Integrity Of Backbone Research Articles

Integrity of xylan backbone affects plant responses to drought.

Drought is a major factor affecting crops, thus efforts are needed to increase plant resilience to this abiotic stress. The overlapping signaling pathways between drought and cell wall integrity maintenance responses create a possibility of increasing drought resistance by modifying cell walls. Here, using herbaceous and woody plant model species, Arabidopsis and hybrid aspen, respectively, we investigated how the integrity of xylan in secondary walls affects the responses of plants to drought stress. Plants, in which secondary wall xylan integrity was reduced by expressing fungal GH10 and GH11 xylanases or by affecting genes involved in xylan backbone biosynthesis, were subjected to controlled drought while their physiological responses were continuously monitored by RGB, fluorescence, and/or hyperspectral cameras. For Arabidopsis, this was supplemented with survival test after complete water withdrawal and analyses of stomatal function and stem conductivity. All Arabidopsis xylan-impaired lines showed better survival upon complete watering withdrawal, increased stomatal density and delayed growth inhibition by moderate drought, indicating increased resilience to moderate drought associated with modified xylan integrity. Subtle differences were recorded between xylan biosynthesis mutants (irx9, irx10 and irx14) and xylanase-expressing lines. irx14 was the most drought resistant genotype, and the only genotype with increased lignin content and unaltered xylem conductivity despite its irx phenotype. Rosette growth was more affected by drought in GH11- than in GH10-expressing plants. In aspen, mild downregulation of GT43B and C genes did not affect drought responses and the transgenic plants grew better than the wild-type in drought and well-watered conditions. Both GH10 and GH11 xylanases strongly inhibited stem elongation and root growth in well-watered conditions but growth was less inhibited by drought in GH11-expressing plants than in wild-type. Overall, plants with xylan integrity impairment in secondary walls were less affected than wild-type by moderately reduced water availability but their responses also varied among genotypes and species. Thus, modifying the secondary cell wall integrity can be considered as a potential strategy for developing crops better suited to withstand water scarcity, but more research is needed to address the underlying molecular causes of this variability.

Effect of high-temperature thermo-oxidation on the mechanical and electrical properties of phenylethynyl-terminated polyimide/carbon fiber composite

The thermal degradation behaviors of phenylethynyl-terminated polyimide and their carbon fiber reinforced composites (CFRC) are significant to be evaluated comprehensively as their high-temperature applications are gradually expanded. It is found that ether-containing thermoset polyimide (PI) film shows unique degradation behaviors near glass transition temperature (Tg). Though the triethylamine (Et3N) chemical imidization method is applied, the PI backbone shows thermal stability at 371℃ thermo-oxidation condition and the main degradation parts site at the curing structure. More aliphatic (CC) linkages will be formed in the thermo-oxidation process and they are ascribed to the further crosslinking behaviors of polyene structure. However, the PI film shows stable mechanical properties after thermo-oxidation. Meanwhile, the formation of an amorphous carbon structure is found after thermo-oxidation treatment, which endows the composite with enhanced electrical properties. Though the micro-cracking phenomenon happens at the matrix/CF interface of composite surface and thus degrades its mechanical properties, the in-plane longitudinal and transverse conductivity of the composite perform an increase, factors of 1.13 to 2.56×104 S/m and 2.28 to 454.8 S/m, respectively. Mechanism study indicates that the formation of amorphous carbon components is caused by the structural rearrangement of the degrading polyene curing structure and becomes more in the thermo-oxidation process, not affecting the backbone integrity and mechanical stability of the PI matrix. As a result, thermo-oxidation treatment degrades the mechanical properties of the composite, whereas the formed amorphous carbon component acts as a self-growing carbon-based nano-filling structure at the PI/CF interface and enhances the electrical contact of the carbon fibers.

Optimizing outcomes in heart failure: 2022 and beyond.

