Modification of phenolic metabolism in soybean hairy roots through down regulation of chalcone synthase or isoflavone synthase

Soybean hairy roots, transformed with the soybean chalcone synthase (CHS6) or isoflavone synthase (IFS2) genes, with dramatically decreased capacity to synthesize isoflavones were produced to determine what effects these changes would have on susceptibility to a fungal pathogen. The isoflavone and coumestrol concentrations were decreased by about 90% in most lines apparently due to gene silencing. The IFS2 transformed lines had very low IFS enzyme activity in microsomal fractions as measured by the conversion of naringenin to genistein. The CHS6 lines with decreased isoflavone concentrations had 5 to 20-fold lower CHS enzyme activities than the appropriate controls. Both IFS2 and CHS transformed lines accumulated higher concentrations of both soluble and cell wall bound phenolic acids compared to controls with higher levels found in the CHS6 lines indicating alterations in the lignin biosynthetic branch of the pathway. Induction of the soybean phytoalexin glyceollin, of which the precursor is the isoflavone daidzein, by the fungal pathogen Fusarium solani f. sp. glycines (FSG) that causes soybean sudden death syndrome (SDS) showed that the low isoflavone transformed lines did not accumulate glyceollin while the control lines did. The (iso)liquritigenin content increased upon FSG induction in the IFS2 transformed roots indicating that the pathway reactions before this point can control isoflavonoid synthesis. The lowest fungal growth rate on hairy roots was found on the FSG partially resistant control roots followed by the SDS sensitive control roots and the low isoflavone transformants. The results indicate the importance of phytoalexin synthesis in root resistance to the pathogen. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Regeneration of Transgenic Soybean (Glycine max) Plants from Electroporated Protoplasts

Transgenic soybean (Glycine max [L.] Merr.) plants were regenerated from calli derived from protoplasts electroporated with plasmid DNA-carrying genes for a selectable marker, neomycin phosphotransferase (NPTII), under the control of the cauliflower mosaic virus 35-Svedberg unit promoter, linked with a nonselectable mannityl opine synthesis marker. Following electroporation and culture, the protoplast-derived colonies were subjected to kanamycin selection (50 micrograms per milliliter) beginning on day 15 for 6 weeks. Approximately, 370 to 460 resistant colonies were recovered from 1 × 10⁶ electroporated protoplasts, giving an absolute transformation frequency of 3.7 to 4.6 × 10⁻⁴. More than 80% of the kanamycin-resistant colonies showed NPTII activity, and about 90% of these also synthesized opines. This indicates that the linked marker genes were co-introduced and co-expressed at a very high frequency. Plants were regenerated from the transformed cell lines. Southern blot analysis of the transformed callus and leaf DNA demonstrated the integration of both genes. Single-plant assays performed with different plant parts showed that both shoot and root tissues express NPTII activity and accumulate opines. Experiments with NPTII and mannityl opine synthesis marker genes on separate plasmids resulted in a co-expression rate of 66%. These results indicate that electroporation can be used to introduce both linked and unlinked genes into the soybean to produce transformed plants.     

A simple and efficient method for obtaining transgenic soybean callus tissues

In the present study, a simple and efficient method for obtaining transgenic callus tissues of soybean [Glycine max (L.) Merr.] was developed based on Agrobacterium-mediated transformation. Hypocotyl segments of soybean were used as the starting material. Several factors such as soybean genotype, Agrobacterium concentration, inoculation time, co-cultivation period and addition of antioxidants in co-cultivation medium affecting the transformation efficiency were examined. The explants were cultured on callus induction medium containing 0.5 mg L^?1 6-benzylaminopurine and 2.0 mg L^?1, 2,4-Dichlorophenoxyacetic acid for callus induction. Callus tissues were induced at both the acropetal and basipetal ends. CaMV35S::GUS and CaMV35S::GFP transgenic callus tissues were obtained using the optimized protocol. The average transformation efficiency reached up to 87.7 % based on GUS detection. From inoculation with Agrobacterium to obtaining transgenic soybean callus will take about 3 weeks. In order to validate this method for gene function investigation, GVG::GmSARK transgenic soybean callus tissues were obtained and their senescence-associated phenotypes were assessed. To our knowledge, this is the first report using hypocotyl segments as starting materials to obtain transgenic callus, and this system provides a method for high-throughput screening of functional genes of interest in transformed soybean callus.     

