Characterization of Photothermal Flowering Responses in Maturity Isolines of Soyabean [Glycine max (L.) Merrill] cv. Clark

All eight isolines of three maturity genes (E₁/e₁, E₂ /e₂, andE₃ /e₃) of soyabean [Glycine max (L.) Merrill] cv. Clark weregrown in widely different combinations of photoperiod and temperature.Under the more inductive conditions, i.e. in a warm mean temperature(30°C) when daylengths were less than the critical value(i.e. less than about 13 h), the isolines flowered at similartimes (23-24 d). The responses of all isolines to temperaturewere also similar, if not identical. Increase in daylength abovethe critical photoperiod progressively delayed flowering untilthe time taken to flower (f) reached a maximum at the ceilingphotoperiod. The relations between the rate of progress towardsflowering (1/f) and photoperiod (between the critical and ceilingvalues) were linear. The coefficient characterizing the slopeof the response (photoperiod sensitivity) varied amongst theisolines. These responses could be grouped into three categoriesof increasing sensitivity: (1) least sensitive, e₁e₂e₃ , e₁E₂e₃, e₁e₂E₃ ; (2) intermediate, E₁e₂e₃ , e₁E₂E₃ ; and (3) mostsensitive, E₁E₂e₃, E₁e₂E₃ , E₁E₂E₃ . Thus, in the Clark cultivargenetic background, E₁ induces greater photoperiod sensitivitybut neither E₂ nor E₃ on their own have any effect. However,both E₂ and E₃ together induce photoperiod sensitivity comparableto that induced by E₁ alone. Furthermore, in addition to thisepistasis, either E₂ or E₃ has considerable epistatic effecton E₁, further increasing photoperiod sensitivity. The effectsof these genes and their epistasis were also reflected in theextent of the maximum delays to flowering which occur when theceiling photoperiod is exceeded, and also possibly in earlinessin circumstances when photoperiods were below the critical value.Copyright1994, 1999 Academic Press Glycine max (L.) Merrill, soyabean, maturity genes, flowering, photoperiod, temperature     

Variation in the Durations of the Photoperiod-sensitive and Photoperiod-insensitive Phases of Post-first Flowering Development in Maturity Isolines of Soyabean [Glycine max(L.) Merrill] 'Clark'

Plants of eight isolines of soyabean [Glycine max(L.) Merrill],comprising all combinations of two alleles at the three lociE₁/e₁,E₂/e₂andE₃/e₃inthe cultivar ‘Clark’ background, were transferredafter different periods following first flowering from longdays (LD, 14 h d^-1) to short days (SD, 12 h d^-1) andvice versaina reciprocal-transfer experiment in a plastic house maintainedat 30/24 °C (day/night). Photoperiod (0.10>P>0.05),transfer time (P<0.001),>isoline (P<0.001), and theirinteractions (P<0.001) all affected flowering duration, i.e.the period from first flowering until the appearance of thelast flower. The flowering duration comprised two distinct phases:a photoperiod-sensitive phase beginning at first flowering,and a subsequent photoperiod-insensitive phase. The durationof the photoperiod-sensitive phase varied much more among theisolines in LD than in SD. Only the dominant alleleE₁increasedthe sensitivity of the photoperiod-sensitive phase of floweringduration to photoperiod singly, but positive epistatic effectswere detected betweenE₁andE₂,E₁andE₃, and especially among allthree dominant alleles. The increases in flowering durationresulting from the combined effects of gene and environment(i.e. photoperiod) were associated with considerable increasesin biomass and seed yield at harvest maturity.Copyright 1998Annals of Botany Company. Glycine max(L.) Merrill, soyabean, maturity genes, flowering, photoperiod, reciprocal transfer, yield.     

Variation in the Durations of the Photoperiod-sensitive and Photoperiod-insensitive Phases of Development to Flowering Among Eight Maturity Isolines of Soyabean [Glycine max (L.) Merrill]

