Host avian species and environmental conditions influence the microbial ecology of brood parasitic brown-headed cowbird nestlings: What rules the roost?

The role of species interactions, as well as genetic and environmental factors, all likely contribute to the composition and structure of the gut microbiome; however, disentangling these independent factors under field conditions represents a challenge for a functional understanding of gut microbial ecology. Avian brood parasites provide unique opportunities to investigate these questions, as brood parasitism results in parasite and host nestlings being raised in the same nest, by the same parents. Here we utilized obligate brood parasite brown-headed cowbird nestlings (BHCO; Molothrus ater) raised by several different host passerine species to better understand, via 16S rRNA sequencing, the microbial ecology of brood parasitism. First, we compared faecal microbial communities of prothonotary warbler nestlings (PROW; Protonotaria citrea) that were either parasitized or non-parasitized by BHCO and communities among BHCO nestlings from PROW nests. We found that parasitism by BHCO significantly altered both the community membership and community structure of the PROW nestling microbiota, perhaps due to the stressful nest environment generated by brood parasitism. In a second dataset, we compared faecal microbiotas from BHCO nestlings raised by six different host passerine species. Here, we found that the microbiota of BHCO nestlings was significantly influenced by the parental host species and the presence of an inter-specific nestmate. Thus, early rearing environment is important in determining the microbiota of brood parasite nestlings and their companion nestlings. Future work may aim to understand the functional effects of this microbiota variability on nestling performance and fitness.

Is "memory-for-when" universal? Group and individual variability in temporal position memory for words, faces, and classrooms.

Many contemporary theories of memory assume that everyone automatically stores temporal contextual information about all types of encountered information, yet most studies on this topic have used words and ignored individual differences. Five experiments accumulated evidence that explicit storage of temporal context information does not appear to occur automatically for all people and types of memoranda. We collected judgments of temporal position (memory-for-when) for words (Experiments 1 & 3), faces (Experiments 2A, 3, 4, and 5), and classrooms (Experiments 2B & 3). At the group level, for each of these memoranda memory-for-when was sensitive to the original input position and showed a temporal primacy effect reflecting better memory for position for items near the beginning of the list, indicating some automatic storage of temporal context information. However, memory-for-when was significantly better for words than classrooms, with faces in the middle. Moreover, individuals varied dramatically in their ability to indicate memory-for-when, especially for classrooms where many people performed at or near chance. Taken together, the data suggest that explicit memory-for-when may be dissociable from the more implicit use of temporal contextual information that is theorised to occur during free recall.

Phenotypic signatures of urbanization? Resident, but not migratory, songbird eye size varies with urban-associated light pollution levels.

Urbanization now exposes large portions of the earth to sources of anthropogenic disturbance, driving rapid environmental change and producing novel environments. Changes in selective pressures as a result of urbanization are often associated with phenotypic divergence; however, the generality of phenotypic change remains unclear. In this study, we examined whether morphological phenotypes in two residential species (Carolina Wren [Thryothorus ludovicianus] and Northern Cardinal [Cardinalis cardinalis]) and two migratory species (Painted Bunting [Passerina ciris], and White-eyed Vireo [Vireo griseus]), differed between urban core and edge habitats in San Antonio, Texas, USA. More specifically, we examined whether urbanization, associated sensory pollution (light and noise) and brightness (open, bright areas cause by anthropogenic land use) influenced measures of avian body (mass and frame size) and lateral eye size. We found no differences in body size between urban core and edge habitats for all species except the Painted Bunting, in which core-urban individuals were smaller. Rather than a direct effect of urbanization, this was due to differences in age structure between habitats, with urban-core areas consisting of higher proportions of younger buntings which are, on average, smaller than older birds. Residential birds inhabiting urban-core areas had smaller eyes compared to their urban-edge counterparts, resulting from a negative association between eye size and light pollution and brightness across study sites; notably, we found no such association in the two migratory species. Our findings demonstrate how urbanization may indirectly influence phenotypes by altering population demographics and highlight the importance of accounting for age when assessing factors driving phenotypic change. We also provide some of the first evidence that birds may adapt to urban environments through changes in their eye morphology, demonstrating the need for future research into relationships among eye size, ambient light microenvironment use, and disassembly of avian communities as a result of urbanization.

Parental benefits and offspring costs reflect parent-offspring conflict over the age of fledging among songbirds.