Genetic transformation and full recovery of alfalfa plants via secondary somatic embryogenesis

A genetic transformation method via secondary somatic embryogenesis was developed for alfalfa (Medicago sativa L.). Mature somatic embryos of alfalfa were infected by Agrobacterium strain GV3101 containing the binary vector pCAMBIA2301. pCAMBIA2301 harbors the uidA Gus reporter gene and npt II acts as the selectable marker gene. Infected primary embryos were placed on SH2K medium containing plant growth regulators to induce cell dedifferentiation and embryogenesis under 75 mg/L kanamycin selection. The induced calli were transferred to plant medium free of plant growth regulators for embryo formation while maintaining selection. Somatic embryos germinated normally upon transfer to a germination medium. Plants were recovered and grown in a tissue culture room before transfer to a greenhouse. Histochemical analysis showed high levels of GUS activity in secondary somatic embryos and in different organs of plants recovered from secondary somatic embryos. The presence and stable integration of transgenes in recovered plants were confirmed by polymerase chain reaction using transgene-specific primers and Southern blot hybridization using the npt II gene probe. The average transformation efficiency achieved via secondary somatic embryogenesis was 15.2%. The selection for transformation throughout the cell dedifferentiation and embryogenic callus induction phases was very effective, and no regenerated plants escaped the selection procedure. Alfalfa transformation is usually achieved through somatic embryogenesis using different organs of developed plants. Use of somatic embryos as explants for transformation can avoid the plant development phase, providing a faster procedure for introduction of new traits and facilitates further engineering of previously transformed lines.     

Modification of isoflavones in soybean seeds via expression of multiple phenolic biosynthetic genes

To modify the level and composition of isoflavones, the important bioactive constituents of soybean seeds, soybean was transformed via co-bombardment of embryogenic cultures with three DNA cassettes containing the CHS6-chalcone synthase and IFS2-isoflavone synthase genes, and a fragment of PAL5-phenylalanine ammonia-lyase gene, all in sense orientation under the lectin promoter mixed with the selectable marker gene, HPT (hygromycin phosphotransferase) under the 35S promoter. Four of six fertile lines produced integrated all four genes.Isoflavone levels were lower in T1 mature seeds of 5 of the 6 lines compared to the control. Transgene segregation was found in one selected line, with formation of additional sublines with different transgene composition found also in the homozygous plants. Decreased isoflavone concentrations (by about 70%) were found in T4 homozygous seeds of the two lines studied in detail here. The embryo axes accumulated most of the glycitein and contained a higher isoflavone concentration than the cotyledons. Expression of transgenes driven by the lectin promoter reduced the isoflavone concentration only in the cotyledons and not in embryo axes, indicating that this promoter is preferably active in cotyledons.     

Quantitative trait loci analysis of individual and total isoflavone contents in soybean seeds

Soybean isoflavones play diverse roles in human health, including cancers, osteoporosis, heart disease, menopausal symptoms and pabulums. The objective of this study was to identify the quantitative trait loci (QTL) associated with the isoflavones daidzein (DC), genistein (GeC), glycitein (GlC) and total isoflavone contents (TIC) in soybean seeds. A population of 184 F_21:0 recombinant inbred lines derived from a ‘Xiaoheidou’ בGR8836’ cross was planted in pot and field conditions to evaluate soybean isoflavones. Twenty-one QTL were detected by composite interval mapping. Several QTL were associated with the traits for DC, GeC, GlC and TIC only. QDGeGlTIC4_1 and QDGlTIC12_1 are reported first in this study and were associated with the DC, GeC, GlC and TIC traits simultaneously. The QTL identified have potential value for marker-assisted selection to develop soybean varieties with desirable isoflavone content.     

