Agrobacterium-Mediated Transformation of the Dwarf Soybean MiniMax.

This study aims to establish an Agrobacterium-mediated transformation system for use with the 'MiniMax'soybean cultivar. MiniMax is a mutant soybean whose growth cycle is around 90 days, half that of most other soybean varieties, making it an optimal model cultivar to test genes of interest before investing in modification of elite lines. We describe an efficient protocol for Agrobacterium-mediated transformation using MiniMax seeds. It uses a modified 'half seed' regeneration protocol for transgenic soybean production, utilizing the rapid generation MiniMax variety to obtain T1 seeds in approximately 145 days. Addition of phloroglucinol (PG) to the regeneration protocol was key to obtaining high-efficiency rooting of the regenerated shoots. Transfer to soil was accomplished using an organic soil amendment containing nutrients and mycorrhiza for plants to thrive in the greenhouse. This combination of genotype and stimulants provides a transformation protocol to genetically engineer MiniMax seeds with a transgenic lab-to-greenhouse production efficiency of 4.0%. This is the first report of MiniMax soybean whole plant transformation and heritable T1 transmission. This protocol provides an ideal resource for enhancing the genetic transformation of any soybean cultivar.

Photosynthetically active radiation is required for seedling growth promotion by volcanic dacitic tuff breccia (Azomite).

A plant's growth and development are shaped by its genome and the capacity to negotiate its environment for access to light, water, and nutrients. There is a vital need to understand the interactions between the plant, its physical environment, and the fertilizers used in agriculture. In this study, a commercially available volcanic ash fertilizer, Azomite®, characterized as dacitic (rhyolitic) tuff breccia, was tested for its effect on promoting early seedling vigor. Early growth and photomorphogenesis processes are well studied in Arabidopsis. Seedling assays under different light conditions were used to dissect the underlying mechanisms involved. These assays are well established and can be translated to agriculturally important crop plants. The volcanic ash fertilizer was tested at different concentrations on seedlings grown on basic media lacking sucrose either in continuous darkness (Dc), continuous Red (Rc), Far-Red (FRc), or White Light (WLc). Micronutrients in the volcanic ash significantly increased seedling growth under Rc and WLc, but not under Dc and FRc, indicating that photosynthetically active radiation was required for the observed growth increase. Furthermore, red-light photoreceptor mutant, phyB-9, lacked the growth response, and higher amount of fertilizer reduced growth in all conditions tested. These data suggest that light triggers the ability of the seedling to utilize micronutrients in volcanic ash in a dose-dependent manner. The methods described here can be used to establish mechanisms of activity of various nutrient inputs and, coupled with whole-genome expression profiling, can lead to better insights into optimizing nutrient field applications to improve crop production.

Phyllosphere microbial associations improve plant reproductive success.

The above-ground (phyllosphere) plant microbiome is increasingly recognized as an important component of plant health. We hypothesized that phyllosphere bacterial recruitment may be disrupted in a greenhouse setting, and that adding a bacterial amendment would therefore benefit the health and growth of host plants. Using a newly developed synthetic phyllosphere bacterial microbiome for tomato (Solanum lycopersicum), we tested this hypothesis across multiple trials by manipulating microbial inoculation of leaves and measuring subsequent plant growth and reproductive success, comparing results from plants grown in both greenhouse and field settings. We confirmed that greenhouse-grown plants have a relatively depauperate phyllosphere bacterial microbiome, which both makes them an ideal system for testing the impact of phyllosphere communities on plant health and important targets for microbial amendments as we move towards increased agricultural sustainability. We find that the addition of the synthetic microbial community early in greenhouse growth leads to an increase in fruit production in this setting, implicating the phyllosphere microbiome as a key component of plant fitness and emphasizing the role that these bacterial microbiomes likely play in the ecology and evolution of plant communities.

Temporally Selective Modification of the Tomato Rhizosphere and Root Microbiome by Volcanic Ash Fertilizer Containing Micronutrients.

