Isolation of novel citrus and plum fruit promoters and their functional characterization for fruit biotechnology.

BACKGROUND: Promoters that confer expression in fruit tissues are important tools for genetic engineering of fruit quality traits, yet few fruit-specific promoters have been identified, particularly for citrus fruit development. RESULTS: In this study, we report five citrus fruit-specific/preferential promoters for genetic engineering. Additionally, we have characterized a novel fruit-preferential promoter from plum. Genes specifically expressed in fruit tissues were selected and their isolated promoter regions were fused with the GUSPlus reporter gene for evaluation in transgenic plants. Stable transformation in Micro-Tom tomato demonstrated that the candidate promoter regions exhibit differing levels of expression and with varying degrees of fruit specificity. CONCLUSIONS: Among the five candidate citrus promoters characterized in this study, the CitSEP promoter showed a fruit-specific expression pattern, while the CitWAX and CitJuSac promoters exhibited high fruit-preferential expression with strong activity in the fruit, weak activity in floral tissues and low or undetectable activity in other tissues. The CitVO1, CitUNK and PamMybA promoters, while exhibiting strong fruit-preferential expression, also showed consistent weak but detectable activity in leaves and other vegetative tissues. Use of these fruit specific/preferential promoters for genetic engineering can help with precise expression of beneficial genes and help with accurate prediction of the activity of new genes in host fruit plants.

A versatile and robust Agrobacterium-based gene stacking system generates high-quality transgenic Arabidopsis plants.

Biotechnology provides a means for the rapid genetic improvement of plants. Although single genes have been important in engineering herbicide and pest tolerance traits in crops, future improvements of complex traits like yield and nutritional quality will likely require the introduction of multiple genes. This research reports a system (GAANTRY; Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) for the flexible, in vivo stacking of multiple genes within an Agrobacterium virulence plasmid Transfer-DNA (T-DNA). The GAANTRY system utilizes in vivo transient expression of unidirectional site-specific recombinases and an alternating selection scheme to sequentially assemble multiple genes into a single transformation construct. To demonstrate GAANTRY's capabilities, 10 cargo sequences were sequentially stacked together to produce a 28.5-kbp T-DNA, which was used to generate hundreds of transgenic events. Approximately 90% of the events identified using a dual antibiotic selection screen exhibited all of the introduced traits. A total of 68% of the tested lines carried a single copy of the selection marker transgene located near the T-DNA left border, and only 8% contained sequence from outside the T-DNA. The GAANTRY system can be modified to easily accommodate any method of DNA assembly and generate high-quality transgenic plants, making it a powerful, yet simple to use tool for plant genetic engineering.

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.

Small serine recombination systems ParA-MRS and CinH-RS2 perform precise excision of plastid DNA.

Selectable marker genes (SMGs) are necessary for selection of transgenic plants. However, once stable transformants have been identified, the marker gene is no longer needed. In this study, we demonstrate the use of the small serine recombination systems, ParA-MRS and CinH-RS2, to precisely excise a marker gene from the plastid genome of tobacco. Transplastomic plants transformed with the pTCH-MRS and pTCH-RS2 vectors, containing the visual reporter gene DsRed flanked by directly oriented MRS and RS2 recognition sites, respectively, were crossed with nuclear-genome transformed tobacco plants expressing plastid-targeted ParA and CinH recombinases, respectively. One hundred per cent of both types of F1 hybrids exhibited excision of the DsRed marker gene. PCR and Southern blot analyses of DNA from F2 plants showed that approximately 30% (CinH-RS2) or 40% (ParA-MRS) had lost the recombinase genes by segregation. The postexcision transformed plastid genomes were stable and the excision events heritable. The ParA-MRS and CinH-RS2 recombination systems will be useful tools for site-specific manipulation of the plastid genome and for generating marker-free plants, an essential step for reuse of SMG and for addressing concerns about the presence of antibiotic resistance genes in transgenic plants.

Precise excision of plastid DNA by the large serine recombinase Bxb1.