Although the development of therapies and tools for the improved management of heart failure (HF) continues apace, day-to-day management in clinical practice is often far from ideal. A Cardiovascular Round Table workshop was convened by the European Society of Cardiology (ESC) to identify barriers to the optimal implementation of therapies and guidelines and to consider mitigation strategies to improve patient outcomes in the future. Key challenges identified included the complexity of HF itself and its treatment, financial constraints and the perception of HF treatments as costly, failure to meet the needs of patients, suboptimal outpatient management, and the fragmented nature of healthcare systems. It was discussed that ongoing initiatives may help to address some of these barriers, such as changes incorporated into the 2021 ESC HF guideline, ESC Heart Failure Association quality indicators, quality improvement registries (e.g. EuroHeart), new ESC guidelines for patients, and the universal definition of HF. Additional priority action points discussed to promote further improvements included revised definitions of HF 'phenotypes' based on trial data, the development of implementation strategies, improved affordability, greater regulator/payer involvement, increased patient education, further development of patient-reported outcomes, better incorporation of guidelines into primary care systems, and targeted education for primary care practitioners. Finally, it was concluded that overarching changes are needed to improve current HF care models, such as the development of a standardized pathway, with a common adaptable digital backbone, decision-making support, and data integration, to ensure that the model 'learns' as the management of HF continues to evolve.

Single-Atom Catalysts on Covalent Organic Frameworks for CO2 Reduction

The long-unresolved issue of CO2 release and the resulting atmospheric change can be solved through the application of effective catalysts. Thus, single-atom catalysts (SACs) have been rapidly developed for the CO2 reduction reaction (CO2RR), as they show improved catalytic metrics and enable the generation of C2+ products. Among numerous novel SACs, such as those based on graphene, metal–organic frameworks, and covalent organic frameworks (COFs), the COF-based SACs are the most promising owing to their high stability, porosity, and designability. Considering this, we describe two synthesis methods of COF-based SACs: ligand coordination and macrocycle backbone integration, and explore the pros and cons of each. We also propose routes for designing superior COF-based SACs and evaluate the factors influencing CO2RRs over COF-based SACs, such as metal loading and ligand types.

CTREP-finder: A web service for quick identification and visualization of clean transgenic and genome-edited plants

To feed 10 billion people in 30 years from now will heavily depend on new higher yield, yet safe, biotech crop varieties, which are developed using biotechnologies, particularly genetic transformation and genome editing. A clean transgenic plant (CTP) contains only the defined transgene, whilst a clean edited plant (CEP) is transgene-free. Currently, it is difficult and time-consuming to identify and locate transgenes within plant genomes due to the complex nature of transfer DNA (T-DNA) or plasmid backbone integration, which has led to a major obstacle in mass scale production of clean transgenic/edited plants (CTREPs). Here we have built a web-based portal, CTREP-finder, for fast detection, characterization and visualization of foreign DNA fragments in plants, based on next-generation whole genome sequencing data. We invented a novel bioinformatics strategy to handle three scenarios of transgene integration and successfully identified CTREPs by testing 132 samples. Furthermore, we compared the CTREP-finder with three public programs, FED, transgeneR and TDNAscan, and it showed that the CTREP-finder outperforms those programs particularly on the localization of transgene integration. The use of CTREP-finder needs little requirement for bioinformatics expertise. Therefore, it is a user-friendly web-based portal that could be used by plant breeders and regulatory agencies for the rapid selection/detection of CTREPs for crop breeding and production.

Studies on Pure Mlb® (Multiple Left Border) Technology and Its Impact on Vector Backbone Integration in Transgenic Cassava.

Imperfect T-DNA processing is common during Agrobacterium-mediated transformation, which integrates vector backbone sequences into the plant genome. However, regulatory restrictions prevent such transgenic plants from being developed for commercial deployment. The binary vector pCAMBIA2300 was modified by incorporating multiple left border (Mlb®) repeats and was tested in BY2 cells, tobacco, and cassava plants to address this issue. PCR analyses confirmed a twofold increase in the vector backbone free events in the presence of triple left borders in all three systems tested. Vector backbone read-through past the LB was reduced significantly; however, the inclusion of Mlbs® did not effectively address the beyond right border read-through. Also, Mlbs® increased the frequency of single-copy and vector backbone free events (clean events) twice compared to a single LB construct. Here, we briefly narrate the strength and limitations of using Mlb® technology and reporter genes in reducing the vector backbone transfer in transgenic events.

