Agrobacterium-mediated Soybean Transformation Research Articles

Aztreonam is a novel chemical inducer that promotes Agrobacteium transformation and lateral root development in soybean.

Agrobacterium-mediated soybean transformation is the simplest method of gene transfer. However, the low transformation due to the intractable nature of soybean genotypes hinders this process. The use of biochemicals (acetosyringone, cinnamic acid, flavonoids, etc.) plays an important role in increasing soybean transformation. These biochemicals induce chemotaxis and virulence gene activation during the infection process. Here we identified a biochemical, aztreonam (a monobactam), for high agrobacterium-mediated transformation in soybean. The soybean explants from three genotypes were inoculated with A. tumefaciens (GV3101) harboring the pMDC32 vector containing hpt or the GmUbi-35S-GUS vector containing the GUS gene during two separate events. High transient GUS expression was obtained during cotyledon explant culture on MS media supplemented with 2.5 mg/L aztreonam. The aztreonam-treated explants showed high efficiency in transient and stable transformation as compared to the untreated control. The transformation of aztreonam-treated explants during seed imbibition resulted in an average of 21.1% as compared to 13.2% in control by using the pMDC32 vector and 28.5 and 20.7% while using the GUS gene cassette, respectively. Based on these findings, the metabolic analysis of the explant after aztreonam treatment was assessed. The high accumulation of flavonoids was identified during an untargeted metabolic analysis. The quantification results showed a significantly high accumulation of the four compounds, i.e., genistein, apigenin, naringenin, and genistin, in cotyledon explants after 18 hours of aztreonam treatment. Alongside this, aztreonam also had some surprising effects on root elongation and lateral root formation when compared to indole-3-butyric acid (IBA). Our findings were limited to soybeans. However, the discovery of aztreonam and its effect on triggering flavonoids could lead to the potential role of aztreonam in the agrobacterium-mediated transformation of different crops.

CRISPR/Cas9-Based Gene Editing in Soybean.

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR associated Cas9)-based gene editing is a robust tool for functional genomics research and breeding programs in various crops. In soybean, a number of laboratories have obtained mutants by CRISPR/Cas9 system; however, there has been not yet a detailed method for the CRISPR/Cas9-based gene editing in soybean. Here, we describe the procedures for constructing the CRISPR/Cas9 plasmid suitable for soybean gene editing and the modified protocols for Agrobacterium-mediated soybean transformation and regeneration from cotyledonary node explants containing the Cas9/sgRNA (single guide RNA) transgenes.

5-Azacytidine promotes shoot regeneration during Agrobacterium-mediated soybean transformation

Agrobacterium-mediated soybean transformation has been greatly improved in recent years, however the transformation efficiency is still low and highly genotype-dependent when compared to other species. Here, we characterized seventeen soybean genotypes based on their genetic transformation efficiencies, i.e., high and low, during Agrobacterium-mediated transformation. To reveal the molecular basis of this transformation difference, we constructed a highly efficient transient transgene expression system using soybean cotyledon protoplasts and then assess the methylation levels of promoter and coding regions of an EYFP (enhanced yellow fluorescent protein) gene introduced into the protoplast cultures of various soybean genotypes using BSP (bisulfite sequencing PCR). Increased methylation was found to be associated with the considerably decreased transfection efficiency (as percentage of EYFP fluorescent protoplasts) in low-efficacy genotypes as compared with those in high-efficacy on three DAT (day after transfection). 5-Azacytidine (5-Azac), a demethylating reagent commonly applied in epigenetic researches, significantly improved the transient transfection efficiency and transgene expression level in low-efficiency genotypes. Furthermore, the shoot regeneration efficiency in low-efficiency genotypes was substantially increased by 5-Azac treatment in an Agrobacterium-mediated soybean transformation system. Taken together, we concluded that lower methylation level in transgene contributed to enhanced shoot regeneration in Agrobacterium-mediated soybean transformation.