Food crops are grown with fertilizers containing nitrogen, phosphorus, and potassium (macronutrients) along with magnesium, calcium, boron, and zinc (micronutrients) at different ratios during their cultivation. Soil and plant-associated microbes have been implicated to promote plant growth, stress tolerance, and productivity. However, the high degree of variability across agricultural environments makes it difficult to assess the possible influences of nutrient fertilizers on these microbial communities. Uncovering the underlying mechanisms could lead us to achieve consistently improved food quality and productivity with minimal environmental impacts. For this purpose, we tested a commercially available fertilizer (surface-mined volcanic ash deposit Azomite) applied as a supplement to the normal fertilizer program of greenhouse-grown tomato plants. Because this treatment showed a significant increase in fruit production at measured intervals, we examined its impact on the composition of below-ground microbial communities, focusing on members identified as "core taxa" that were enriched in the rhizosphere and root endosphere compared to bulk soil and appeared above their predicted neutral distribution levels in control and treated samples. This analysis revealed that Azomite had little effect on microbial composition overall, but it had a significant, temporally selective influence on the core taxa. Changes in the composition of the core taxa were correlated with computationally inferred changes in functional pathway enrichment associated with carbohydrate metabolism, suggesting a shift in available microbial nutrients within the roots. This finding exemplifies how the nutrient environment can specifically alter the functional capacity of root-associated bacterial taxa, with the potential to improve crop productivity. IMPORTANCE Various types of soil fertilizers are used routinely to increase crop yields globally. The effects of these treatments are assessed mainly by the benefits they provide in increased crop productivity. There exists a gap in our understanding of how soil fertilizers act on the plant-associated microbial communities. The underlying mechanisms of nutrient uptake are widely complex and, thus, difficult to evaluate fully but have critical influences on both soil and plant health. Here, we presented a systematic approach to analyzing the effects of fertilizer on core microbial communities in soil and plants, leading to predictable outcomes that can be empirically tested and used to develop simple and affordable field tests. The methods described here can be used for any fertilizer and crop system. Continued effort in advancing our understanding of how fertilizers affect plant and microbe relations is needed to advance scientific understanding and help growers make better-informed decisions.

Draft Genome Sequence of Agrobacterium fabrum Strain 1D1104.

This work reports the draft genome sequence of Agrobacterium fabrum strain 1D1104. The assembled genome is composed of a 2,774,783-bp circular chromosome, a 2,110,112-bp linear chromosome, an AT plasmid of 133,577 bp, and four unassembled contigs of 5,389,544 bp, 42,391 bp, 41,768 bp, and 35,476 bp.

Draft Genome Sequence of Agrobacterium tumefaciens Strain 1D1526.

This work reports the draft genome sequence of Agrobacterium tumefaciens strain 1D1526. The assembled genome is composed of a 2,881,823-bp circular chromosome, a 2,235,711-bp linear chromosome, and a 44,582-bp unassembled contig.

Transgene stacking in potato using the GAANTRY system.

OBJECTIVE: GAANTRY (Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) is a flexible and effective system for stably stacking multiple genes within an Agrobacterium virulence plasmid Transfer-DNA (T-DNA). We examined the ability of the GAANTRY Agrobacterium rhizogenes ArPORT1 '10-stack' strain to generate transgenic potato plants. RESULTS: The 28.5 kilobase 10-stack T-DNA, was introduced into Lenape potato plants with a 32% transformation efficiency. Molecular and phenotypic characterization confirmed that six of the seven tested independent transgenic lines carried the entire desired construct, demonstrating that the GAANTRY 10-stack strain can be used can be used in a tissue culture-based callus transformation method to efficiently generate transgenic potato plants. Analysis using droplet digital PCR showed that most of the characterized events carry one or two copies of the 10-stack transgenes and that 'backbone' DNA from outside of the T-DNA was absent in the transgenic plants. These results demonstrate that the GAANTRY system efficiently generates high quality transgenic potato plants with a large construct of stacked transgenes.

Complete Genome Sequence of Agrobacterium fabrum Strain 1D159.

This work reports the draft genome sequence of Agrobacterium fabrum strain 1D159 (also known as ATCC strain 27912). The assembled genome is composed of a 2,861,352-bp circular chromosome, a 2,058,040-bp linear chromosome, a 519,735-bp AT plasmid, and the 223,394-bp Ti virulence plasmid. The wild nondisarmed strain produces small gall-like structures in citrus.

Into the weeds: Matching importation history to genetic consequences and pathways in two widely used biological control agents.

