Amino acids induce high seed-specific expression driven by a soybean (Glycine max) glycinin seed storage protein promoter.

KEY MESSAGE: We characterize GFP expression driven by a soybean glycinin promoter in transgenic soybean. We demonstrate specific amino acid-mediated induction of this promoter in developing soybean seeds in vitro. In plants, gene expression is primarily regulated by promoter regions which are located upstream of gene coding sequences. Promoters allow transcription in certain tissues and respond to environmental stimuli as well as other inductive phenomena. In soybean, seed storage proteins (SSPs) accumulate during seed development and account for most of the monetary and nutritional value of this crop. To better study the regulatory functions of a SSP promoter, we developed a cotyledon culture system where media and media addenda were evaluated for their effects on cotyledon development and promoter activity. Stably transformed soybean events containing a glycinin SSP promoter regulating the green fluorescent protein (GFP) were generated. Promoter activity, as visualized by GFP expression, was only observed in developing in planta seeds and in vitro-cultured isolated embryos and cotyledons from developing seeds when specific media addenda were included. Asparagine, proline, and especially glutamine induced glycinin promoter activity in cultured cotyledons from developing seeds. Other amino acids did not induce the glycinin promoter. Here, we report, for the first time, induction of a reintroduced glycinin SSP promoter by specific amino acids in cotyledon tissues during seed development.

Host-derived gene silencing of parasite fitness genes improves resistance to soybean cyst nematodes in stable transgenic soybean.

KEY MESSAGE: Soybean expressing small interfering RNA of SCN improved plant resistance to SCN consistently, and small RNA-seq analysis revealed a threshold of siRNA expression required for resistance ability. Soybean cyst nematode (SCN), Heterodera glycines, is one of the most destructive pests limiting soybean production worldwide, with estimated losses of $1 billion dollars annually in the USA alone. RNA interference (RNAi) has become a powerful tool for silencing gene expression. We report here that the expression of hairpin RNAi constructs, derived from two SCN genes related to reproduction and fitness, HgY25 and HgPrp17, enhances resistance to SCN in stably transformed soybean plants. The analyses of T3 to T5 generations of stable transgenic soybeans by molecular strategies and next-generation sequencing confirmed the presence of specific short interfering RNAs complementary to the target SCN genes. Bioassays performed on transgenic soybean lines targeting SCN HgY25 and HgPrp17 fitness genes showed significant reductions (up to 73%) for eggs/g root in the T3 and T4 homozygous transgenic lines. Targeted mRNAs of SCN eggs collected from the transgenic soybean lines were efficiently down-regulated, as confirmed by quantitative RT-PCR. Based on the small RNA-seq data and bioassays, it is our hypothesis that a threshold of small interfering RNA molecules is required to significantly reduce SCN populations feeding on the host plants. Our results demonstrated that host-derived gene silencing of essential SCN fitness genes could be an effective strategy for enhancing resistance in crop plants.

Changes to the core and flanking sequences of G-box elements lead to increases and decreases in gene expression in both native and synthetic soybean promoters.

Cis-regulatory elements in promoters are major determinants of binding specificity of transcription factors (TFs) for transcriptional regulation. To improve our understanding of how these short DNA sequences regulate gene expression, synthetic promoters consisting of both classical (CACGTG) and variant G-box core sequences along with different flanking sequences derived from the promoters of three different highly expressing soybean genes, were constructed and used to regulate a green fluorescent protein (gfp) gene. Use of the classical 6-bp G-box provided information on the base level of GFP expression while modifications to the 2-4 flanking bases on either side of the G-box influenced the intensity of gene expression in both transiently transformed lima bean cotyledons and stably transformed soybean hairy roots. The proximal 2-bp sequences on either flank of the G-box significantly affected G-box activity, while the distal 2-bp flanking nucleotides also influenced gene expression albeit with a decreasing effect. Manipulation of the upstream 2- to 4-bp flanking sequence of a G-box variant (GACGTG), found in the proximal region of a relatively weak soybean glycinin promoter, significantly enhanced promoter activity using both transient and stable expression assays, if the G-box variant was first converted into a classical G-box (CACGTG). In addition to increasing our understanding of regulatory element composition and structure, this study shows that minimal targeted changes in native promoter sequences can lead to enhanced gene expression, and suggests that genome editing of the promoter region can result in useful and predictable changes in native gene expression.

Transient Transformation Using Particle Bombardment for Gene Expression Analysis.