Biomimetic crosslinking of collagen gels by energetic electrons: The role of L-lysine

Energetic electrons have recently evolved as a powerful tool for crosslinking bio-derived hydrogels without the need for adding potentially hazardous reagents. Application of this approach allows for synthesis of biomimetic collagen-derived networks of highly tunable properties and functionalization. Yet, the underlying reaction kinetics are still not sufficiently established at this point. While hydroxyl radicals are generated by energetic electron-induced hydrolysis of water and play a key role in introducing covalent bonds between network fibers, a detailed mechanistic understanding would significantly increase applicability. We present a comprehensive analysis of central aspects of the reactivity between the hydroxyl radical (•OH) and collagen, elastin, glycine (Gly) and l-lysine (Lys). Pulse radiolysis (PR), solid state nuclear magnetic resonance (NMR), ultraviolet-visible absorption spectroscopy (UV/VIS) and electron spray ionization mass spectrometry (ESI-MS) shine light on distinct features of the crosslinking process. These highlight retained protein backbone integrity in collagen and elastin whilst Lys's ability to form several imine bonded Lys-Lys-species suggests striking similarities to crosslinking via lysyl oxidase catalysis in vivo. Thus, energetic electron based crosslinking opens the venue for customized hybrid gels of outstanding biomimicry and –compatibility. Statement of significanceEnergetic electron beam treatment constitutes a highly attractive approach to establish chemical bonds between (bio) molecules. Although a convincing number of publications showed the versatility regarding crosslinking of bioderived hydrogels, insights into the underlying chemistry are still unestablished at this point. The present work unravels the mechanistics of energetic electron induced processes in collagen and elastin hydrogels as well as several abundant amino acids in aqueous solution. As key finding we demonstrate, that i) the connection between polymer chains is dominated by amino acid side chain interaction and ii) two single l-lysine molecules form an imine bond between the terminal amino group of one molecule and the delta carbon of the second molecule. We also consider the formation of H-bonds as a second crosslinking pathway. These findings open up for advanced, optionally spatially resolved biomaterials design.

A rare transgenic event of rice with Agrobacterium binary vector backbone integration at the right T-DNA border junction

Agrobacterium tumefaciens precisely transfers the transferred DNA (T-DNA) between the right border (RB) and the left border (LB) and integrates it in the plant genome. However, transfer of the binary vector backbone (BVB) at the LB junction has been frequently reported. Here, we describe a complex T-DNA integration in the rice transgenic event CG27 with BVB at the RB junction. Amplification of an RB-plant DNA junction fragment by genome walking and its sequencing revealed that the T-DNA portion ended at the 1st bp of RB, but is followed by a binary vector backbone (BVB) along with an RB-flanking T-DNA portion, both in an inverse orientation. This is followed by a 5-bp filler sequence and the rice chromosome 1 sequence. The configuration of the BVB integration at the RB junction was independently confirmed by PCR. Disruption of native chromosome 1 sequence was confirmed by PCR with chromosome 1-specific primers designed from either side of the T-DNA integration site. BVB sequence close to the RB is transferred and integrated into the CG27 plant genome by a mechanism that does not involve LB-skipping and RB read-through. Therefore, a check for BVB sequences at both LB and RB junctions in a transgenic crop is important in biosafety evaluation.

Vector backbone integration in transgenic cassava is significantly correlated to T-DNA copy number

Multiple T-DNA integrations often occur with transgenic technology, resulting in complex integration patterns and transgene silencing. This study, investigates the correlation coefficient of T-DNA copy number on vector backbone (VBB) integration in transgenic cassava using Dot blot and PCR analysis. Thirty-nine, fifty-one and thirty-eight transgenic cassava plant lines recovered from transformations of cassava friable embryogenic callus with A. tumefaciens strain LBA4404 independently carrying p8016, p8052, and p900 were randomly selected and evaluated for VBB integration and T-DNA copy number. The occurrences of events with low (1-2) and high (≥ 3) T-DNA copy numbers were correlated with the presence and absence of VBB integration. Seventy-two to ninety-eight percent of VBB-free events were low copy number events while 2 to 28% of same where high copy number events. Correlation coefficient of the data revealed that the number of VBB-free events showed a significant positive correlation (r = 0.821, n = 9, p = 0.01) for events with low T-DNA copy number and a significant negative correlation (r = -0.739, n =9, p = 0.02) for high copy number events. This shows that the recovery of events with low T-DNA copy number increases the chances of recovering VBB-free events thereby enhancing the production of quality transgenic events.
 Key words: Copy number, DsRed, T-DNA, transformation, transgenic cassava, vector backbone integration.