In soyabean [Glycine max (L.) Merrill] the period between sowingand flowering is comprised of three successive developmentalphases—pre-inductive, inductive and post-inductive—inwhich the rate of development is affected, respectively, bytemperature only, by photoperiod and temperature, and then againby temperature only. A reciprocal-transfer experiment (carriedout at a mean temperature of 25°C) in which cohorts of plantswere transferred successively between short and long photoperiodsand vice-versa showed that eight combinations of three pairsof maturity alleles (E₁/e₁, E₂ /e₂, E₃ /e₃) had their greatesteffect on the duration of the inductive phase in long days.This phase was increased with the increasing photoperiod sensitivityinduced by the different gene combinations, and ranged fromabout 27 to 54 d according to genotype. In a short day regime(11·5 h d^-1), less than the critical photoperiod, theduration of the inductive phase was brief—requiring about11 photoperiodic cycles in the less photoperiod-sensitive genotypesand only about seven cycles in the more sensitive ones. Thematurity genes also affected the duration of the two photoperiod-insensitivephases; these durations were positively correlated with thephotoperiod-sensitivity potential of the gene combinations.The largest effect was on the pre-inductive phase which variedfrom 3 to 11 d, while the post-inductive phase varied from about13 to 18 d. As a consequence of these non-photoperiodic effectsof the maturity genes, even in the most inductive regimes (daylengthsless than the critical photoperiod) the time taken to flowerby the less photoperiod-sensitive combinations of maturity geneswas somewhat less than in the more sensitive combinations—rangingfrom about 28 to 34 d. The genetic and practical implicationsof these findings are discussed.Copyright 1994, 1999 AcademicPress Glycine max (L.) Merrill, soyabean, maturity genes, isolines, flowering, photoperiod     

Effects of Photoperiod and Maturity Genes on Plant Growth, Partitioning, Radiation Use Efficiency, and Yield in Soyabean [Glycine max(L.) Merrill] 'Clark'

Plants of four isolines of soyabean [Glycine max(L.) Merrill]‘Clark’, viz‘L71-920’ (maturity genecomplemente₁e₂e₃ ), ‘L80-5914’ (E₁e₂e₃), ‘Clark’(e₁E₂E₃), and ‘L65-3366’ (E₁E₂E₃), were grown inshort (12.25 h d ^{- 1}natural light) and long days (12.25 h d^{- 1}natural light supplemented with 2.75 h d ^{- 1}low-irradianceartificial light) from first flowering to maturity in a polythenetunnel maintained at 30/24°C (day/night). Whereas therewere few differences among the isolines grown in short days,in long days the dominant alleles increased crop duration, biomassand seed yield substantially. Increases in biological and economicyield were not solely a consequence of longer crop duration:the dominant alleles also increased crop growth rate and radiationuse efficiency in long days (from 1.3 g MJ ^{- 1}total radiationine₁e₂e₃ to 2.8 g MJ ^{- 1}inE₁E₂E₃ ). Greater radiation use efficiencyresulted from a relatively longer leaf area duration, betterdistribution and orientation of a larger mass of leaves withinthe canopy, and smaller partitioning of assimilates to reproductivestructures. The work reveals the substantial effects of thethree lociE₁ / e₁, E₂/ e₂and E₃/e₃ on the response of plantgrowth, as well as development, to environment. Their relevanceto crop adaptation is discussed. Copyright 2000 Annals of BotanyCompany Glycine max(L.) Merrill, soyabean, maturity genes, flowering, phenology, growth, yield     

Durations of the Photoperiod-sensitive and Photoperiod-insensitive Phases of Development to Flowering in Four Cultivars of Soyabean [Glycine max (L.) Merrill]

Four cultivars of soyabean [Glycine max (L.) Merill] of diverseorigin were grown in pots in a plastic-house maintained at day/nighttemperatures of 30/20°C. Plants were transferred at varioustimes after sowing from short (11·5 h d^-1) to long (13·5h d^-1) days and vice versa. The times from sowing to first floweringfor control plants grown continuously in short days varied from38 to 53 d, whereas the flowering of plants grown continuouslyin long days was delayed by about 20 d in each cultivar. Theduration of the initial photoperiod-insensitive phase (oftencalled the juvenile phase) varied three-fold between cultivars,i.e. from 11 to 33 d. As expected, the duration of the photoperiod-sensitivephase was greater in long days, but there was comparativelylittle genetic variation in photoperiod-sensitivity as definedin terms of days delay in time to flowering per hour increasein photoperiod (9-11 d h^-1). Similarly, there was little variationin the photoperiod-insensitive post-inductive phase; it rangedfrom 15 to 20 d. In consequence, the duration of the initialphotoperiod-insensitive phase was a strong determinant of timeto first flowering in these cultivars. The importance of thisso-called juvenile trait is discussed in terms of preventingthe premature flowering of USA-adapted cultivars when grownin short tropical daylengths and thus improving the adaptationof the crop to the lower latitudes.Copyright 1993, 1999 AcademicPress Glycine max (L.) Merill, soyabean, photoperiodism, juvenility, flowering     

Control of Seed Growth in Soya Beans [Glycine max (L.) Merrill]