The intentional introduction of exotic species through classical biological control programs provides unique opportunities to examine the consequences of population movement and ecological processes for the genetic diversity and population structure of introduced species. The weevils Neochetina bruchi and N. eichhorniae (Coleoptera: Curculionidae) have been introduced globally to control the invasive floating aquatic weed, Eichhornia crassipes, with variable outcomes. Here, we use the importation history and data from polymorphic microsatellite markers to examine the effects of introduction processes on population genetic diversity and structure. We report the first confirmation of hybridization between these species, which could have important consequences for the biological control program. For both species, there were more rare alleles in weevils from the native range than in weevils from the introduced range. N. eichhorniae also had higher allelic richness in the native range than in the introduced range. Neither the number of individuals initially introduced nor the number of introduction steps appeared to consistently affect genetic diversity. We found evidence of genetic drift, inbreeding, and admixture in several populations as well as significant population structure. Analyses estimated two populations and 11 sub-clusters for N. bruchi and four populations and 23 sub-clusters for N. eichhorniae, indicating divergence of populations during and after introduction. Genetic differentiation and allocation of introduced populations to source populations generally supported the documented importation history and clarified pathways in cases where multiple introductions occurred. In populations with multiple introductions, genetic admixture may have buffered against the negative effects of serial bottlenecks on genetic diversity. The genetic data combined with the introduction history from this biological control study system provide insight on the accuracy of predicting introduction pathways from genetic data and the consequences of these pathways for the genetic variation and structure of introduced species.

Draft Genome Sequence of Serratia sp. 1D1416.

This work reports the draft genome of Serratia sp. 1D1416. The assembled genome contains a 5,552,016-bp circular chromosome. The strain was discovered in a mixed culture from a gall isolated from Euonymus japonicas.

Accurate measurement of transgene copy number in crop plants using droplet digital PCR.

Genetic transformation is a powerful means for the improvement of crop plants, but requires labor- and resource-intensive methods. An efficient method for identifying single-copy transgene insertion events from a population of independent transgenic lines is desirable. Currently, transgene copy number is estimated by either Southern blot hybridization analyses or quantitative polymerase chain reaction (qPCR) experiments. Southern hybridization is a convincing and reliable method, but it also is expensive, time-consuming and often requires a large amount of genomic DNA and radioactively labeled probes. Alternatively, qPCR requires less DNA and is potentially simpler to perform, but its results can lack the accuracy and precision needed to confidently distinguish between one- and two-copy events in transgenic plants with large genomes. To address this need, we developed a droplet digital PCR-based method for transgene copy number measurement in an array of crops: rice, citrus, potato, maize, tomato and wheat. The method utilizes specific primers to amplify target transgenes, and endogenous reference genes in a single duplexed reaction containing thousands of droplets. Endpoint amplicon production in the droplets is detected and quantified using sequence-specific fluorescently labeled probes. The results demonstrate that this approach can generate confident copy number measurements in independent transgenic lines in these crop species. This method and the compendium of probes and primers will be a useful resource for the plant research community, enabling the simple and accurate determination of transgene copy number in these six important crop species.

Biological differences that distinguish the 2 major stages of wound healing in potato tubers.

The two stages of potato tuber wound healing, closing layer formation (CLF) and wound periderm formation (WPF), have critical biological differences. The first stage, CLF, involves early induction of DNA synthesis and nuclear division in the absence of cell division. The transition phase from CLF to the second stage, WPF, is marked by a transient decrease in expression of suberin-specific genes. The second stage involves cell division. Although biologically active cytokinins (CKs) are not present in quantifiable amounts during this stage, the presence of precursor and catabolic products suggest the presence of trace amounts of active CKs that, in conjunction with increased auxin (indole acetic acid), provide necessary signals for meristematic activity. Augmenting these putative trace amounts with exogenous biologically active CK inhibits WPF; this suggests that the CK requirements for meristematic activity are finely controlled and sensitive to extremely low concentrations. Evidence is discussed for separate biological processes and signals that distinguish the 2 stages of wound healing.

Impact of light-exposure on the metabolite balance of transgenic potato tubers with modified glycoalkaloid biosynthesis.