Mechanically Gated Degradable Polymers.

Degradable polymers are desirable for the replacement of conventional organic polymers that persist in the environment, but they often suffer from the unintentional scission of the degradable functionalities on the polymer backbone, which diminishes polymer properties during storage and regular use. Herein, we report a strategy that combats unintended degradation in polymers by combining two common degradation stimuli-mechanical and acid triggers-in an "AND gate" fashion. A cyclobutane (CB) mechanophore is used as a mechanical gate to regulate an acid-sensitive ketal that has been widely employed in acid degradable polymers. This gated ketal is further incorporated into the polymer backbone. In the presence of an acid trigger alone, the pristine polymer retains its backbone integrity, and delivering high mechanical forces alone by ultrasonication degrades the polymer to an apparent limiting molecular weight of 28 kDa. The sequential treatment of ultrasonication followed by acid, however, leads to a further 11-fold decrease in molecular weight to 2.5 kDa. Experimental and computational evidence further indicate that the ungated ketal possesses mechanical strength that is commensurate with the conventional polymer backbones. Single molecule force spectroscopy (SMFS) reveals that the force necessary to activate the CB molecular gate on the time scale of 100 ms is approximately 2 nN.

World Flora Online (WFO) - Quality control workflow for an evolving taxonomic backbone

Initially populated with information gathered from The Plant List, the WFO’s taxonomic backbone will be augmented and modernized by newer taxonomic sources delivered by global plant Taxonomic Expert Networks (TENs). At the same time, descriptive data from floras, such as text descriptions, images, geographic distributions, keys, and trait data will be linked to this evolving taxonomic backbone. Semi-automated workflows have to be built to efficiently pull together the work of the TENs and digital floras and monographs into a single source: the WFO portal. These semi-automated workflows will have to ensure multiple functionalities: format check of the data submitted by the different backbone data providers (TENs or descriptive data providers), integrity and quality checks of the data provided, comparison of these data with the current WFO’s taxonomic backbone (matching with the WFO ID’s and with the names, check at the level of the family and the taxonomic status attribution, etc.). Applications will also be developed as Decision Making Helper for the TENs: thanks to these applications, TENs will be able to make decisions concerning the resolution of names not present in the current backbone or on the questions of conflicts at the level of the belonging to a family or the taxonomic status for a proposed name. At the end of these processes, new versions of the taxonomic backbone will regularly be delivered to be integrated into the WFO portal.

Critical Sites of DNA Backbone Integrity for Damaged Base Removal by Formamidopyrimidine-DNA Glycosylase.

DNA glycosylases, the enzymes that initiate base excision DNA repair, recognize damaged bases through a series of precisely orchestrated movements. Most glycosylases sharply kink the DNA axis at the lesion site and extrude the target base from the DNA double helix into the enzyme's active site. Little attention has been paid so far to the role of the physical continuity of the DNA backbone in allowing the required conformational distortion. Here, we analyze base excision by formamidopyrimidine-DNA glycosylase (Fpg) from substrates keeping all phosphates but containing a nick within three nucleotides of the lesion in either DNA strand. Four phosphoester linkages at the damaged nucleotide and two nucleotides 3' to it were essential for Fpg activity, while the breakage of the others, even at the same critical phosphates, had no effect or even stimulated the reaction. Reduction of the likelihood of hydrogen bonding at the nicks by using dideoxynucleotides as their 3'-terminal groups was more detrimental for the activity. All phosphoester bonds in the complementary strand were dispensable for base excision, but nicks close to the orphaned nucleotide caused early termination of damaged strand cleavage. Elastic network analysis of Fpg-DNA structures showed that the vibrational motions of the critical phosphates are strongly correlated, in part due to the presence of the protein. Overall, our results suggest that mechanical forces propagating along the DNA backbone play a critical role in the correct conformational distortion of DNA by Fpg and possibly by other target base-everting DNA glycosylases.