Mapping Isoflavone QTL with Main,Epistatic and QTL × Environment Effects in Recombinant Inbred Lines of Soybean

Soybean (Glycine max (L.) Merr.) isoflavone is important for human health and plant defense system. To identify novel quantitative trait loci (QTL) and epistatic QTL underlying isoflavone content in soybean, F_5:6, F_5:7 and F_5:8 populations of 130 recombinant inbred (RI) lines, derived from the cross of soybean cultivar ‘Zhong Dou 27′ (high isoflavone) and ‘Jiu Nong 20′ (low isoflavone), were analyzed with 95 new SSR markers. A new linkage map including 194 SSR markers and covering 2,312 cM with mean distance of about 12 cM between markers was constructed. Thirty four QTL for both individual and total seed isoflavone contents of soybean were identified. Six, seven, ten and eleven QTL were associated with daidzein (DZ), glycitein (GC), genistein (GT) and total isoflavone (TI), respectively. Of them 23 QTL were newly identified. The qTIF_1 between Satt423 and Satt569 shared the same marker Satt569 with qDZF_2, qGTF_1 and qTIF_2. The qGTD2_1 between Satt186 and Satt226 was detected in four environments and explained 3.41%-10.98% of the phenotypic variation. The qGTA2_1, overlapped with qGCA2_1 and detected in four environments, was close to the previously identified major QTL for GT, which were responsible for large a effects. QTL (qDZF_2, qGTF_1 and qTIF_2) between Satt144-Satt569 were either clustered or pleiotropic. The qGCM_1, qGTM_1 and qTIM_1 between Satt540-Sat_244 explained 2.02%–9.12% of the phenotypic variation over six environments. Moreover, the qGCE_1 overlapped with qGTE_1 and qTIE_1, the qTIH_2 overlapped with qGTH_1, qGCI_1 overlapped with qDZI_1, qTIL_1 overlapped with qGTL_1, and qTIO_1 overlapped with qGTO_1. In this study, some of unstable QTL were detected in different environments, which were due to weak expression of QTL, QTL by environment interaction in the opposite direction to a effects, and/or epistasis. The markers identified in multi-environments in this study could be applied in the selection of soybean cultivars for higher isoflavone content and in the map-based gene cloning.     

Effects of medium components and light on callus induction, growth, and frond regeneration in Lemna gibba (Duckweed)

Summary Basal media, plant growth regulator type and concentration, sucrose, and light were examined for their effects on duckweed (Lemna gibba) frond proliferation, callus induction and growth, and frond regeneration. Murashige and Skoog medium proved best for callus induction and growth, while Schenk and Hildebrandt medium proved best for frond proliferation. The ability of auxin to induce callus was associated with the relative strength of the four auxins tested, with 20 or 50 μM 2,4-dichlorophenoxyacetic acid giving the highest frequency (10%) of fronds producing callus. Auxin combinations did not improve callus induction frequency. Auxin in combination with other plant growth regulators was needed for long-term callus growth; the two superior plant growth regulator combinations were 10 μM naphthaleneacetic acid, 10 μM gibberellic acid, and 2 μM benzyladenine with either 1 or 20 μM 2,4-dichlorophenoxyacetic acid. Three percent sucrose was best for callus induction and growth. Callus induction and growth required light. Callus that proliferated from each frond’s meristematic zone contained a mixture of dedifferentiated and somewhat organized cell masses. Continual callus selection was required to produce mostly dedifferentiated, slow-growing callus cell lines. Frond regeneration occurred on Schenk and Hildebrandt medium without plant growth regulators but was promoted by 1 μM benzyladenine. Callus maintained its ability to regenerate fronds for at least 10 mo. Regenerated fronds showed a slower growth rate than normal fronds and a low percentage of abnormal morphologies that reverted to normal after one or two subcultures.     