The seed is the primary sink for photosynthate during reproductivegrowth and an understanding of the mechanisms controlling therate of seed growth is necessary to understand completely theyield production process. The growth rate of individual seedsof seven soya bean [Glycine max (L.) Merrill] cultivars withgenetic differences in seed size varied from 10.8 to 3.9 mgseed^–1 day^–1. The growth rates were highly correlatedwith final seed size. The growth rate of cotyledons culturedin a complete nutrient medium was highly correlated with thegrowth rate of seeds developing on the plant and with finalseed size. The number of cells per seed in the cotyledons variedfrom 10.2 to 5.7 x 10⁶ across the seven cultivars. The numberof cells per seed in the cotyledons was significantly correlatedwith final seed size and the seed growth rate both on the plantand in the culture medium. The data suggest that genetic differencesin seed growth rates are controlled by the cotyledons and thenumber of cells in the cotyledons may be the mechanism of control. Glycine max L., soya bean, seed size, growth rate, cell number, sink activity     

Rhizobium inoculant for soybean [Glycine max (L.) Merrill] in Mekong Delta

Summary Rhizobial inoculation trials were conducted in an acid heavy clay soil in Mekong Delta, Viet Nam, using peat based inoculants produced locally and the commercial granular product of Nitragin CCo., Wisconsin, USA. The pH of these soils ranged from 4.5 to 5.1. Two soybean cultivars, MTD₆ and MTD₁₀, were tested as host plants. There were no significant differences between locally made inoculant treated plants and the uninoculated controls in both cultivars. But, the Nitragin inoculation improved all plant characteristics examined in both cultivars. Grain yields of Nitragin inoculated plants of cultivar MTD₆ and cultivar MTD₁₀ were 6.5 and 5.5 times as much as those of the controls; protein content of grain increased 11 and 16 percent, respectively. Well nodulated plants had shorter life cycles, flowering durations, and days to flowering. The Rhizobium symbiosis resulted in an additional 153 kg grain-N/ha. These studies show that a surface coated commercial multistrain inoculant can be used to successfully grow soybeans in the acid, heavy clay soils of the Mekong Delta.     

Apical proliferation of embryogenic tissue of soybean [Glycine max (L.) Merrill]

Somatic embryos and embryogenic tissues were initiated from immature zygotic embryos of soybean [Glycine max (L.) Merrill cv. Fayette]. Zygotic embryos were placed on a medium containing 40 mg/l of 2,4-dichlorophenoxyacetic acid and 6% sucrose. Somatic embryos were first seen 4 weeks after cultures were initiated. Following transfer, secondary somatic embryos proliferated directly from the apical or terminal portions of the older primary somatic embryos. Single somatic embryos or clusters of embryos were seen growing directly from the top of older somatic embryos. Light microscopy revealed that these embryos were of surface or subsurface origin. The apical soybean somatic embryo tissue may represent cotyledonary tissue (which has been shown to be most responsive) at a very young and manipulatable state.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid Salaries and research support were provided by state and federal funds appropriated to OARDC-OSU. Journal Article No. 131-87     

Diallel analysis for aluminium tolerance in tropical soybeans [Glycine max (L.) Merrill]

The soybean is a major crop in the agricultural systems of the Brazilian Cerrados (Savannahs), whose soils are acidic, devoid of nutrients and need to be amended before they are cultivated. However, below the ploughed layer there is a scarcity of nutrients and toxic aluminium (Al). These limit root growth, subsequently causing nutritional imbalance and drought stress. Our aim in the investigation described here was to identify genetic differences in the aluminium tolerance of soybeans by a 9 × 9 diallel cross among contrasting varieties grown in high-Al areas and in hydroponics. Combining ability analysis indicated predominantly additive gene effects, and the additive-dominance model explained most of the genetic differences in this germ plasm for mineral element absorption and root growth under aluminium stress. The relationship between the two factors suggest that conjugation hydroponics and field evaluations in breeding programmes would further improve soybeans with respect to yield stability under tropical cultivation conditions.     

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     

Characterization of novel cysteine proteases from germinating cotyledons of soybean [Glycine max (L.) Merrill].

The enzymatic properties of novel cysteine proteases D3-alpha and beta which were purified from germinating soybean cotyledons were investigated. The enzyme activities were exhibited in the presence of a thiol reagent, such as 2-mercaptoethanol, and apparently inhibited by E-64, a cysteine protease inhibitor. Hydrolytic activities toward carbobenzoxy-Phe-Arg-MCA were detected at a pH above 4.0. The optimum temperature for activities was about 40 degrees C. The isoelectric point of D3-alpha and beta was 4.4 and 4. 7, respectively. The molecular mass of D3-alpha and beta, measured by MALDI/TOF mass spectrometry, was 26,178 and 26,429 Da, respectively. The substrate specificities of the enzymes were examined using peptide-MCAs and peptides, and cathepsin L-like broad specificity was observed at pH 4.0. These results demonstrated that these enzymes are cysteine endopeptidases [EC 3.4.22.-] like papain [EC 3.4.22.2].     