Transient transformation or transient expression results in rapid and fleeting gene expression. This approach is often used as a first-tier screening tool for evaluation of components that affect gene expression. Here, we describe the use of particle bombardment of lima bean cotyledons with constructs containing the green fluorescent protein (gfp) coding region for evaluation of promoter components that influence gene expression. Although this approach is conceptually quite simple, this lima bean transient expression system may not work well, if our methods and notes are not carefully read and followed. Our laboratory has successfully optimized this method over the past 10 years, resulting in a transient expression system, which works like no other that we have seen.

Challenges and opportunities for improving food quality and nutrition through plant biotechnology.

Plant biotechnology has been around since the advent of humankind, resulting in tremendous improvements in plant cultivation through crop domestication, breeding and selection. The emergence of transgenic approaches involving the introduction of defined DNA sequences into plants by humans has rapidly changed the surface of our planet by further expanding the gene pool used by plant breeders for plant improvement. Transgenic approaches in food plants have raised concerns on the merits, social implications, ecological risks and true benefits of plant biotechnology. The recently acquired ability to precisely edit plant genomes by modifying native genes without introducing new genetic material offers new opportunities to rapidly exploit natural variation, create new variation and incorporate changes with the goal to generate more productive and nutritious plants.

Synthetic introns help identify sequences in the 5' UTR intron of the Glycine max polyubiquitin (Gmubi) promoter that give increased promoter activity.

MAIN CONCLUSION: Specific sequences within the leader intron of a soybean polyubiquitin gene stimulated gene expression when placed either within a synthetic intron or upstream of a core promoter. The intron in the 5' untranslated region of the soybean polyubiquitin promoter, Gmubi, seems to contribute to the high activity of this promoter. To identify the stimulatory sequences within the intron, ten different sequential intronic sequences of 40 nt were isolated, cloned as tetrameric repeats and placed upstream of a minimal cauliflower mosaic virus 35S (35S) core promoter, which was used to control expression of the green fluorescent protein. Intron fragment tetramers were also cloned within a modified, native intron, creating a Synthetic INtron Cassette (SINC), which was then placed downstream of Gmubi and 35S core promoters. Intron fragment tetramers and SINC constructs were evaluated using transient expression in lima bean cotyledons and stable expression in soybean hairy roots. Intron fragments, used as tetramers upstream of the 35S core promoter, yielded up to 80 times higher expression than the core promoter in transient expression analyses and ten times higher expression in stably transformed hairy roots. Tetrameric intronic fragments, cloned downstream of the Gmubi and 35S core promoters and within the synthetic intron, also yielded increased transient and stable GFP expression that was up to 4 times higher than Gmubi alone and up to 40 times higher than the 35S core promoter alone. These intron fragments contain sequences that seem to act as promoter regulatory elements and may contribute to the increased expression observed with this native strong promoter. Intron regulatory elements and synthetic introns may provide additional tools for increasing transgene expression in plants.

Establishment of in vitro soybean aphids, Aphis glycines (Hemiptera: Aphididae): a tool to facilitate studies of aphid symbionts, plant-insect interactions and insecticide efficacy.

BACKGROUND: Studies on plant-insect interactions of the soybean aphid, Aphis glycines (Matsumura), can be influenced by environmental fluctuations, status of the host plant and variability in microbial populations. Maintenance of aphids on in vitro-grown plants minimizes environmental fluctuations, provides uniform host materials and permits the selective elimination of aphid-associated microbes for more standardized controls in aphid research. RESULTS: Aphids were reared on sterile, in vitro-grown soybean seedlings germinated on plant tissue culture media amended with a mixture of antimicrobials. For initiation and maintenance of in vitro aphid colonies, single aphids were inoculated onto single in vitro seedlings. After three rounds of transfer of 'clean' aphids to fresh in vitro seedlings, contamination was no longer observed, and aphids performed equally well when compared with those reared on detached leaves. The addition of the insecticides thiamethoxam and chlorantraniliprole to the culture medium confirmed uptake and caused significant mortality to the in vitro aphids. The use of the antimicrobial mixture removed the associated bacteria Arsenophonus but retained Buchnera and Wolbachia within the in vitro aphids. CONCLUSION: The in vitro aphid system is a novel and highly useful tool to understand insecticidal efficacy and expand our knowledge of tritrophic interactions among plants, insects and symbionts. © 2016 Society of Chemical Industry.

