Glycine max (L.) Merrill] and peanut (Arachis hypogaea L.) to be determined. The two zygosity assays, based on TaqMan technology that uses a fluorogenic probe which hybridizes to a PCR target sequence flanked by primers, were both accurate and reproducible in the determination of the number of transgenes present in a cell line. In the first assay, in which TaqMan assays were performed on increasing amounts of a plasmid containing the transgene of interest, a linear relationship between the level of fluorescence and the template amount was produced. Using the resultant linear relationships as standard curves, we were able to determine the zygosity of both soybeans segregating for the cry1Ac transgene and that of a T₁ peanut segregating for the hph transgene. In the second assay, a relative determination of copy number (referred to as comparative Ct) was performed on transgenic soybeans by comparing the amplification efficiency of the transgene of interest to that of an endogenous gene in a multiplexed PCR reaction. Both methods proved to be sufficiently sensitive to differentiate between homozygotes and hemizygotes. These assays have numerous potential applications in plant genetic engineering and tissue culture, including the hastening of the identification of transgenic tissue, selecting transformation events with a low number of transgenes and the monitoring of the transmission of transgenes in subsequent crosses.