Novel major quantitative trait loci regulating the content of isoflavone in soybean seeds

Despite their medicinal, pharmaceutical, and nutritional importance of isoflavones, the genetic basis controlling the amounts of isoflavones in soybean seeds is still not well understood. The main obstacle is the great variability in the content of isoflavone in seeds harvested from different environments. In this study, quantitative trait loci (QTL) for the content of different isoflavones including daidzein, genistein, and glycitein were investigated in a population of recombinant inbred lines derived from the cross of “Hwangkeum” (Glycine max) by “IT182932” (Glycine soja). Seeds analyzed were harvested in three different experimental environments. QTL analyses for isoflavone content were conducted by composite interval mapping across a genomewide genetic map. Two major QTL were mapped to soybean chromosomes 5 and 8, which were designated QDZGT1 and QDZGT2, respectively. Both loci have not been previously reported in other isoflavone sources. The results from this study will be useful in cloning genes that can control the contents of isoflavones in soybean and for the development of soybean lines containing a high or low isoflavone content.     

Tissue culture specificity of the tobacco ASA2 promoter driving hpt as a selectable marker for soybean transformation selection

This study was carried out to determine if the tobacco anthranilate synthase ASA2 2.3 kb promoter drives tissue culture specific expression and if it is strong enough to drive hpt (hygromycin phosphotransferase) gene expression at a level sufficient to allow selection of transformed soybean embryogenic culture lines. A number of transformed cell lines were selected showing that the promoter was strong enough. Northern blot analysis of plant tissues did not detect hpt mRNA in the untransformed control or in the ASA2-hpt plants except in developing seeds while hpt mRNA was detected in all tissues of the CaMV35S-hpt positive control line plants. However, when the more sensitive RT-PCR assay was used all tissues of the ASA2-hpt plants except roots and mature seeds were found to contain detectable hpt mRNA. Embryogenic tissue cultures initiated from the ASA2-hpt plants contained hpt mRNA detectable by both northern and RT-PCR analysis and the cultures were hygromycin resistant. Friable callus initiated from leaves of ASA2-hpt plants did in some cases contain hpt mRNA that was only barely detectable by northern hybridization even though the callus was very hygromycin resistant. Thus the ASA2 promoter is strong enough to drive sufficient hpt expression in soybean embryogenic cultures for hygromycin selection and only very low levels of expression were found in most plant tissues with none in mature seeds.     

Salt stress induced soybean <Emphasis Type="Italic">GmIFS1</Emphasis> expression and isoflavone accumulation and salt tolerance in transgenic soybean cotyledon hairy roots and tobacco

Effects of isoflavones on plant salt tolerance were investigated in soybean (Glycine max L. Merr. cultivar N23674) and tobacco (Nicotiana tabacum L.). Leaf area, fresh weight, net photosynthetic rate (Pn), and transpiration rate (Tr) of soybean N23674 plants treated with 80 mM NaCl were significantly reduced, while a gene (GmIFS1) encoding for 2-hydroxyisoflavone synthase was highly induced, and isoflavone contents significantly increased in leaves and seeds. To test the impact of isoflavones to salt tolerance, transgenic soybean cotyledon hairy roots expressing GmIFS1 (hrGmIFS1) were produced. Salt stress slightly increased isoflavone content in hairy roots of the transgenic control harboring the empty vector but substantially reduced the maximum root length, root fresh weight, and relative water content (RWC). The isoflavone content in hrGmIFS1 roots, however, was significantly higher, and the above-mentioned root growth parameters decreased much less. The GmIFS1 gene was also transformed into tobacco plants; plant height and leaf fresh weight of transgenic GmIFS1 tobacco plants were much greater than control plants after being treated with 85 mM NaCl. Leaf antioxidant capacity of transgenic tobacco was significantly higher than the control plants. Our results suggest that salt stress-induced GmIFS1 expression increased isoflavone accumulation in soybean and improved salt tolerance in transgenic soybean hairy roots and tobacco plants.     