A Leader Intron of a Soybean Elongation Factor 1A (eEF1A) Gene Interacts with Proximal Promoter Elements to Regulate Gene Expression in Synthetic Promoters.

Introns, especially the first intron in the 5' untranslated region (5'UTR), can significantly impact gene expression via intron-mediated enhancement (IME). In this study, we demonstrate the leader intron of a soybean elongation factor 1A (eEF1A) gene (GmScreamM8) was essential for the high activity of the native promoter. Furthermore, the interaction of the GmScreamM8 leader intron with regulatory element sequences from several soybean eEF1A promoters was studied using synthetic promoters, which consisted of element tetramers upstream of a core promoter used to regulate a green fluorescent protein (gfp) reporter gene. Element tetramers, placed upstream of a GmScreamM8 core promoter, showed very high activity using both transient expression in lima bean cotyledons and stable expression in soybean hairy roots, only if the native leader intron was included, suggesting an interaction between intronic sequences and promoter elements. Partial deletions of the leader intron showed that a 222 bp intronic sequence significantly contributed to very high levels of GFP expression. Generation of synthetic intron variants with a monomeric or trimeric repeat of the 222 bp intronic sequence, yielded almost two-fold higher expression compared to the original intron, while partial deletion of the 222 bp intronic repeated sequence significantly decreased gene expression, indicating that this intronic sequence was essential for the intron-element interaction enhancement.

Low Agrobacterium tumefaciens inoculum levels and a long co-culture period lead to reduced plant defense responses and increase transgenic shoot production of sunflower (Helianthus annuus L.).

Agrobacterium-mediated plant transformation is typically conducted by inoculating plant tissues with an Agrobacterium suspension containing approximately 108-109 bacteria mL-1, followed by a 2-3-d co-culture period. Use of longer co-culture periods could potentially increase transformation efficiencies by allowing more time for Agrobacterium to interact with plant cells, but bacterial overgrowth is likely to occur, leading to severe tissue browning and reduced transformation and regeneration. Low bacterial inoculum levels were therefore evaluated as a means to reduce the negative outcomes associated with long co-culture. The use of low inoculum bacterial suspensions (approximately 6 × 102 bacteria mL-1) followed by long co-culture (15 d) led to the production of an average of three transformed sunflower shoots per explant while the use of high inoculum (approximately 6 × 108 bacteria mL-1) followed by short co-culture (3 d) led to no transformed shoots. Low inoculum and long co-culture acted synergistically, and both were required for the improvement of sunflower transformation. Gene expression analysis via qRT-PCR showed that genes related to plant defense response were generally expressed at lower levels in the explants treated with low inoculum than those treated with high inoculum during 15 d of co-culture, suggesting that low inoculum reduced the induction of plant defense responses. The use of low inoculum with long co-culture (LI/LC) led to large increases in sunflower transformation efficiency. This method has great potential for improving transformation efficiencies and expanding the types of target tissues amenable for transformation of different plant species.

Isolation and Characterization of Agrobacterium Strains from Soil: A Laboratory Capstone Experience.

In this investigation, the students' goal was to isolate and characterize Agrobacterium strains from soil. Following selection and enrichment on 1A-t medium, putative Agrobacterium isolates were characterized by Gram stain reaction and biochemical tests. Isolates were further evaluated using polymerase chain reaction (PCR) with different primer sets designed to amplify specific regions of bacterial deoxyribonucleic acid (DNA). Primer sets included AGRH to identify isolates that were members of the Rhizobiaceae, BIOVAR1 primers to identify members of Agrobacterium biovar group I, and a third set, VIRG, to determine presence of virG (only present in pathogenic Agrobacterium strains). During the investigation, students applied previously learned techniques including serial dilution, use of selective/differential media, staining protocols, biochemical analysis, molecular analysis via PCR, and electrophoresis. Students also gained practical experience using photo documentation to record data for an eventual mock journal publication of the capstone laboratory experience. Pre- and post-evaluation of class content knowledge related to the techniques, protocols, and learning objectives of these laboratories revealed significant learning gains in the content areas of Agrobacterium-plant interactions (p ≤ 0.001) and molecular biology (p ≤ 0.01). The capstone journal assignment served as the assessment tool to evaluate mastery and application of laboratory technique, the ability to accurately collect and evaluate data, and critical thinking skills associated with experimental troubleshooting and extrapolation. Analysis of journal reports following the capstone experience showed significant improvement in assignment scores (p ≤ 0.0001) and attainment of capstone experience learning outcomes.