Parent-offspring conflict has explained a variety of ecological phenomena across animal taxa, but its role in mediating when songbirds fledge remains controversial. Specifically, ecologists have long debated the influence of songbird parents on the age of fledging: Do parents manipulate offspring into fledging to optimize their own fitness or do offspring choose when to leave? To provide greater insight into parent-offspring conflict over fledging age in songbirds, we compared nesting and postfledging survival rates across 18 species from eight studies in the continental United States. For 12 species (67%), we found that fledging transitions offspring from comparatively safe nesting environments to more dangerous postfledging ones, resulting in a postfledging bottleneck. This raises an important question: as past research shows that offspring would benefit-improve postfledging survival-by staying in the nest longer: Why then do they fledge so early? Our findings suggest that parents manipulate offspring into fledging early for their own benefit, but at the cost of survival for each individual offspring, reflecting parent-offspring conflict. Early fledging incurred, on average, a 13.6% postfledging survival cost for each individual offspring, but parents benefitted through a 14.0% increase in the likelihood of raising at least one offspring to independence. These parental benefits were uneven across species-driven by an interaction between nest mortality risk and brood size-and predicted the age of fledging among species. Collectively, our results suggest that parent-offspring conflict and associated parental benefits explain variation in fledging age among songbird species and why postfledging bottlenecks occur.

Surface Passivation by Quantum Exclusion: On the Quantum Efficiency and Stability of Delta-Doped CCDs and CMOS Image Sensors in Space.

Radiation-induced damage and instabilities in back-illuminated silicon detectors have proved to be challenging in multiple NASA and commercial applications. In this paper, we develop a model of detector quantum efficiency (QE) as a function of Si-SiO2 interface and oxide trap densities to analyze the performance of silicon detectors and explore the requirements for stable, radiation-hardened surface passivation. By analyzing QE data acquired before, during, and after, exposure to damaging UV radiation, we explore the physical and chemical mechanisms underlying UV-induced surface damage, variable surface charge, QE, and stability in ion-implanted and delta-doped detectors. Delta-doped CCD and CMOS image sensors are shown to be uniquely hardened against surface damage caused by ionizing radiation, enabling the stability and photometric accuracy required by NASA for exoplanet science and time domain astronomy.

Developing a rapid and highly efficient cowpea regeneration, transformation and genome editing system using embryonic axis explants.

Cowpea (Vigna unguiculata (L.) Walp.) is one of the most important legume crops planted worldwide, but despite decades of effort, cowpea transformation is still challenging due to inefficient Agrobacterium-mediated transfer DNA delivery, transgenic selection and in vitro shoot regeneration. Here, we report a highly efficient transformation system using embryonic axis explants isolated from imbibed mature seeds. We found that removal of the shoot apical meristem from the explants stimulated direct multiple shoot organogenesis from the cotyledonary node tissue. The application of a previously reported ternary transformation vector system provided efficient Agrobacterium-mediated gene delivery, while the utilization of spcN as selectable marker enabled more robust transgenic selection, plant recovery and transgenic plant generation without escapes and chimera formation. Transgenic cowpea plantlets developed exclusively from the cotyledonary nodes at frequencies of 4% to 37% across a wide range of cowpea genotypes. CRISPR/Cas-mediated gene editing was successfully demonstrated. The transformation principles established here could also be applied to other legumes to increase transformation efficiencies.

Development of an efficient marker-free soybean transformation method using the novel bacterium Ochrobactrum haywardense H1.

We have discovered a novel bacterium, Ochrobactrum haywardense H1 (Oh H1), which is capable of efficient plant transformation. Ochrobactrum is a new host for Agrobacterium-derived vir and T-DNA-mediated transformation. Oh H1 is a unique, non-phytopathogenic species, categorized as a BSL-1 organism. We engineered Oh H1 with repurposed Agrobacterium virulence machinery and demonstrated Oh H1 can transform numerous dicot species and at least one monocot, sorghum. We generated a cysteine auxotrophic Oh H1-8 strain containing a binary vector system. Oh H1-8 produced transgenic soybean plants with an efficiency 1.6 times that of Agrobacterium strain AGL1 and 2.9 times that of LBA4404Thy-. Oh H1-8 successfully transformed several elite Corteva soybean varieties with T0 transformation frequency up to 35%. In addition to higher transformation efficiencies, Oh H1-8 generated high-quality, transgenic events with single-copy, plasmid backbone-free insertion at frequencies higher than AGL1. The SpcN selectable marker gene is excised using a heat shock-inducible excision system resulting in marker-free transgenic events. Approximately, 24.5% of the regenerated plants contained only a single copy of the transgene and contained no vector backbone. There were no statistically significant differences in yield comparing T3 null-segregant lines to wild-type controls. We have demonstrated that Oh H1-8, combined with spectinomycin selection, is an efficient, rapid, marker-free and yield-neutral transformation system for elite soybean.