Marker genes are essential for the selection and identification of rarely occurring transformation events generated in biotechnology. This includes plastid transformation, which requires that multiple copies of the modified chloroplast genome be present to obtain genetically stable transplastomic plants. However, the marker gene becomes dispensable when homoplastomic plants are obtained. Here, we demonstrate the precise excision of attP- and attB-flanked DNA from the plastid genome mediated by the large serine recombinase Bxb1. We transformed the tobacco plastid genome with the pTCH-PB vector containing a stuffer fragment of DNA flanked by directly oriented nonhomologous attP and attB recombinase recognition sites. In the absence of the Bxb1 recombinase, the transformed plastid genomes were stable and heritable. Nuclear-transformed transgenic tobacco plants expressing a plastid-targeted Bxb1 recombinase were crossed with transplastomic pTCH-PB plants, and the T1 hybrids exhibited efficient excision of the target sequence. The Bxb1-att system should prove to be a useful tool for site-specifically manipulating the plastid genome and generating marker-free transplastomic plants.

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.

Complete Genome of Agrobacterium fabrum Strain 1D1416.

This work reports the draft genome of Agrobacterium fabrum strain 1D1416. The assembled genome is composed of a 2,837,379-bp circular chromosome, a 2,043,296-bp linear chromosome, a 519,735-bp AT1 plasmid, a 188,396-bp AT2 plasmid, and a 196,706-bp Ti virulence plasmid. The nondisarmed strain produces gall-like structures in citrus tissue.

The Bxb1 recombination system demonstrates heritable transmission of site-specific excision in Arabidopsis.

BACKGROUND: The mycobacteriophage large serine recombinase Bxb1 catalyzes site-specific recombination between its corresponding attP and attB recognition sites. Previously, we and others have shown that Bxb1 has catalytic activity in various eukaryotic species including Nicotiana tabacum, Schizosaccharomyces pombe, insects and mammalian cells. RESULTS: In this work, the Bxb1 recombinase gene was transformed and constitutively expressed in Arabidopsis thaliana plants harboring a chromosomally integrated attP and attB-flanked target sequence. The Bxb1 recombinase successfully excised the target sequence in a conservative manner and the resulting recombination event was heritably transmitted to subsequent generations in the absence of the recombinase transgene. In addition, we also show that Bxb1 recombinase expressing plants can be manually crossed with att-flanked target transgenic plants to generate excised progeny. CONCLUSION: The Bxb1 large serine recombinase performs site-specific recombination in Arabidopsis thaliana germinal tissue, producing stable lines free of unwanted DNA. The precise site-specific deletion produced by Bxb1 in planta demonstrates that this enzyme can be a useful tool for the genetic engineering of plants without selectable marker transgenes or other undesirable exogenous sequences.

The effects of edema and self-adherent wrap on the work of flexion in a cadaveric hand.

PURPOSE: Early motion protocols after flexor tendon repair often require hand therapy in edematous digits. Self-adherent wraps are used in the postoperative period to reduce edema. The purpose of this study was to determine whether the presence of a self-adherent wrap affected the work of flexion during early motion protocols. METHODS: In an unpreserved cadaveric hand, the flexor digitorum profundus and flexor pollicis longus tendons were identified and attached to a tensile testing machine to measure work of flexion (WoF). We simulated subcutaneous edema by injecting normal saline into the digits. Moderate and severe edema was simulated by 10% and 20% increases in circumference of the digits, respectively. We evaluated 2 commonly used products: 2.5-cm Coban self-adherent wrap (3M, St. Paul, MN) and 2.5-cm Co-Wrap cohesive bandage (Hartmann, Rock Hill, SC). Statistical analyses include analysis of variance, 95% confidence intervals for average responses, and graphical display of both data and model predictions. RESULTS: In digits without edema or wraps, WoF ranged from 0.0114 J (small finger) to 0.0710 J (thumb). Without wraps, simulated moderate and severe edema was predicted to increase WoF by an average of 23% and 71%, respectively. Application of self-adherent wrap increased WoF values significantly in all digits. In the majority of conditions tested, application of self-adherent wrap increased WoF more significantly than moderate edema did. The effects of edema and self-adherent wrap were additive, producing WoF values 4 times the baseline values. CONCLUSIONS: Edema and self-adherent wrap increased WoF in this model. Therapists and surgeons should be aware of increased stress placed on tendons when early motion protocols are initiated in the presence of edema and self-adherent wrap. CLINICAL RELEVANCE: We recommend removal of self-adherent wrap before starting a therapy session.