A novel cis-acting element in the GmERF3 promoter contributes to inducible gene expression in soybean and tobacco after wounding.

KEY MESSAGE: Using in silico and functional analyses, we cloned and validated the expression profile of an inducible soybean promoter (GmERF3) along with its novel wound-induced and delayed expression (WIDE) element. Promoters and their contributing promoter elements are the main regulators of gene expression at the transcriptional level. Although the Ethylene Response Factor (ERF) gene family is one of the most well-studied stress-responsive gene families in plants, their promoter regions have received little attention. In this study, we investigated the expression patterns driven by the soybean (Glycine max) GmERF3 promoter and its cis-acting elements in soybean and tobacco. Transcriptomic data revealed that the native GmERF3 gene was differentially expressed in organs and tissues of plants. In transgenic soybeans containing a 1.3 kb GmERF3 promoter fused to the green fluorescent protein (gfp) gene, organ- and tissue-specificity was observed in untreated plants while mechanical wounding led to induction of GFP expression. Further in silico and in planta analyses of the GmERF3 promoter sequence in soybean revealed different cis-acting elements, including a novel cis-acting element, which contributed to increased expression, 1-2 days after mechanical wounding. We have named this DNA motif the wound-induced and delayed expression element (GGATTCAAGTTTAACC). A synthetic promoter containing a tetrameric repeat of this element showed high but late wound-induced GFP expression in leaves of transgenic tobacco. Our study expands the toolbox of inducible promoters and promoter elements for potential use in basic and applied research.

Feeding Behavior of Soybean Aphid (Hemiptera: Aphididae) Biotype 2 on Resistant and Susceptible Soybean.

Host plant resistance to the soybean aphid, Aphis glycines Matsumura, is an effective means of controlling populations of this introduced pest species in the United States. Rag (Resistance to Aphis glycines) genes identified in soybean germplasm have been incorporated into commercial cultivars, but differential responses by soybean aphid biotypes to the Rag genes have made understanding mechanisms underlying resistance associated with Rag genes increasingly important. We compared the behavior of biotype 2 aphids on the resistant soybean line PI243540, which is a source of Rag2, and the susceptible cultivar Wyandot. Scanning electron microscopy revealed that the abaxial surface of leaves from resistant plants had a higher density of both long and glandulartrichomes, which might repel aphids, on veins. Time-lapse animation also suggested a repellent effect of resistant plants on aphids. However, electropenatography (EPG) indicated that the time to first probe did not differ between aphids feeding on the resistant and susceptible lines. EPG also indicated that fewer aphids feeding on resistant plants reached the phloem, and the time before reaching the phloem was much longer relative to susceptible soybean. For aphids that reached the phloem, there was no difference in either number of feedings or their duration in phloem. However, aphids feeding on resistant soybean had fewer prolonged phases of active salivation (E1) and many more pathway activities and non-probing intervals. Together, the feeding behavior of aphids suggested that Rag2 resistance has strong antixenosis effects, in addition to previously reported antibiosis, and was associated with epidermal and mesophyll tissues.

Generation of Transgenic Soybean (Glycine max) via Particle Bombardment of Embryogenic Cultures.

This protocol describes one method for generating transgenic soybean (Glycine max) using particle bombardment of embryogenic cultures. Embryogenic cultures consist of proliferating masses of somatic embryos and are initially obtained from the cotyledons of immature seeds. Embryogenic cultures are bombarded with small DNA-coated particles and the cells that receive, incorporate and express the DNA are selected, based on a co-delivered antibiotic resistance gene. For recovery of whole plants from the antibiotic resistant embryogenic tissues, the tissues are first placed on a medium conducive to embryo development. Mature embryos are then dried for a short period and placed on a germination medium. Plantlets are then gradually exposed to ambient light and humidity conditions, prior to transfer to a greenhouse. Operators need to be constantly monitoring the protocol and observant of all outcomes. These procedures also are mostly descriptive and need to be precisely followed to be successful. © 2016 by John Wiley & Sons, Inc.

Isolation and characterization of "GmScream" promoters that regulate highly expressing soybean (Glycine max Merr.) genes.