An efficient Agrobacterium-mediated soybean transformation method using green fluorescent protein as a selectable marker

ABSTRACTGenetic transformation plays a vital role in gene functional study and molecular breeding of soybean. Conventional soybean transformation methods using chemical selectable markers, such as antibiotic or herbicide resistance genes, rely on the identification of positive transgenic lines at advanced developmental stages, making selection procedure labor intensive and time consuming. Utilization of a visual maker to track the transgene would avoid the uncertainty and blindness in the transformation process. In this research, we used green fluorescent protein (GFP) as the selectable marker to detect transgenics at early stages of soybean development. Positive transformants were detected recurrently during each stage of the process based on visualization of the green fluorescence signal, which help us to discard the non-transgenic ones in each stage to reduce the unnecessary experimental cost and lab space. In addition, the positive transgenic seeds can be identified before planting for early detection of transgene and obtain homozygous lines in advance. The method established in this study is also a useful reference for other plant species.

Confirmation of Drought Tolerance of Ectopically Expressed AtABF3 Gene in Soybean.

Soybean transgenic plants with ectopically expressed AtABF3 were produced by Agrobacterium-mediated transformation and investigated the effects of AtABF3 expression on drought and salt tolerance. Stable Agrobacterium-mediated soybean transformation was carried based on the half-seed method (Paz et al. 2006). The integration of the transgene was confirmed from the genomic DNA of transformed soybean plants using PCR and the copy number of transgene was determined by Southern blotting using leaf samples from T2 seedlings. In addition to genomic integration, the expression of the transgenes was analyzed by RT-PCR and most of the transgenic lines expressed the transgenes introduced. The chosen two transgenic lines (line #2 and #9) for further experiment showed the substantial drought stress tolerance by surviving even at the end of the 20-day of drought treatment. And the positive relationship between the levels of AtABF3 gene expression and drought-tolerance was confirmed by qRT-PCR and drought tolerance test. The stronger drought tolerance of transgenic lines seemed to be resulted from physiological changes. Transgenic lines #2 and #9 showed ion leakage at a significantly lower level (P < 0.01) than non-transgenic (NT) control. In addition, the chlorophyll contents of the leaves of transgenic lines were significantly higher (P < 0.01). The results indicated that their enhanced drought tolerance was due to the prevention of cell membrane damage and maintenance of chlorophyll content. Water loss by transpiration also slowly proceeded in transgenic plants. In microscopic observation, higher stomata closure was confirmed in transgenic lines. Especially, line #9 had 56% of completely closed stomata whereas only 16% were completely open. In subsequent salt tolerance test, the apparently enhanced salt tolerance of transgenic lines was measured in ion leakage rate and chlorophyll contents. Finally, the agronomic characteristics of ectopically expressed AtABF3 transgenic plants (T2) compared to NT plants under regular watering (every 4 days) or low rate of watering condition (every 10 days) was investigated. When watered regularly, the plant height of drought-tolerant line (#9) was shorter than NT plants. However, under the drought condition, total seed weight of line #9 was significantly higher than in NT plants (P < 0.01). Moreover, the pods of NT plants showed severe withering, and most of the pods failed to set normal seeds. All the evidences in the study clearly suggested that overexpression of the AtABF3 gene conferred drought and salt tolerance in major crop soybean, especially under the growth condition of low watering.

Comparison of transformation frequency using the bar gene regulated by the CaMV 35S or NOS promoter in Agrobacterium-mediated soybean (Glycine max L.) transformation

In Agrobacterium tumefaciens-mediated plant transformation, the promoter chosen to drive a selectable marker gene has an effect on transformation frequency. The objective of this work was to compare the effect on soybean transformation of different promoter and regulator combinations driving a selectable marker gene using mature seeds as explants. Two commonly used promoters, double cauliflower mosaic virus 35S (CaMV 35S) and nopaline synthase (NOS), and one regulator, tobacco etch virus (TEV) translational enhancer, were tested. Four different promoter/enhancer combinations were constructed to drive a bialaphos resistance gene (bar) and used for multiple independent transformation experiments. Herbicide application and PCR analysis were used to confirm the inheritance of the bar gene in the T 1 generation. Quantitative real-time PCR (qRT-PCR) was used to estimate transgene copy number in T 1 transgenic events. The average transformation frequency from combining NOS with the TEV enhancer (3.5% across 12 replications) was significantly higher than the frequencies obtained from the double CaMV 35S without an enhancer (1.6% across 16 replications), double CaMV 35S with an enhancer (1.4% across 8 replications), and NOS without an enhancer (1.0% across 12 replications). The bar transcript levels in T 1 transgenic leaf tissue did not correlate with transformation frequencies achieved by the different constructs. No significant differences were identified between constructs in the average transgene copy number across events. These data show that a selectable marker system comprised of the bar gene regulated by the NOS promoter in combination with the TEV enhancer is preferred in Agrobacterium-mediated soybean transformation.