The influence of the rolC gene on isoflavonoid production in callus cultures of Maackia amurensis

The polyphenolic complex of Maackia amurensis, as well as a complex of isoflavonoids from M. amurensis callus cultures, display strong hepatoprotective effects in experimental animal and human studies. To increase the yield of polyphenols in cultures of M. amurensis, calli were transformed with the rolC gene as well as with an empty vector that was used as a control. HPLC analysis revealed that the transgenic cultures produced the same complex of isoflavonoids. The complex consisted of 20 compounds, including isoflavones and their glucosides as well as pterocarpans and their glucosides. The cultures transformed with either the empty vector or the rolC gene construct produced on average 1.22 % dry weight (DW) and 1.39 % DW of isoflavonoids, respectively. Isoflavonoid production in the transformed callus lines carrying the empty vector and the rolC gene construct reached 106 and 146 mg/L, respectively. Moreover, the rolC gene construct promoted cell growth and overall cell productivity. The transgenic callus lines expressing the rolC gene exhibited higher levels of the following six isoflavonoids: daidzein, calycosin, formononetin, 4′-Ο-β-glucopyranosyldaidzin, maackiain and 6′-O-malonyl-3-O-β-D-glucopyranosylmaackiain. However, lower levels of genistin were observed in rolC calli than in those carrying the empty vector.     

Stable transformation and actin visualization in callus cultures of dodder (Cuscuta europaea)

Agrobacterium tumefaciens-mediated transformation of callus culture, combined with a visual selection of GFP-tagged fimbrin actin binding domain (FABD₂) expression is described for parasitic species (Cuscuta europaea). The conditions for callus induction from 1 cm-long explants from the basal part of 7-day-old dodder seedlings were defined. We obtained light-green calli, which were transformed with A. tumefaciens bacterial strain GV3101 carrying plasmid pCB302 (35S::ABD₂:gfp) with neomycin phosphotransferase (nptII) gene. The limitations of selection procedures based on antibiotics were avoided using green fluorescent protein (GFP) detection, as a visual selection marker subcellularly targeted to the actin cytoskeleton. Fluorescence microscopy analyses demonstrated a network of nucleus-associated actin arrays and dense cortical actin arrangements in stably transformed Cuscuta callus cells. RT-PCR analyses confirmed gfp expression in transformed calli 7, 14 and 21 days after transformation. Although the GFP fluorescence associated with the actin cytoskeleton has retained for at least six months without silencing, no shoot regeneration was observed. It can be concluded that, C. europaea callus cells are competent for transformation, but under given conditions, these cells failed to realize their morphogenic and regeneration potentials.     

QTL underlying the resistance to soybean aphid (<Emphasis Type="Italic">Aphis glycines</Emphasis> Matsumura) through isoflavone-mediated antibiosis in soybean cultivar ‘Zhongdou 27’

Soybean aphid (Aphis glycines Matsumura) results in severe yield loss of soybean in many soybean-growing countries of the world. A few loci have been previously identified to be associated with the aphid resistance in soybean. However, none of them was via isoflavone-mediated antibiosis process. The aim of the present study was to conduct genetic analysis of aphid resistance and to identify quantitative trait loci (QTL) underlying aphid resistance in a Chinese soybean cultivar with high isoflavone content. One hundred and thirty F_5:6 derived recombinant inbred lines from the ‘Zhongdou 27’ × ‘Jiunong 20’ cross were used. Two QTL were directly associated with resistance to aphid as measured by aphid damage index. qRa_1, close to Satt470 on soybean linkage group (LG) A2 (chromosome 8), was consistently detected for 3- and 4-week ratings and explained a large portion of phenotypic variations ranging from 25 to 35%. qRa_2, close to Satt144 of LG F (chromosome 13), was detected for 3- and 4-week ratings and could explain 7 and 11% of the phenotypic variation, respectively. These two QTL were highly associated with high isoflavone content and both positive alleles were derived from ‘Zhongdou 27’, a cultivar with higher isoflavone content. The results revealed that higher individual or total isoflavones contents in soybean lines could protect soybean against aphid attack. These two QTL detected jointly provide potential for marker-assisted selection to improve the resistance of soybean cultivars to aphid along with the increase of isoflavone content.     