Tissue-specific expression of Ruby in Mexican lime (C. aurantifolia) confers anthocyanin accumulation in fruit.

Tissue specific promoters are important tools for the precise genetic engineering of crop plants. Four fruit-preferential promoters were examined for their ability to confer a novel fruit trait in transgenic Mexican lime (Citrus aurantifolia). The Ruby transcription factor activates fruit anthocyanin accumulation within Moro blood orange and has been shown to function in activating anthocyanin accumulation in heterologous plant species. Although the CitVO1, CitUNK, SlE8, and PamMybA promoters were previously shown to confer strong fruit-preferential expression in transgenic tomato, they exhibited no detectable expression in transgenic Mexican lime trees. In contrast, the CitWax promoter exhibited high fruit-preferential expression of Ruby, conferring strong anthocyanin accumulation within the fruit juice sac tissue and moderate activity in floral/reproductive tissues. In some of the transgenic trees with high levels of flower and fruit anthocyanin accumulation, juvenile leaves also exhibited purple coloration, but the color disappeared as the leaves matured. We show that the CitWax promoter enables the expression of Ruby to produce anthocyanin colored fruit desired by consumers. The production of this antioxidant metabolite increases the fruits nutritional value and may provide added health benefits.

Transgene excision in pollen using a codon optimized serine resolvase CinH-RS2 site-specific recombination system.

Transgene escape, a major environmental and regulatory concern in transgenic crop cultivation, could be alleviated by removing transgenes from pollen, the most frequent vector for transgene flow. A transgene excision vector containing a codon optimized serine resolvase CinH recombinase (CinH) and its recognition sites RS2 were constructed and transformed into tobacco (Nicotiana tabacum cv. Xanthi). CinH recombinase recognized 119 bp of nucleic acid sequences, RS2, in pollen and excised the transgene flanked by the RS2 sites. In this system, the pollen-specific LAT52 promoter from tomato was employed to control the expression of CinH recombinase. Loss of expression of a green fluorescent protein (GFP) gene under the control of the LAT59 promoter from tomato was used as an indicator of transgene excision. Efficiency of transgene excision from pollen was determined by flow cytometry (FCM)-based pollen screening. While a transgenic event in the absence of CinH recombinase contained about 70% of GFP-synthesizing pollen, three single-copy transgene events contained less than 1% of GFP-synthesizing pollen based on 30,000 pollen grains analyzed per event. This suggests that CinH-RS2 recombination system could be effectively utilized for transgene biocontainment.

Recombinase technology: applications and possibilities.

The use of recombinases for genomic engineering is no longer a new technology. In fact, this technology has entered its third decade since the initial discovery that recombinases function in heterologous systems (Sauer in Mol Cell Biol 7(6):2087-2096, 1987). The random insertion of a transgene into a plant genome by traditional methods generates unpredictable expression patterns. This feature of transgenesis makes screening for functional lines with predictable expression labor intensive and time consuming. Furthermore, an antibiotic resistance gene is often left in the final product and the potential escape of such resistance markers into the environment and their potential consumption raises consumer concern. The use of site-specific recombination technology in plant genome manipulation has been demonstrated to effectively resolve complex transgene insertions to single copy, remove unwanted DNA, and precisely insert DNA into known genomic target sites. Recombinases have also been demonstrated capable of site-specific recombination within non-nuclear targets, such as the plastid genome of tobacco. Here, we review multiple uses of site-specific recombination and their application toward plant genomic engineering. We also provide alternative strategies for the combined use of multiple site-specific recombinase systems for genome engineering to precisely insert transgenes into a pre-determined locus, and removal of unwanted selectable marker genes.