To increase our understanding of the regulatory components that control gene expression, it is important to identify, isolate and characterize new promoters. In this study, a group of highly expressed soybean (Glycine max Merr.) genes, which we have named "GmScream", were first identified from RNA-Seq data. The promoter regions were then identified, cloned and fused with the coding region of the green fluorescent protein (gfp) gene, for introduction and analysis in different tissues using 3 tools for validation. Approximately half of the GmScream promoters identified showed levels of GFP expression comparable to or higher than the Cauliflower Mosaic Virus 35S (35S) promoter. Using transient expression in lima bean cotyledonary tissues, the strongest GmScream promoters gave over 6-fold higher expression than the 35S promoter while several other GmScream promoters showed 2- to 3-fold higher expression. The two highest expressing promoters, GmScreamM4 and GmScreamM8, regulated two different elongation factor 1A genes in soybean. In stably transformed soybean tissues, GFP driven by the GmScreamM4 or GmScreamM8 promoter exhibited constitutive high expression in most tissues with preferentially higher expression in proliferative embryogenic tissues, procambium, vascular tissues, root tips and young embryos. Using deletion analysis of the promoter, two proximal regions of the GmScreamM8 promoter were identified as contributing significantly to high levels of gene expression.

The intron and 5' distal region of the soybean Gmubi promoter contribute to very high levels of gene expression in transiently and stably transformed tissues.

KEY MESSAGE: An extended version of an intron-containing soybean polyubiquitin promoter gave very high levels of gene expression using three different validation tools. The intron-containing Glycine max polyubiquitin promoter (Gmubi) is able to regulate expression levels five times higher than the widely used CaMV35S promoter. In this study, eleven Gmubi derivatives were designed and evaluated to determine which regions contributed to the high levels of gene expression, observed with this promoter. Derivative constructs regulating GFP were evaluated using transient expression in lima bean cotyledons and stable expression in soybean hairy roots. With both expression systems, removal of the intron in the 5'UTR led to reduced levels of gene expression suggesting a role of the intron in promoter activity. Promoter constructs containing an internal intron duplication and upstream translocations of the intron resulted in higher and similar expression levels to Gmubi, respectively, indicating the presence of enhancers within the intron. Evaluation of 5' distal extensions of the Gmubi promoter resulted in significantly higher levels of GFP expression, suggesting the presence of upstream regulatory elements. A twofold increase in promoter strength was obtained when Gmubi was extended 1.5 kb upstream to generate GmubiXL (2.4 kb total length). In stably transformed soybean plants containing GFP regulated by CaMV35S, Gmubi and GmubiXL, the GmubiXL promoter clearly produced the highest levels of gene expression, with especially high GFP fluorescence in the vascular tissue and root tips. Use of GmubiXL leads to very high levels of gene expression in soybean and represents a native soybean promoter, which may be useful for regulating transgene expression for both basic and applied research.

Development and optimization of agroinfiltration for soybean.

KEY MESSAGE: Agroinfiltration is an efficient method to study transgene expression in plant tissue. In this study, sonication followed by vacuum infiltration is shown to increase agroinfiltration-mediated GUS expression in soybean. Agroinfiltration, a valuable tool for rapid analysis of gene function, has been used extensively on leaf tissue of Nicotiana benthamiana and several other plant species. However, the application of this approach for gene functionality studies in soybean has been largely unsuccessful. Improvements in agroinfiltration of many plants have been achieved through a variety of approaches to allow better delivery, penetration and infection of Agrobacterium to interior leaf tissues. In this work, an agroinfiltration approach was developed for transient expression in soybean utilizing sonication followed by vacuum infiltration of intact seedlings. The optimal infiltration buffer, sonication time, and vacuum conditions for agroinfiltration of soybean were evaluated by monitoring expression of an introduced ß-glucuronidase (GUS) reporter gene. The developed method included the use of an infiltration buffer (10 mM 2-(N-morpholino)ethanesulfonic acid sodium salt, 10 mM MgCl2, 100 µM acetosyringone) supplemented with the reducing agent dithiothreitol, with 30 s sonication followed by vacuum infiltration. These techniques were further applied to evaluate five different Agrobacterium strains and six different plant genetic backgrounds. Among the Agrobacterium strains examined, J2 produced the highest levels of GUS activity and 'Peking' was the most responsive genotype.

Identification and validation of promoters and cis-acting regulatory elements.