Physiological role of nitrate under anaerobic stress in Saccharum officinarum callus cells tolerant and sensitive to anoxia

The physiological role of nitrate as a protective factor against anaerobic stress was studied in experiments with tolerant to anoxia sugarcane (Saccharum officibarum L.) callus lines obtained by in vitro selection in the absence of exogenous carbohydrates. Original cell lines, which were not subjected to selection and therefore more sensitive to oxygen shortage, served as a control. In these lines, anaerobic stress was created in the presence or absence of nitrate in nutrient medium. The presence of nitrate in nutrient medium increased markedly tolerance to anaerobic stress of both lines differing in their sensitivity to anaerobiosis. However, the degree of tolerance differed substantially in compared lines. In the presence of exogenous nitrate, in tolerant cells there were no signs of mitochondrial membrane destruction or degradation even after 72 h of anoxia, whereas in control cells 48-h anaerobic incubation led to the complete degradation of mitochondrial membranes and membranes of other organelles. It is concluded that significant increase in the tolerance of S. officinarum cells in the process of in vitro selection most likely occurred due to induction and stimulation of not only the processes of glycolysis and fermentation, but also nitrate and maybe nitrite utilization.     

Agrobacterium-mediated stable transformation of cell suspension cultures of barley (Hordeum vulgare)

Barley (Hordeum vulgare L. cvs. Igri and Dissa) cell suspension cultures, which had been initiated from immature embryo-derived (IED) and microspore-derived (MSD) callus, were co-cultivated with various Agrobacterium tumefaciens strains. The T-DNA vectors contained visually-detectable marker genes (C1/Lc orgusA-intron), as reporters of transient T-DNA transfer, and also drug resistance genes (hph or bar) to facilitate selection of stably transformed cell lines. A set of normal binary vectors in a super-virulent Agrobacterium strain [EHA101(pBECKS)] and also a super-binary vector [LBA4404(pTOK233)] were used in this study. Cells of the suspension cultures which received T-DNA were able to proliferate under selection regimes and a number of hygromycin- or phosphinothricin-resistant barley callus lines were isolated which expressed a co-transferred gusA gene. To ensure homogeneity of the cell lines, prolonged tissue culture regimes were used but these resulted in a loss of the capacity to regenerate plants from the transgenic callus lines. The frequency of recovery of transformed callus lines ranged from 0.3% to 2.9%. Southern blot analyses of the transformed callus lines confirmed the presence of the marker genes and demonstrated them to be associated with DNA which was distinct from that of the original Agrobacterium plasmid. Furthermore, independent transgenic lines showed diverse patterns of hybridising bands. These data suggest that the T-DNA fragment was stably maintained through integration into the genomes of the barley cell lines. This revised version was published online in June 2006 with corrections to the Cover Date.     

Callus culture and in vitro biosynthesis of cardiac glycosides from Calotropis gigantea (L.) Ait