Enhanced Soybean Infection by the Legume “Super-Virulent”Agrobacterium tumefaciensStrain KAT23

Agrobacterium tumefaciens KAT23 harbors a nopaline-type Ti plasmid and is "super-virulent" to soybean (Glycine max) and other leguminous plants. The right and left border sequences of the essential cis-element for T-DNA transfer were removed in order to utilize the high infectivity of this strain in an Agrobacterium-mediated soybean transformation system. The resulting strain, named Soy2, showed no oncogenic activity. After inoculation with disarmed Soy2 harboring binary vector pIG121-Hm and pCAMBIA-WR, soybean epicotyls exhibited high beta-glucuronidase activities, with efficiencies higher than EHA105, an A. tumefaciens strain widely used in making transgenic plants.

Advances in Agrobacterium-mediated plant transformation with enphasys on soybean

Soybean is one of humanity's major sources of plant protein. It is also very important for animal feed and as industrial raw material. Great advances have recently been achieved in its genetic transformation. This review provides a comprehensive discussion of important factors affecting Agrobacterium-mediated soybean transformation including target tissues, plant tissue health, wounding methods, regeneration systems, selectable markers and reporter genes.

Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation

Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation

Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation

The utility of transformation for soybean improvement requires an efficient system for production of stable transgenic lines. We describe here an improved cotyledonary node method using an alternative explant for Agrobacterium tumefaciens-mediated soybean transformation. We use the term "half-seed" to refer to this alternative cotyledonary explant that is derived from mature seed of soybean following an overnight imbibition and to distinguish it from cotyledonary node derived from 5-7-day-old seedlings. Transformation efficiencies using half-seed explants ranged between 1.4 and 8.7% with an overall efficiency of 3.8% based on the number of transformed events that have been confirmed in the T1 generation by phenotypic assay using the herbicide Liberty (active ingredient glufosinate) and by Southern analysis. This efficiency is 1.5-fold higher than the cotyledonary node method used in our laboratory. Significantly, the half-seed system is simple and does not require deliberate wounding of explants, which is a critical and technically demanding step in the cotyledonary node method.

Assessment of conditions affecting Agrobacterium-mediated soybean transformation using the cotyledonary node explant

Selection for lodging resistant cultivars in cereal breeding programs is difficult due to the challenge of screening for this trait under natural field conditions. The identification of easily measurable culm traits related to lodging resistance would simplify the selection process. The present study was conducted to determine if differences in culm anatomy exist among modern wheat genotypes differing in lodging susceptibility, and to determine the association between culm characters and lodging. From a 2-year field study conducted in Edmonton, Alberta, 13 spring wheat cultivars were chosen based on predetermined susceptibility to artificially induced lodging. Morphological and anatomical culm measurements were made visually and with an environmental scanning electron microscope. Genotypes differed (P < 0.05) for plant height, number of internodes per culm, basal internode length and diameter, culm wall thickness and the number of vascular bundles, but not for adventitious root frequency, lumen diameter or sclerenchyma ring thickness. Mean genotype field scores for artificially induced lodging were correlated (P < 0.05) with plant height (r= 0.51) and the length of the fourth basal internode (r= 0.51). Short, wide basal internodes and thick culm walls were characteristic of three lodging tolerant genotypes: Kohika, Sapphire and Olso. Nevertheless, despite such apparent genotype specific association between culm anatomy and field lodging, general applicable associations were not observed for most traits. The most practical and easily selectable trait for lodging resistance within a wheat breeding program remains plant height.