Diallel analysis for mineral element absorption in tropical adapted soybeans [Glycine max (L.) Merrill]

The Brazilian tropical adapted soybeans contains, in addition to superior morphological characters, genetic factors for tolerance to cultivation in acidic, mineral-stressed soils. However, the selection process for these hindrances has been empirical, and information on the genetics of mineral element uptake by the plant is necessary. The objective of this investigation was to identify the mode of inheritance for the absorption of phosphorus, potassium, calcium, magnesium, iron, aluminium, manganese, zinc and copper in a 9 × 9 diallel cross. General combining ability (GCA) was higher than specific combining ability (SCA), with the exception of copper, manganese and zinc, indicating predominantly additive effects. The ratios of GCA/SCA varied between 3.4 (calcium) and 8.5 (magnesium). The regression of covariance (W_r) on variance (V_r) showed that the additive-dominance model explained the genetic differences in this germ plasm. However, the detection of overdominance could be related to possible heterozygosity in the parental varieties for mineral absorption. Broad-sense heritability values were higher than narrow sense heritability values for aluminium, iron, potassium, calcium and magnesium, being in the range of 67.9–86.9% and 42.0–56.6%, respectively. This is an indication that soybeans can be further improved to efficient utilisation of nutrients and to tolerate toxic factors in the soil.     

Nitric oxide donor regulates Agrobacterium-mediated genetic transformation efficiency in soybean [Glycine max (L.) Merrill]

The present study investigates the potentiality of Sodium nitroprusside (SNP) to enhance the efficiency of genetic transformation in soybean. Half-seeds cultured on co-cultivation [4.44 μM N⁶-benzyl adenine (BA) and 30 μM SNP]; shoot induction (4.44 μM BA and 30 μM SNP) and rooting medium [4.93 μM indole 3-butyric acid (IBA) and 30 μM SNP] exhibited improved transformation efficiency (34.6%) in contrast to the regeneration system devoid of SNP (23%). The putatively transformed plants were evaluated by GUS assay and molecular analysis like PCR and Southern hybridization. Furthermore, the transformation system developed herein entails a shorter period (75-days) for developing plantlets from half-seeds of soybean. The outcome of this study revealed that the addition of SNP increased regeneration efficiency of plants, which translated to improved transformation efficiency in soybean.

     

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.     

Transformation of soybean [Glycine max (L.) Merrill] using proliferative embryogenic tissue maintained on semi-solid medium

Summary Transgenic soybean can be efficiently produced by particle bombardment of embryogenic suspension culture material. Unfortunately, the time required to obtain a transformation-competent soybean suspension culture line is often lengthy and can result in reduced fertility of regenerated plants. In addition, establishment and maintenance of embryogenic suspension cultures can be very difficult. The objective of this work was to minimize the time required to obtain transformation-competent embryogenic tissue and optimize DNA delivery into that tissue. Somatic embryos were induced from immature cotyledons of soybean [Glycine max (L.) Merrill cv ‘Jack’] by placement of cotyledons, adaxial side up, on a MS-based induction medium containing 40 mg (181 μM) 2,4-dichlorophenoxyacetic acid (2,4-D) per 1 and 6% sucrose. Embryogenic tissues, which formed from the surface of the cotyledons within 2–4 wk, were transferred to an embryo proliferation medium containing 20 mg (90 μM) 2,4-D per 1 and 3% sucrose. After 4 wk, proliferative embryogenic tissue could be used for transformation via particle bombardment. Desiccation of target tissue, period of subculture prior to bombardment, and the number of bombardments per target tissue were evaluated for enhancement of transient β-glucuronidase (GUS) expression. The highest number of blue foci was observed when the target tissue was desiccated for 10 min in an uncovered Petri plate containing proliferation medium, subcultured on the same day of bombardment, and bombarded three times on a single day. For stable transformation, selection was started 20 d after bombardment using 9 mg hygromycin per 1 for 4 wk, and 18 mg per 1 thereafter. Stably transformed clones were obtained from tissue bombarded once and twice on a single day. GUS assays and Southern hybridization analysis of DNA from putative clones confirmed stable integration of the introduced genes. Fertile transgenic plants were obtained in 11–12 mo following culture initiation.     