Calotropis gigantea (L.) Ait., belonging to the family Asclepiadaceae, is a source of many cardiac glycosides (CGs) and their steroidal moieties (genins). These CGs have been reported to have anti-proliferative activity on tumor cell lines and are potential targets for cancer chemotherapy. However, the abundance of CGs in wild plants is particularly restricted and it is difficult to isolate the desired compound in required quantities. This study is the first attempt to standardize the induction and proliferation of callus from various explants of C. gigantea specifically for the production of CGs. Callus growth was accompanied by CG measurement using high-performance liquid chromatography-tandem mass spectrometry. Murashige and Skoog (MS) and modified Murashige and Skoog (MMS) media were optimized with various combinations and concentrations of auxin and cytokinin for induction and growth of calli from a range of explant sources. While leaves and stem explants resulted in greatest callus induction, MMS medium was found to be optimal. However, no CG was produced from callus grown on this medium. In contrast, the induction and proliferation of callus on MS medium were optimum at primary stages, but growth slowed during the third subculture. Therefore, calli were transferred to MMS medium to promote callus proliferation and production of CGs. As a result, three CGs and two genins were biosynthesized. Furthermore, the callus induction data in MS medium indicated that among different auxins, 2,4-dichlorophenoxyacetic acid was the best for callus induction compared to 1-naphthylacetic acid and indole-3-acetic acid. The data also revealed that the cytokinin/auxin ratio was critical rather than their independent presence for the induction of callus. Thus, the in vitro biosynthesis of targeted CGs may offer an alternative pathway for new source of anti-proliferative agents in required quantities.     

Fine-mapping of QTLs for individual and total isoflavone content in soybean (<Emphasis Type="Italic">Glycine max L.</Emphasis>) using a high-density genetic map

Key message

Fifteen stable QTLs were identified using a high-density soybean genetic map across multiple environments. One major QTL, qIF5-1, contributing to total isoflavone content explained phenotypic variance 49.38, 43.27, 46.59, 45.15 and 52.50%, respectively.

Abstract

Soybeans (Glycine max L.) are a major source of dietary isoflavones. To identify novel quantitative trait loci (QTL) underlying isoflavone content, and to improve the accuracy of marker-assisted breeding in soybean, a valuable mapping population comprised of 196 F_7:8–10 recombinant inbred lines (RILs, Huachun 2 × Wayao) was utilized to evaluate individual and total isoflavone content in plants grown in four different environments in Guangdong. A high-density genetic linkage map containing 3469 recombination bin markers based on 0.2 × restriction site-associated DNA tag sequencing (RAD-seq) technology was used to finely map QTLs for both individual and total isoflavone contents. Correlation analyses showed that total isoflavone content, and that of five individual isoflavone, was significantly correlated across the four environments. Based on the high-density genetic linkage map, a total of 15 stable quantitative trait loci (QTLs) associated with isoflavone content across multiple environments were mapped onto chromosomes 02, 05, 07, 09, 10, 11, 13, 16, 17, and 19. Further, one of them, qIF5-1, localized to chromosomes 05 (38,434,171–39,045,620 bp) contributed to almost all isoflavone components across all environments, and explained 6.37–59.95% of the phenotypic variance, especially explained 49.38, 43.27, 46.59, 45.15 and 52.50% for total isoflavone. The results obtained in the present study will pave the way for a better understanding of the genetics of isoflavone accumulation and reveals the scope available for improvement of isoflavone content through marker-assisted selection.

     

Selection and Inbreeding of Heterodera glycines on Glycine max

Few soybean cyst nematodes (SCN), Heterodera glycines, of a diverse gene pool developed into females on soybeans PI 89772 or PI 209332. Nematodes surviving the selection pressure were then inbred for nine generations by single cyst transfers on the same selecting soybean line. These nematodes appeared to tolerate concurrent selection and inbreeding. Effects of selection-inbreeding, selection only, and secondary selection were evaluated by relative ability to produce cysts on 11 soybean lines. The genetic differences of PI 89772 (also Peking and Pickett 71) and PI 209332 were reaffirmed. The random effects of inbreeding indicated that Ilsoy and Williams may have genes for resistance different from those in PI 89772 or PI 209332. Egg inoculum obtained from soil resulted in very few cysts in some tests. Fresh egg inoculum (from cysts on 27-30-day-old plants) generally resulted in more cysts and more consistent results. Concurrent with the change in inoculum, there was a large increase in relative numbers of cysts on several soybean lines but no change on other lines; the true cause of this large interaction is unknown. Secondary selection of two inbreds was effective and suppressed cyst numbers on the line on which one inbred was selected initially. These results are consistent with the allelism linkage of some SCN genes reported previously.