Metabolite profiling (liquid chromatography-mass spectrometry (LC-MS) and gas chromatography (GC-MS)) was used to assess the impact of light on the composition of transgenic potato (Solanum tuberosum L. cv. Desirée) with reduced glycoalkaloid content via the down-regulation of the SGT1 gene. Transgenic tubers exhibited an almost complete knock-out of α-solanine production and light had little impact on its accumulation. Levels of α-chaconine increased significantly in the peel of both the control and transgenic lines when exposed to light, particularly in the transgenic line. Major differences in metabolite profiles existed between outer and inner tuber tissues, and between light and dark-treated tubers. Many of the light-induced changes are explicable in terms of pathways known to be affected by stress responses. The impact of transgenesis on profiles was much less than that of tissue type or light and most differences were explicable in terms of the modification to the glycoalkaloid pathway.

Modifying glycoalkaloid content in transgenic potato--Metabolome impacts.

Metabolite profiling has been used to assess the potential for unintended composition changes in potato (Solanum tuberosum L. cv. Desirée) tubers, which have been genetically modified (GM) to reduce glycoalkaloid content, via the independent down-regulation of three genes SGT1, SGT2 and SGT3 known to be involved in glycoalkaloid biosynthesis. Differences between the three groups of antisense lines and control lines were assessed using liquid chromatography-mass spectrometry (LC-MS) and gas chromatography (GC)-MS, and data analysed using principal component analysis and analysis of variance. Compared with the wild-type (WT) control, LC-MS revealed not only the expected changes in specific glycoalkaloid levels in the GM lines, but also significant changes in several other metabolites, some of which were explicable in terms of known pathways. Analysis of polar and non-polar metabolites by GC-MS revealed other significant (unintended) differences between SGT lines and the WT, but also between the WT control and other control lines used.

A family of small cyclic amphipathic peptides (SCAmpPs) genes in citrus.

BACKGROUND: Citrus represents a crop of global importance both in economic impact and significance to nutrition. Citrus production worldwide is threatened by the disease Huanglongbing (HLB), caused by the phloem-limited pathogen Candidatus Liberibacter spp.. As a source of stable HLB-resistance has yet to be identified, there is considerable interest in characterization of novel disease-associated citrus genes. RESULTS: A gene family of Small Cyclic Amphipathic Peptides (SCAmpPs) in citrus is described. The citrus genomes contain 100-150 SCAmpPs genes, approximately 50 of which are represented in the citrus EST database. These genes encode small ~50 residue precursor proteins that are post-translationally processed, releasing 5-10 residue cyclic peptides. The structures of the SCAmpPs genes are highly conserved, with the small coding domains interrupted by a single intron and relatively extended untranslated regions. Some family members are very highly transcribed in specific citrus tissues, as determined by representation in tissue-specific cDNA libraries. Comparison of the ESTs of related SCAmpPs revealed an unexpected evolutionary profile, consistent with targeted mutagenesis of the predicted cyclic peptide domain. The SCAmpPs genes are displayed in clusters on the citrus chromosomes, with apparent association with receptor leucine-rich repeat protein arrays. This study focused on three SCAmpPs family members with high constitutive expression in citrus phloem. Unexpectedly high sequence conservation was observed in the promoter region of two phloem-expressed SCAmpPs that encode very distinct predicted cyclic products. The processed cyclic product of one of these phloem SCAmpPs was characterized by LC-MS-MS analysis of phloem tissue, revealing properties consistent with a K(+) ionophore. CONCLUSIONS: The SCAmpPs amino acid composition, protein structure, expression patterns, evolutionary profile and chromosomal distribution are consistent with designation as ribosomally synthesized defense-related peptides.

Compositional and toxicological analysis of a GM potato line with reduced α-solanine content--a 90-day feeding study in the Syrian Golden hamster.