Studies of promoters that largely regulate gene expression at the transcriptional level are crucial for improving our basic understanding of gene regulation and will expand the toolbox of available promoters for use in plant biotechnology. In this review, we present a comprehensive analysis of promoters and their underlying mechanisms in transcriptional regulation, including epigenetic marks and chromatin-based regulation. Large-scale prediction of promoter sequences and their contributing cis-acting elements has become routine due to recent advances in transcriptomic technologies and genome sequencing of several plants. However, predicted regulatory sequences may or may not be functional and demonstration of the contribution of the element to promoter activity is essential for confirmation of regulatory sequences. Synthetic promoters and introns provide useful approaches for functional validation of promoter sequences. The development and improvement of gene expression tools for rapid, efficient, predictable, and high-throughput analysis of promoter components will be critical for confirmation of the functional regulatory element sequences identified through transcriptomic and genomic analyses.

WRKY transcription factors: key components in abscisic acid signalling.

WRKY transcription factors (TFs) are key regulators of many plant processes, including the responses to biotic and abiotic stresses, senescence, seed dormancy and seed germination. For over 15 years, limited evidence has been available suggesting that WRKY TFs may play roles in regulating plant responses to the phytohormone abscisic acid (ABA), notably some WRKY TFs are ABA-inducible repressors of seed germination. However, the roles of WRKY TFs in other aspects of ABA signalling, and the mechanisms involved, have remained unclear. Recent significant progress in ABA research has now placed specific WRKY TFs firmly in ABA-responsive signalling pathways, where they act at multiple levels. In Arabidopsis, WRKY TFs appear to act downstream of at least two ABA receptors: the cytoplasmic PYR/PYL/RCAR-protein phosphatase 2C-ABA complex and the chloroplast envelope-located ABAR-ABA complex. In vivo and in vitro promoter-binding studies show that the target genes for WRKY TFs that are involved in ABA signalling include well-known ABA-responsive genes such as ABF2, ABF4, ABI4, ABI5, MYB2, DREB1a, DREB2a and RAB18. Additional well-characterized stress-inducible genes such as RD29A and COR47 are also found in signalling pathways downstream of WRKY TFs. These new insights also reveal that some WRKY TFs are positive regulators of ABA-mediated stomatal closure and hence drought responses. Conversely, many WRKY TFs are negative regulators of seed germination, and controlling seed germination appears a common function of a subset of WRKY TFs in flowering plants. Taken together, these new data demonstrate that WRKY TFs are key nodes in ABA-responsive signalling networks.

High level transgenic expression of soybean (Glycine max) GmERF and Gmubi gene promoters isolated by a novel promoter analysis pipeline.

BACKGROUND: Although numerous factors can influence gene expression, promoters are perhaps the most important component of the regulatory control process. Promoter regions are often defined as a region upstream of the transcriptional start. They contain regulatory elements that interact with regulatory proteins to modulate gene expression. Most genes possess their own unique promoter and large numbers of promoters are therefore available for study. Unfortunately, relatively few promoters have been isolated and characterized; particularly from soybean (Glycine max). RESULTS: In this research, a bioinformatics approach was first performed to identify members of the Gmubi (G.max ubiquitin) and the GmERF (G. max Ethylene Response Factor) gene families of soybean. Ten Gmubi and ten GmERF promoters from selected genes were cloned upstream of the gfp gene and successfully characterized using rapid validation tools developed for both transient and stable expression. Quantification of promoter strength using transient expression in lima bean (Phaseolus lunatus) cotyledonary tissue and stable expression in soybean hairy roots showed that the intensity of gfp gene expression was mostly conserved across the two expression systems. Seven of the ten Gmubi promoters yielded from 2- to 7-fold higher expression than a standard CaMV35S promoter while four of the ten GmERF promoters showed from 1.5- to 2.2-times higher GFP levels compared to the CaMV35S promoter. Quantification of GFP expression in stably-transformed hairy roots of soybean was variable among roots derived from different transformation events but consistent among secondary roots, derived from the same primary transformation events. Molecular analysis of hairy root events revealed a direct relationship between copy number and expression intensity; higher copy number events displayed higher GFP expression. CONCLUSION: In this study, we present expression intensity data on 20 novel soybean promoters from two different gene families, ubiquitin and ERF. We also demonstrate the utility of lima bean cotyledons and soybean hairy roots for rapid promoter analyses and provide novel insights towards the utilization of these expression systems. The soybean promoters characterized here will be useful for production of transgenic soybean plants for both basic research and commercial plant improvement.

Robotics and dynamic image analysis for studies of gene expression in plant tissues.

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.