Ultrahigh-strength, nonflammable and high-wettability separators based on novel polyimide-core@polybenzimidazole-sheath nanofibers for advanced and safe lithium-ion batteries

Herein, a novel core@sheath nanofibrous membrane with ultrahigh strength, superior fire resistance and high wettability is prepared for lithium-ion batteries separator via self-bonding and self-compression technique, using the polyimide nanofiber as the core material and polybenzimidazole as the reinforced sheath material. The self-bonding and self-compression make the polyimide@polybenzimidazole-reinforced nanofibrous membrane possess ultrahigh strength up to 59 MPa and more compact porous structure, which is an exciting finding. The as-synthesized nanofibrous membrane exhibits super high thermal dimensional stability up to 300 °C and satisfying wettability. Furthermore the good thermal stability of as-synthesized membrane enables it to preserve the integrity of backbone at temperatures as high as 540 °C. More significantly, the unique fire resistance of polybenzimidazole also ensures the high security of batteries. Notably, the battery using the as-synthesized separator displays much higher capability (130.2 mAh g−1, 5 C) than that with Celgard separator (95.4 mAh g−1, 5 C), and it can work steadily at 120 °C. Therefore, we believe that this work is really a significant breakthrough for the polyimide nonwoven separators, especially the ultrahigh mechanical strength, indicating the promising applications for the next generation high-safety and high-performance lithium-ion batteries.

Fluorescent Gene Tagging of Transcriptionally Silent Genes in hiPSCs.

SummaryWe describe a multistep method for endogenous tagging of transcriptionally silent genes in human induced pluripotent stem cells (hiPSCs). A monomeric EGFP (mEGFP) fusion tag and a constitutively expressed mCherry fluorescence selection cassette were delivered in tandem via homology-directed repair to five genes not expressed in hiPSCs but important for cardiomyocyte sarcomere function: TTN, MYL7, MYL2, TNNI1, and ACTN2. CRISPR/Cas9 was used to deliver the selection cassette and subsequently mediate its excision via microhomology-mediated end-joining and non-homologous end-joining. Most excised clones were effectively tagged, and all properly tagged clones expressed the mEGFP fusion protein upon differentiation into cardiomyocytes, allowing live visualization of these cardiac proteins at the sarcomere. This methodology provides a broadly applicable strategy for endogenously tagging transcriptionally silent genes in hiPSCs, potentially enabling their systematic and dynamic study during differentiation and morphogenesis.

Coexpression of octopine and succinamopine Agrobacterium virulence genes to generate high quality transgenic events in maize by reducing vector backbone integration.

Agrobacterium-mediated transformation is a complex process that is widely utilized for generating transgenic plants. However, one of the major concerns of this process is the frequent presence of undesirable T-DNA vector backbone sequences in the transgenic plants. To mitigate this deficiency, a ternary strain of A. tumefaciens was modified to increase the precision of T-DNA border nicking such that the backbone transfer is minimized. This particular strain supplemented the native succinamopine VirD1/VirD2 of EHA105 with VirD1/VirD2 derived from an octopine source (pTi15955), the same source as the binary T-DNA borders tested here, residing on a ternary helper plasmid containing an extra copy of the succinamopine VirB/C/G operons and VirD1. Transformation of maize immature embryos was carried out with two different test constructs, pDAB101556 and pDAB111437, bearing the reporter YFP gene and insecticidal toxin Cry1Fa gene, respectively, contained in the VirD-supplemented and regular control ternary strains. Molecular analyses of ~ 700 transgenic events revealed a significant 2.6-fold decrease in events containing vector backbone sequences, from 35.7% with the control to 13.9% with the VirD-supplemented strain for pDAB101556 and from 24.9% with the control to 9.3% with the VirD-supplemented strain for pDAB111437, without compromising transformation efficiency. In addition, while the number of single copy events recovered was similar, there was a 24-26% increase in backbone-free events with the VirD-supplemented strain compared to the control strain. Thus, supplementing existing VirD1/VirD2 genes in Agrobacterium, to recognize diverse T-DNA borders, proved to be a useful tool to increase the number of high quality events in maize.