Discrete-space continuous-time models of marine mammal exposure to Navy sonar.

Assessing the patterns of wildlife attendance to specific areas is relevant across many fundamental and applied ecological studies, particularly when animals are at risk of being exposed to stressors within or outside the boundaries of those areas. Marine mammals are increasingly being exposed to human activities that may cause behavioral and physiological changes, including military exercises using active sonars. Assessment of the population-level consequences of anthropogenic disturbance requires robust and efficient tools to quantify the levels of aggregate exposure for individuals in a population over biologically relevant time frames. We propose a discrete-space, continuous-time approach to estimate individual transition rates across the boundaries of an area of interest, informed by telemetry data collected with uncertainty. The approach allows inferring the effect of stressors on transition rates, the progressive return to baseline movement patterns, and any difference among individuals. We apply the modeling framework to telemetry data from Blainville's beaked whale (Mesoplodon densirostris) tagged in the Bahamas at the Atlantic Undersea Test and Evaluation Center (AUTEC), an area used by the U.S. Navy for fleet readiness training. We show that transition rates changed as a result of exposure to sonar exercises in the area, reflecting an avoidance response. Our approach supports the assessment of the aggregate exposure of individuals to sonar and the resulting population-level consequences. The approach has potential applications across many applied and fundamental problems where telemetry data are used to characterize animal occurrence within specific areas.

Host community-wide patterns of post-fledging behavior and survival of obligate brood parasitic brown-headed cowbirds.

The antagonistic arms races between obligate brood parasites and their hosts provide critical insights into coevolutionary processes and constraints on the evolution of life history strategies. In avian brood parasites-a model system for examining host-parasite dynamics-research has primarily focused on the egg and nestling stage, while far less is known about the behavior and ecology of fledgling and juvenile brood parasites. To provide greater insights into the post-fledging period of generalist brood parasites, we used handheld and automated telemetry systems to examine the behavior and survival of fledgling brown-headed cowbirds (Molothrus ater). Our host community-wide analysis (data on cowbirds fledged from different host species were pooled) shows that fledgling cowbirds' follow patterns of movement and survival found across the post-fledging literature on parental passerine species. Cowbird fledgling survival was lowest during the first 3 days post-fledging, whereas daily rates of survival neared 100% after about 16 days post-fledging. Cowbird daytime post-fledging activity rates, perch heights, and distance from the natal area all increased with fledging age and young generally gained independence from host parents at 3-4 weeks post-fledging, with approximately the same latency as has been observed in studies on fledglings of cowbird host species. Our research demonstrates how automated telemetry systems can overcome past methodological limitations in post-fledging research and provides an important foundation for future studies examining adaptations that cowbirds and other brood parasites use to exploit hosts during the post-fledging period.

Characterization of the Percival detector with soft X-rays.

In this paper the back-side-illuminated Percival 2-Megapixel (P2M) detector is presented, along with its characterization by means of optical and X-ray photons. For the first time, the response of the system to soft X-rays (250 eV to 1 keV) is presented. The main performance parameters of the first detector are measured, assessing the capabilities in terms of noise, dynamic range and single-photon discrimination capability. Present limitations and coming improvements are discussed.

Pre- to post-fledging carryover effects and the adaptive significance of variation in wing development for juvenile songbirds.