Steroidal glycoalkaloids (GAs) are toxins, produced by plants of the Solanaceae family. The potato plant (Solanum tuberosum L.) and its tubers predominantly contain the two GAs α-chaconine and α-solanine. These compounds are believed to act in synergy, and the degree of toxicity may therefore depend on their ratio in the potato. To determine the influence of α-solanine: α-chaconine ratio in potatoes on toxicity, a GM potato line (SGT 9-2) with reduced α-solanine content, and the parental control line (Desirée wild-type) having a traditional α-solanine: α-chaconine ratio were (1) studied for compositional similarity by analysing for a range of potato constituents, and (2) used in a 90-day feeding trial with the Syrian Golden hamster to study differential toxicity. The animal feeding study used diets with up to 60% freeze-dried potato powder from either line. Whilst data indicated some compositional differences between the GM line and its wildtype control these did not raise concerns related to nutritional value or safety. Results of the feeding trials showed a low number of significant differences between potato lines with different α-solanine: α-chaconine ratio but none were considered to raise safety concerns with regard to human (or animal) consumption.

pBINPLUS/ARS: an improved plant transformation vector based on pBINPLUS.

Binary plant transformation vectors are widely used for introduction of transgenes into plants via Agrobacterium tumefaciens-mediated transformation. We report the construction of a binary plant vector pBINPLUS/ARS based on the pBINPLUS vector. Improvements introduced into pBINPLUS/ARS include the use of nonproprietary (ubiquitin-3 gene of Solanum tuberosum) promoter and terminator sequences for transcription of the NptII selectable marker and introduction of rare 8-bp restriction enzyme sites flanking both the NptII coding sequence (PmeI) and the entire selectable marker gene (FseI). This vector offers all of the advantages of its predecessor pBINPLUS and its helper plasmid pUCAP, which use the proprietary nopaline synthase promoter and terminator, while allowing for facile modification of selectable marker sequences in complex binary vector constructs. pBINPLUS/ARS has been used to introduce transgenes into potato and other crop species and is available to all researchers in academic, government, and industrial laboratories for proof-of-principle and commercial applications.

Potato glycosterol rhamnosyltransferase, the terminal step in triose side-chain biosynthesis.

Steroidal glycoalkaloids (SGAs) are potentially harmful specialty metabolites found in Solanaceous plants. Two tri-glycosylated alkaloids, alpha-chaconine and alpha-solanine accumulate in potato tubers. Expressed sequence tags (ESTs) were identified in the available database by searching for protein homology to the Sgt1 (SOLtu:Sgt1) steriodalalkaloid galactosyltransferase. The EST sequence data was used to isolate Sgt3 cDNA sequences by polymerase chain reaction (PCR) from a wounded potato tuber cDNA library. The resulting 1515bp open reading frame of Sgt3, encodes a predicted SGT3 amino acid sequence that is 18 residues longer than, 45% identical to, and 58% homologous to the SGT1 protein. The amino-terminal region of the Sgt3 cDNA was used to create an antisense transgene under control of the granule bound starch synthase, GBSS6, promoter and the ubiquitin, Ubi3, polyadenylation signal. Analysis of SGA metabolites in selected transgenic tubers revealed a dramatic decrease in the accumulation of alpha-chaconine and alpha-solanine. This decrease was compensated by an increase in beta-solanine and beta-chaconine with minor accumulation of alpha-SGAs. These results allowed the identification of the function for SGT3 as the beta-solanine/beta-chaconine rhamnosyl transferase, the terminal step in formation of the potato glycoalkaloid triose side chains.

The primary in vivo steroidal alkaloid glucosyltransferase from potato.

To provide tools for breeders to control the steroidal glycoalkaloid (SGA) pathway in potato, we have investigated the steroidal alkaloid glycosyltransferase (Sgt) gene family. The committed step in the SGA pathway is the glycosylation of solanidine by either UDP-glucose or UDP-galactose leading to alpha-chaconine or alpha-solanine, respectively. The Sgt2 gene was identified by deduced protein sequence homology to the previously identified Sgt1 gene. SGT1 has glucosyltransferase activity in vitro, but in vivo serves as the UDP-galactose:solanidine galactosyltransferase. Two alleles of the Sgt2 gene were isolated and its function was established with antisense transgenic lines and in vitro assays of recombinant protein. In tubers of transgenic potato (Solanum tuberosum) cvs. Lenape and Desirée expressing an antisense Sgt2 gene construct, accumulation of alpha-solanine was increased and alpha-chaconine was reduced. Studies with recombinant SGT2 protein purified from yeast show that SGT2 glycosylation activity is highly specific for UDP-glucose as a sugar donor. This data establishes the function of the gene product (SGT2), as the primary UDP-glucose:solanidine glucosyltransferase in vivo.