Plant tissue culture independent Agrobacterium tumefaciens mediated In-planta transformation strategy for upland cotton (Gossypium hirsutum).

A new method of transgenic development called “In-planta” transformation method, where Agrobacterium is used to infect the plantlets but the steps of in vitro regeneration of plants is totally avoided. In this study, we have reported a simple In-planta method for efficient transformation of diploid cotton Gossypium hirsutum cv LRK-516 Anjali using Agrobacterium tumefaciens EHA-105 harbouring recombinant binary vector plasmid pBinAR with Arabidopsis At-NPR1 gene. Four day old plantlets were used for transformation. A vertical cut was made at the junction of cotyledonary leaves, moderately bisecting the shoot tip and exposing meristem cells at apical meristem. This site was infected with Agrobacterium inoculum. The transgenic events obtained were tested positive for the presence of At-NPR1 gene with promoter nptII gene. They are also tested negative for vector backbone integration and Agrobacterium contamination in T0 events. With this method a transformation frequency of 6.89% was reported for the cv LRK-516.

The Functionality of Minimal PiggyBac Transposons in Mammalian Cells.

Minimal piggyBac vectors are a modified single-plasmid version of the classical piggyBac delivery system that can be used for stable transgene integration. These vectors have a truncated terminal domain in the delivery cassette and thus, integrate significantly less flanking transposon DNA into host cell chromatin than classical piggyBac vectors. Herein, we test various characteristics of this modified transposon. The integration efficiency of minimal piggyBac vectors was inversely related to the size of both the transposon and the entire plasmid, but inserts as large as 15 kb were efficiently integrated. Open and super-coiled vectors demonstrated the same integration efficiency while DNA methylation decreased the integration efficiency and silenced the expression of previously integrated sequences in some cell types. Importantly, the incidence of plasmid backbone integration was not increased above that seen in nontransposon control vectors. In BALB/c mice, we demonstrated prolonged expression of two transgenes (intracellular mCherry and secretable Gaussia luciferase) when delivered by the minimal piggyBac that resulted in a more sustained antibody production against the immunogenic luciferase than when delivered by a transient (nontransposon) vector plasmid. We conclude that minimal piggyBac vectors are an effective alternative to other integrative systems for stable DNA delivery in vitro and in vivo.

120. Generation of Transgene-Only Gene-Modified Human Cells Via Transposition

DNA transposons such as piggyBac and Sleeping Beauty employ a cut-and-paste mechanism of transposition that has proven effective for numerous gene transfer and cellular reprogramming applications. Therapeutic transposons are carried into cells usually on plasmid DNA vectors either on the same plasmid as the transposase expression cassette in “cis” or on another plasmid in “trans.” We examined the fate of the remaining plasmid backbone after transposition. It has been assumed that the recircularized plasmid backbone remains episomal and is quickly lost from dividing cells. We delivered transposons containing a neomycin resistance gene and the transposase in either “cis” or “trans” to human embryonic kidney (HEK-293) cells, then selected the cells for 8 weeks and evaluated transposase expression by immunoblot. All of the piggyBac and 5 out of 6 of the Sleeping Beauty “cis” transfections resulted in transposase-positive clones. In contrast, none of the piggyBac “trans” transfections resulted in transposase-positive clones. We were concerned that the prolonged expression of the transposase might result in some instability of the transgenes delivered “cis” in vivo; however, we found that mice that received a piggyBac “cis” luciferase transposon vector by hydrodynamic injection had long-term luciferase values that were not statistically different from mice receiving the plasmids in “trans.” We next evaluated if piggyBac “trans” delivery could be modified to reduce the amount of backbone integration and found that clones that were transfected with lower amounts of transposase showed a decrease in the frequency of backbone integrations. We found no evidence that transposition increases the frequency of integration of co-delivered linear DNA. We sequenced backbone integration events and found that that the majority of the time, once the transposon is excised from its parent plasmid, the plasmid backbone is repaired by re-ligation and integrated into the genome at a site other than the excision site. Alternatively, a rare event may occur in which the linearized plasmid backbone can be integrated without excision repair. None of the integration events occurred at the TTAA tetranucleotide sequence, indicating that they are not directly mediated by piggyBac transposase. We next devised a strategy to measure and eliminate the backbones from human cells. We created a plasmid containing the neomycin-resistance gene in the transposon portion of the plasmid and the eGFP marker in the backbone. Transfected HEK-293 cells were selected with geneticin and sorted for eGFP. While the selected cells contained approximately ten copies of the transposon per diploid cell, unsorted cells typically had one to two copies of plasmid backbone per diploid genome. eGFP sorting effectively eliminated recovery of backbone-negative transgene-only cells. We expect further improvements to improve recovery of transgene-only cells. We will also investigate the extent of backbone integration in therapeutically relevant cell types and in vivo.