Evolutionary ecologists have long been interested in the adaptive significance of morphological traits across stages of animal life. In some cases, traits that are not adaptive in one life stage may be adaptive in a subsequent stage. As such, morphological traits may generate important carryover effects, whereby conditions experienced during one life-history stage influence fitness during subsequent stages. Carryover effects are particularly relevant in young animals, as early life stages are thought to be critical with respect to animal life-history evolution and population dynamics. In songbirds, pre- to post-fledging carryover effects operating within species may be critical for survival and shape life histories among species, but remain poorly understood. Among potential songbird traits, wing development and its associated flight ability may be the most important for post-fledging survival. Thus, to assess the adaptive significance of wing development for juvenile songbirds under Arnold's (Integrative and Comparative Biology, 23, 1983, 347) classic performance-morphology-fitness paradigm, we tested for pre- to post-fledging carryover effects among 20 coexisting species (nine focal species) of an avian community in east-central Illinois, USA. We found evidence for pre- to post-fledging carryover effects of wing development in all species, by which individuals with less developed wings exhibited poorer flight ability and experienced higher rates of mortality after fledging. Furthermore, our findings suggest that carryover effects operating at the species level ultimately help shape patterns of life-history variation among species. Specifically, we found that species with higher rates of nest predation had shorter nestling periods, fledged young with less developed wings and exhibited higher rates of post-fledging mortality. Our results highlight the adaptive significance of wing development as a key factor generating pre- to post-fledging carryover effects among songbirds, and demonstrate how morphological traits, locomotor performance, and age-specific survival may trade-off and interact across juvenile life stages to shape animal life histories.

High efficiency Agrobacterium-mediated site-specific gene integration in maize utilizing the FLP-FRT recombination system.

An efficient Agrobacterium-mediated site-specific integration (SSI) technology using the flipase/flipase recognition target (FLP/FRT) system in elite maize inbred lines is described. The system allows precise integration of a single copy of a donor DNA flanked by heterologous FRT sites into a predefined recombinant target line (RTL) containing the corresponding heterologous FRT sites. A promoter-trap system consisting of a pre-integrated promoter followed by an FRT site enables efficient selection of events. The efficiency of this system is dependent on several factors including Agrobacterium tumefaciens strain, expression of morphogenic genes Babyboom (Bbm) and Wuschel2 (Wus2) and choice of heterologous FRT pairs. Of the Agrobacterium strains tested, strain AGL1 resulted in higher transformation frequency than strain LBA4404 THY- (0.27% vs. 0.05%; per cent of infected embryos producing events). The addition of morphogenic genes increased transformation frequency (2.65% in AGL1; 0.65% in LBA4404 THY-). Following further optimization, including the choice of FRT pairs, a method was developed that achieved 19%-22.5% transformation frequency. Importantly, >50% of T0 transformants contain the desired full-length site-specific insertion. The frequencies reported here establish a new benchmark for generating targeted quality events compatible with commercial product development.

Morphogenic Regulators Baby boom and Wuschel Improve Monocot Transformation.

While transformation of the major monocot crops is currently possible, the process typically remains confined to one or two genotypes per species, often with poor agronomics, and efficiencies that place these methods beyond the reach of most academic laboratories. Here, we report a transformation approach involving overexpression of the maize (Zea mays) Baby boom (Bbm) and maize Wuschel2 (Wus2) genes, which produced high transformation frequencies in numerous previously nontransformable maize inbred lines. For example, the Pioneer inbred PHH5G is recalcitrant to biolistic and Agrobacterium tumefaciens transformation. However, when Bbm and Wus2 were expressed, transgenic calli were recovered from over 40% of the starting explants, with most producing healthy, fertile plants. Another limitation for many monocots is the intensive labor and greenhouse space required to supply immature embryos for transformation. This problem could be alleviated using alternative target tissues that could be supplied consistently with automated preparation. As a major step toward this objective, we transformed Bbm and Wus2 directly into either embryo slices from mature seed or leaf segments from seedlings in a variety of Pioneer inbred lines, routinely recovering healthy, fertile T0 plants. Finally, we demonstrated that the maize Bbm and Wus2 genes stimulate transformation in sorghum (Sorghum bicolor) immature embryos, sugarcane (Saccharum officinarum) callus, and indica rice (Oryza sativa ssp indica) callus.

Advancing Agrobacterium-Based Crop Transformation and Genome Modification Technology for Agricultural Biotechnology.

The last decade has seen significant strides in Agrobacterium-mediated plant transformation technology. This has not only expanded the number of crop species that can be transformed by Agrobacterium, but has also made it possible to routinely transform several recalcitrant crop species including cereals (e.g., maize, sorghum, and wheat). However, the technology is limited by the random nature of DNA insertions, genotype dependency, low frequency of quality events, and variation in gene expression arising from genomic insertion sites. A majority of these deficiencies have now been addressed by improving the frequency of quality events, developing genotype-independent transformation capability in maize, developing an Agrobacterium-based site-specific integration technology for precise gene targeting, and adopting Agrobacterium-delivered CRISPR-Cas genes for gene editing. These improved transformation technologies are discussed in detail in this chapter.