Pod and Seed Mycoflora on Transgenic and Conventional Soybean [Glycine max (L.) Merrill] cultivars in Mississippi

A 2-year (1999-2000) study was conducted at Starkville and Stoneville, MS to determine if the occurrence of the mycoflora varied on Roundup Ready (transgenic) compared to conventional soybean (Glycine max) cultivars. A total of 7,658 fungal isolates were identified from the pod and seed tissues of four cultivars compared at growth stages R6 and R8. Ninety-nine percent of all fungi isolated were mitosporic fungi and ascomycetes. In both years, total fungal isolates from the two locations were greater from the pod (65%) than from seed (33%) tissues. Isolation frequency from conventional cultivars was 54% compared to 46% for the transgenic cultivars. The most common fungi identified that are reported pathogens of soybean included Alternaria, Cercospora, Cladosporium, Diaporthe, Fusarium and Verticillium spp. When main effects and interactions were compared among the frequency data for the fungal genera, significant differences occurred, but consistent trends were not noted. Isolation frequencies of Diaporthe spp. during the R6 growth stage, were significantly greater on the conventional than on the transgenic cultivars in both years of the study, but only at Starkville. Isolation frequencies from samples taken during the R8 growth stage were similar at both locations in 1999 and 2000. Fusarium spp. isolated at R6 and R8 growth stages from pod and seed tissues were significantly greater on conventional than on transgenic cultivars in 2000. Even though frequencies were often significantly different between the transgenic and conventional cultivars, the data was not consistent between locations, pod and seed tissues, or growth stages. The pod and seed mycoflora of transgenic and conventional soybean cultivars was, therefore, similar in Mississippi.     

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.     

Factors Affecting Shedding of Flowers in Soybean (Glycine max (L.) Merrill)

Flower shedding in soybean, Glycine max (L.) Merrill, was studiedusing cultivar ‘Clark’, isoline E₁t, which has relativelylong racemes for convenient identification and observation ofindividual flowers. On each raceme studied, pod set was greatestat the proximal (basal) positions, whereas shedding was greatestat the most distal positions. When proximal flowers were removedas they reached anthesis, pod set increased at the more distalpositions. Pod set was increased in some instances by applicationof water directly to the ovaries as a drop in the calyx cup.Peroxidase activity changed in parallel with ovary development,increasing rapidly in growing pods but not in shedding flowers.Increases in flower peroxidase was mainly in ovary walls. Flowerstaken at or near anthesis from positions with high percent podset could be grown in vitro with especially good ovary enlargement,whereas ovaries in flowers taken from positions of low pod setdid not enlarge in culture. Unidentified substances were extracted from young pods which,when incorporated into lanolin and tested in an in situ bioassay,could mimic the effect of proximal flowers in inducing sheddingof distal flowers. Indole-3-acetic acid resembled the extractedmaterials in inducing shedding, but differed by eliciting side-effectsthat extracts did not. The growth substances abscisic acid,gibberellic acid, and benzyladenine did not promote sheddingin the in situ test. The evidence was taken to indicate that soybean flower sheddingis induced in distal flowers by substances from the more proximal,fertilized ovaries, and that this is possibly due to interferencewith some of the intense metabolic changes that follow pollinationand fertilization.     

A novel Agrobacterium rhizogenes-mediated transformation method of soybean [Glycine max (L.) Merrill] using primary-node explants from seedlings

A novel Agrobacterium rhizogenes-mediated transformation method using a primary-node explant from Dairyland cultivar 93061 was developed for soybean using the disarmed Agrobacterium strain SHA17. Transformed plants regenerated from explants inoculated with SHA17 were fertile and phenotypically normal. In a comparative experiment, regeneration frequencies were not significantly different between explants inoculated with A. rhizogenes strain SHA17 and Agrobacterium tumefaciens strain AGL1; however, a 3.5-fold increase in transformation efficiency [(number of Southern or TaqMan-positive independent events/total number of explants inoculated) × 100] was found for explants cocultured with SHA17 compared to AGL1 (6.6 and 1.64%, respectively). Southern analysis of 48 T₀ plants suggested that 37.5, 23, and 39.6% of the T₀ plants contained 1, 2, and 3 or more T-DNA fragments integrated into the genome, respectively. Additionally, T₁ progeny analysis of 8 independent events resulted in typical Mendelian inheritance of T-DNA genes. Of seven T₀ plants that had two or more T-DNA fragments, six contained multiple loci segregating in T₁ progenies. Further analysis of four lines confirmed the presence of PAT, GUS, and/or DsRED2 proteins in transgenic plants that were encoded on the T-DNA into the T₂ generation.