High-throughput transformation pipeline for a Brazilian japonica rice with bar gene selection.

The goal of this work was to establish a transformation pipeline for upland Curinga rice (Oryza sativa L. ssp. japonica) with bar gene selection employing bialaphos and phosphinothricin as selection agents. The following genes of interest: AtNCED3, Lsi1, GLU2, LEW2, PLD-alpha, DA1, TOR, AVP1, and Rubisco were cloned into the binary vector p7i2x-Ubi and were transferred into Agrobacterium strain EHA 105. Embryogenic calli derived from the mature embryos were transformed, and transgenic cells and shoots were selected on the medium supplemented with bialaphos or phosphinothricin (PPT) using a stepwise selection scheme. Molecular analyses were established using polymerase chain reaction and Southern blot for the bar gene and the NOS terminator. Overall, 273 putative transgenic plants were analyzed by Southern blot with 134 events identified. In total, 77 events had a single copy of the transgene integrated in the plant genome while 29 events had two copies. We tested backbone integration in 101 transgenic plants from all constructs and found 60 transgenic plants having no additional sequence integrated in the plant genome. The bar gene activity was evaluated by the chlorophenol red test and the leaf painting test using phosphinothricin with several transgenic plants. The majority of T0 plants carrying the single copy of transgene produced T1 seeds in the screen house.

Development of late blight resistant potatoes by cisgene stacking.

BackgroundPhytophthora infestans, causing late blight in potato, remains one of the most devastating pathogens in potato production and late blight resistance is a top priority in potato breeding. The introduction of multiple resistance (R) genes with different spectra from crossable species into potato varieties is required. Cisgenesis is a promising approach that introduces native genes from the crops own gene pool using GM technology, thereby retaining favourable characteristics of established varieties.ResultsWe pursued a cisgenesis approach to introduce two broad spectrum potato late blight R genes, Rpi-sto1 and Rpi-vnt1.1 from the crossable species Solanum stoloniferum and Solanum venturii, respectively, into three different potato varieties. First, single R gene-containing transgenic plants were produced for all varieties to be used as references for the resistance levels and spectra to be expected in the respective genetic backgrounds. Next, a construct containing both cisgenic late blight R genes (Rpi-vnt1.1 and Rpi-sto1), but lacking the bacterial kanamycin resistance selection marker (NPTII) was transformed to the three selected potato varieties using Agrobacterium-mediated transformation. Gene transfer events were selected by PCR among regenerated shoots. Through further analyses involving morphological evaluations in the greenhouse, responsiveness to Avr genes and late blight resistance in detached leaf assays, the selection was narrowed down to eight independent events. These cisgenic events were selected because they showed broad spectrum late blight resistance due to the activity of both introduced R genes. The marker-free transformation was compared to kanamycin resistance assisted transformation in terms of T-DNA and vector backbone integration frequency. Also, differences in regeneration time and genotype dependency were evaluated.ConclusionsWe developed a marker-free transformation pipeline to select potato plants functionally expressing a stack of late blight R genes. Marker-free transformation is less genotype dependent and less prone to vector backbone integration as compared to marker-assisted transformation. Thereby, this study provides an important tool for the successful deployment of R genes in agriculture and contributes to the production of potentially durable late blight resistant potatoes.