Identifying prognostic structural features in tissue sections of colon cancer patients using point pattern analysis.

Diagnosis and prognosis of cancer are informed by the architecture inherent in cancer patient tissue sections. This architecture is typically identified by pathologists, yet advances in computational image analysis facilitate quantitative assessment of this structure. In this article, we develop a spatial point process approach to describe patterns in cell distribution within tissue samples taken from colorectal cancer (CRC) patients. In particular, our approach is centered on the Palm intensity function. This leads to taking an approximate-likelihood technique in fitting point processes models. We consider two Neyman-Scott point processes and a void process, fitting these point process models to the CRC patient data. We find that the parameter estimates of these models may be used to quantify the spatial arrangement of cells. Importantly, we observe characteristic differences in the spatial arrangement of cells between patients who died from CRC and those alive at follow up.

Elevated vitamin E content improves all-trans ß-carotene accumulation and stability in biofortified sorghum.

Micronutrient deficiencies are common in locales where people must rely upon sorghum as their staple diet. Sorghum grain is seriously deficient in provitamin A (ß-carotene) and in the bioavailability of iron and zinc. Biofortification is a process to improve crops for one or more micronutrient deficiencies. We have developed sorghum with increased ß-carotene accumulation that will alleviate vitamin A deficiency among people who rely on sorghum as their dietary staple. However, subsequent ß-carotene instability during storage negatively affects the full utilization of this essential micronutrient. We determined that oxidation is the main factor causing ß-carotene degradation under ambient conditions. We further demonstrated that coexpression of homogentisate geranylgeranyl transferase (HGGT), stacked with carotenoid biosynthesis genes, can mitigate ß-carotene oxidative degradation, resulting in increased ß-carotene accumulation and stability. A kinetic study of ß-carotene degradation showed that the half-life of ß-carotene is extended from less than 4 wk to 10 wk on average with HGGT coexpression.

Increased postoperative dexamethasone and gabapentin reduces opioid consumption after total knee arthroplasty.

PURPOSE: Dexamethasone and gabapentin are used in multimodal pain management protocols to reduce postoperative pain after total knee arthroplasty. For both analgesic adjuvants, the optimal dose regimen to reduce opioid usage is still unclear. METHODS: The opioid consumption of patients undergoing primary TKA before and after a change of the analgesic adjuvant medication in our protocol (old protocol: 4 mg of dexamethasone daily for 2 days, 600 mg gabapentin daily for 1 week; new protocol: 10 mg dexamethasone daily for 2 days, 300 mg gabapentin every 8 h for 1 week) were retrospectively compared. All surgeries were performed under spinal anesthesia. Peri- and postoperative pain medication remained unchanged. RESULTS: A total of 186 patients who received TKA between 11/29/2016 and 06/09/2017 were screened. Six patients who received general anesthesia, 4 patients who underwent simultaneous bilateral TKA, and 16 patients with ongoing opioid consumption at the time of surgery were excluded, leaving 80 patients in each group. Opioid consumption within 24 h [morphine equivalents in mg: mean 50.5, standard deviation (SD) 30.0 (old) vs. 39.8, SD 24.2 (new); P = 0.0470], cumulative consumption over 48 h (97.3, SD 64.4 vs. 70.4, SD 51.2; P = 0.0040) and cumulative consumption over 72 h (108.1, SD 79.5 vs. 82.5, SD 72.6; P = 0.0080), were all significantly lower in the new protocol. CONCLUSION: Increased postoperative administration of dexamethasone and gabapentin after TKA is associated with lower opioid consumption. Within the first 48 h, up to about 25% of opioids can be spared, comparing high-dose to low-dose protocols. LEVEL OF EVIDENCE: Therapeutic Level III.

Perioperative Dietary Restriction of Carbohydrates in the Management of Blood Glucose Levels in Patients Undergoing Total Knee Replacement.