Ideal anthropomorphic values of the female breast: correlation of pluralistic aesthetic evaluations with objective measurements.

BACKGROUND: In determining ideal anthropomorphic values of the female breast, previous studies relied on the aesthetic judgments of 1 plastic surgeon alone or conveyed no aesthetic judgment. METHODS: Anthropomorphic measurements were recorded from the breasts of 109 female volunteers. Images of their breasts were arranged into a computerized survey, and plastic surgeons, cosmetic breast surgery patients, and reconstructive breast surgery patients were interviewed for aesthetic feedback. RESULTS: In all, 252 plastic surgeons, 15 cosmetic patients, and 25 reconstructive patients submitted 4446 evaluations. The ideal sternal notch to nipple distance was 21 to 21.5 cm, the ideal nipple to base distance was 6 cm, and the ideal base to inframammary fold distance was 2 cm. These and other ideal anthropomorphic measurements were calculated and compared with previously published values. Among cosmetic patients, insufficient cleavage had the most negative effect on aesthetic value, and among reconstructive patients, severe asymmetry had the most negative effect. CONCLUSIONS: Ideal anthropomorphic values were similar among plastic surgeons and patients, and these values will be useful in achieving quantitative breast surgery. In cosmetic patients, operative planning should focus more on superomedial fullness of the breast to create cleavage, and in reconstructive patients, postoperative asymmetry should be minimized.

Gene Assembly in Agrobacterium via Nucleic Acid Transfer Using Recombinase Technology (GAANTRY).

Plant biotechnology provides a means for the rapid genetic improvement of crops including the enhancement of complex traits like yield and nutritional quality through the introduction and coordinated expression of multiple genes. 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) region. The system provides a simple and efficient method for assembling and stably maintaining large stacked constructs within the GAANTRY ArPORT1 Agrobacterium rhizogenes strain. The assembly process utilizes unidirectional site-specific recombinases in vivo and an alternating bacterial selection scheme to sequentially assemble multiple genes into a single transformation construct. A detailed description of the procedures used for bacterial transformation, selection, counter selection, and genomic PCR validation with the GAANTRY system are presented. The methods described facilitate the efficient assembly and validation of large GAANTRY T-DNA constructs. This powerful, yet simple to use, technology will be a convenient tool for transgene stacking and plant genetic engineering of rice and other crop plants.

Efficient Gene Stacking in Rice Using the GAANTRY System.

Genetic engineering of rice provides a means for improving rice grain quality and yield, and the introduction and expression of multiple genes can produce new traits that would otherwise be difficult to obtain through conventional breeding. GAANTRY (Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) was previously shown to be a precise and robust system to stably stack ten genes (28 kilobases (kb)) within an Agrobacterium virulence plasmid Transfer-DNA (T-DNA) and obtain high-quality Arabidopsis and potato transgenic events. To determine whether the GAANTRY system can be used to engineer a monocotyledonous crop, two new T-DNA constructs, carrying five (16.9 kb) or eleven (37.4 kb) cargo sequences were assembled and transformed into rice. Characterization of 53 independent transgenic events demonstrated that more than 50% of the plants carried all of the desired cargo sequences and exhibited the introduced traits. Additionally, more than 18% of the lines were high-quality events containing a single copy of the introduced transgenes and were free of sequences from outside of the T-DNA. Therefore, GAANTRY provides a simple, precise and versatile tool for transgene stacking in rice and potentially other cereal grain crops.

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.

PhiC31 recombination system demonstrates heritable germinal transmission of site-specific excision from the Arabidopsis genome.