BACKGROUND: Approximately 75% of the US population over 65 years has prediabetes or diabetes. Despite current evidence for the efficacy of carbohydrate restriction in managing blood glucose, this practice has not been implemented as part of routine perioperative blood sugar management. We hypothesize that a carbohydrate reduced hospital diet (CRD) of 135 g/d may improve blood sugar levels following total knee arthroplasty (TKA) compared to a non-carbohydrate reduced hospital diet (NCRD). METHODS: We randomized non-insulin-dependent prediabetic and diabetic patients undergoing TKA to either an NCRD or a CRD. Sixty-four patients were enrolled in the study and 2 were excluded, leading to 62 patients in the final analysis. The NCRD group included 14 females (47%) and 16 males (53%), with mean age of 68.5 years (±6.3 years). The CRD group included 16 females (50%) and 16 males (50%), with mean age of 68.0 years (±8.0 years). For hemoglobin A1C, the NCRD group had mean 5.8% (±0.6%) and the CRD group had mean 5.7% (±0.8%). For body mass index, the NCRD group had mean 29.3 kg/m2 (±6.3 kg/m2) and the CRD group 32.7 kg/m2 (±5.0 kg/m2). The primary outcome measure was mean blood glucose. RESULTS: Mean blood sugar values during hospital stay were significantly lower in the CRD group with 121.5 mg/dL (±17.1 mg/dL) compared to the NCRD group 141.2 mg/dL (±31.3 mg/dL, P = .0031). CONCLUSION: Blood sugar levels after surgery can be significantly reduced with a CRD. Further research is necessary to study the effect of reduced blood sugar levels on complications and infection rates following TKA surgery. LEVEL OF EVIDENCE: I.

Optimal Timing of Glucose Measurements After Total Joint Arthroplasty.

BACKGROUND: Postoperative glucose levels after total joint arthroplasty are important to monitor as hyperglycemia has been linked to complications such as periprosthetic joint infection. The purposes of this study were to identify how postoperative glucose values vary during the perioperative period and determine the optimal time to check glucose levels to best evaluate for hyperglycemia. METHODS: A retrospective study was conducted from September 2017 to September 2018 on 314 patients who underwent knee and hip arthroplasties. Blood glucose levels were collected immediately preoperatively, immediately postoperatively, at 5 PM on the day of surgery (DOS), at 9 PM on the DOS, and in the morning of postoperative day (POD) 1. The total number of hyperglycemic patients was assessed at 3 glucose thresholds: strict ≥ 126 mg/dL, intermediate ≥ 137 mg/dL, and lenient ≥ 180 mg/dL. Descriptive statistics were performed for each glucose time period, and adjusted comparisons were made between the mean glucose values and number of hyperglycemic patients at all time points. RESULTS: Mean (±95% confidence interval) glucose values were 105.7 ± 2.1 mg/dL preoperatively, 117.3 ± 2.5 mg/dL immediately postoperatively, 138.6 ± 4.3 mg/dL at 5 PM on the DOS, 142.9 ± 4.3 mg/dL at 9 PM on the DOS, and 116.7 ± 3.1 mg/dL in the morning of POD 1. Values measured at 5 PM and 9 PM were significantly higher than those measured at all other time points in both diabetics and nondiabetics (P < .001 for all). For all 3 hyperglycemia thresholds, the highest number of hyperglycemic patients was observed at 9 PM on the DOS: strict = 205 (65.3%) patients, intermediate = 177 (56.4%) patients; and lenient = 90 (28.7%) patients. CONCLUSION: Most patients who underwent total joint arthroplasty are hyperglycemic postoperatively, and 9 PM on the night of surgery may be the most sensitive time for detecting hyperglycemia in both diabetic and nondiabetic patients, in contrast to traditional POD 1 levels.

An improved ternary vector system for Agrobacterium-mediated rapid maize transformation.

KEY MESSAGE: A simple and versatile ternary vector system that utilizes improved accessory plasmids for rapid maize transformation is described. This system facilitates high-throughput vector construction and plant transformation. The super binary plasmid pSB1 is a mainstay of maize transformation. However, the large size of the base vector makes it challenging to clone, the process of co-integration is cumbersome and inefficient, and some Agrobacterium strains are known to give rise to spontaneous mutants resistant to tetracycline. These limitations present substantial barriers to high throughput vector construction. Here we describe a smaller, simpler and versatile ternary vector system for maize transformation that utilizes improved accessory plasmids requiring no co-integration step. In addition, the newly described accessory plasmids have restored virulence genes found to be defective in pSB1, as well as added virulence genes. Testing of different configurations of the accessory plasmids in combination with T-DNA binary vector as ternary vectors nearly doubles both the raw transformation frequency and the number of transformation events of usable quality in difficult-to-transform maize inbreds. The newly described ternary vectors enabled the development of a rapid maize transformation method for elite inbreds. This vector system facilitated screening different origins of replication on the accessory plasmid and T-DNA vector, and four combinations were identified that have high (86-103%) raw transformation frequency in an elite maize inbred.