BACKGROUND: The large serine recombinase phiC31 from broad host range Streptomyces temperate phage, catalyzes the site-specific recombination of two recognition sites that differ in sequence, typically known as attachment sites attB and attP. Previously, we characterized the phiC31 catalytic activity and modes of action in the fission yeast Schizosaccharomyces pombe. RESULTS: In this work, the phiC31 recombinase gene was placed under the control of the Arabidopsis OXS3 promoter and introduced into Arabidopsis harboring a chromosomally integrated attB and attP-flanked target sequence. The phiC31 recombinase excised the attB and attP-flanked DNA, and the excision event was detected in subsequent generations in the absence of the phiC31 gene, indicating germinal transmission was possible. We further verified that the genomic excision was conservative and that introduction of a functional recombinase can be achieved through secondary transformation as well as manual crossing. CONCLUSION: The phiC31 system performs site-specific recombination in germinal tissue, a prerequisite for generating stable lines with unwanted DNA removed. The precise site-specific deletion by phiC31 in planta demonstrates that the recombinase can be used to remove selectable markers or other introduced transgenes that are no longer desired and therefore can be a useful tool for genome engineering in plants.

Phenotypic Examination of Camelina sativa (L.) Crantz Accessions from the USDA-ARS National Genetics Resource Program.

Camelina sativa (L.) Crntz. is a hardy self-pollinated oilseed plant that belongs to the Brassicaceae family; widely grown throughout the northern hemisphere until the 1940s for production of vegetable oil but was later displaced by higher-yielding rapeseed and sunflower crops. However, interest in camelina as an alternative oil source has been renewed due to its high oil content that is rich in polyunsaturated fatty acids, antioxidants as well as its ability to grow on marginal lands with minimal requirements. For this reason, our group decided to screen the existing (2011) National Genetic Resources Program (NGRP) center collection of camelina for its genetic diversity and provide a phenotypic evaluation of the cultivars available. Properties evaluated include seed and oil traits, developmental and mature morphologies, as well as chromosome content. Selectable marker genes were also evaluated for potential use in biotech manipulation. Data is provided in a raw uncompiled format to allow other researchers to analyze the unbiased information for their own studies. Our evaluation has determined that the NGRP collection has a wide range of genetic potential for both breeding and biotechnological manipulation purposes. Accessions were identified within the NGRP collection that appear to have desirable seed harvest weight (5.06 g/plant) and oil content (44.1%). Other cultivars were identified as having fatty acid characteristics that may be suitable for meal and/or food use, such as low (<2%) erucic acid content, which is often considered for healthy consumption and ranged from a high of 4.79% to a low of 1.83%. Descriptive statistics are provided for a breadth of traits from 41 accessions, as well as raw data, and key seed traits are further explored. Data presented is available for public use.

The LP2 leucine-rich repeat receptor kinase gene promoter directs organ-specific, light-responsive expression in transgenic rice.

Biotechnologists seeking to limit gene expression to nonseed tissues of genetically engineered cereal crops have only a few choices of well characterized organ-specific promoters. We have isolated and characterized the promoter of the rice Leaf Panicle 2 gene (LP2, Os02g40240). The LP2 gene encodes a leucine-rich repeat-receptor kinase-like protein that is strongly expressed in leaves and other photosynthetic tissues. Transgenic rice plants containing an LP2 promoter-GUS::GFP bifunctional reporter gene displayed an organ-specific pattern of expression. This expression corresponded to transcript levels observed on RNA blots of various rice organs and microarray gene expression data. The strongest beta-glucuronidase activity was observed in histochemically stained mesophyll cells, but other green tissues and leaf cell types including epidermal cells also exhibited expression. Low or undetectable levels of LP2 transcript and LP2-mediated reporter gene expression were observed in roots, mature seeds, and reproductive tissues. The LP2 promoter is highly responsive to light and only weak expression was detected in etiolated rice seedlings. The specificity and strength of the LP2 promoter suggests that this promoter will be a useful control element for green tissue-specific expression in rice and potentially other plants. Organ-specific promoters like LP2 will enable precise, localized expression of transgenes in biotechnology-derived crops and limit the potential of unintended impacts on plant physiology and the environment.

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.