Showing papers on "Transformation (genetics) published in 2020"

Overexpression of the Transcription Factor GROWTH-REGULATING FACTOR5 Improves Transformation of Dicot and Monocot Species.

Abstract: Successful regeneration of genetically modified plants from cell culture is highly dependent on the species, genotype, and tissue-type being targeted for transformation. Studies in some plant species have shown that when expression is altered, some genes regulating developmental processes are capable of triggering plant regeneration in a variety of plant cells and tissue-types previously identified as being recalcitrant to regeneration. In the present research, we report that developmental genes encoding GROWTH-REGULATING FACTORS positively enhance regeneration and transformation in both monocot and dicot species. In sugar beet (Beta vulgaris ssp. vulgaris), ectopic expression of Arabidopsis GRF5 (AtGRF5) in callus cells accelerates shoot formation and dramatically increases transformation efficiency. More importantly, overexpression of AtGRF5 enables the production of stable transformants in recalcitrant sugar beet varieties. The introduction of AtGRF5 and GRF5 orthologs into canola (Brassica napus L.), soybean (Glycine max L.), and sunflower (Helianthus annuus L.) results in significant increases in genetic transformation of the explant tissue. A positive effect on proliferation of transgenic callus cells in canola was observed upon overexpression of GRF5 genes and AtGRF6 and AtGRF9. In soybean and sunflower, the overexpression of GRF5 genes seems to increase the proliferation of transformed cells, promoting transgenic shoot formation. In addition, the transformation of two putative AtGRF5 orthologs in maize (Zea mays L.) significantly boosts transformation efficiency and resulted in fully fertile transgenic plants. Overall, the results suggest that overexpression of GRF genes render cells and tissues more competent to regeneration across a wide variety of crop species and regeneration processes. This sets GRFs apart from other developmental regulators and, therefore, they can potentially be applied to improve transformation of monocot and dicot plant species.

Wheat gene Sr60 encodes a protein with two putative kinase domains that confers resistance to stem rust

Abstract: Wheat stem rust, caused by Puccinia graminis Pers. f. sp. tritici (Pgt), is a devastating fungal disease threatening global wheat production. The present paper reports the identification of stem rust resistance gene Sr60, a race-specific gene from diploid wheat Triticum monococcum L. that encodes a protein with two putative kinase domains. This gene, designated as WHEAT TANDEM KINASE 2 (WTK2), confers intermediate levels of resistance to Pgt. WTK2 was identified by map-based cloning and validated by transformation of a c.10-kb genomic sequence including WTK2 into susceptible common wheat variety Fielder (Triticum aestivum L.). Transformation of Fielder with WTK2 was sufficient to confer Pgt resistance. Sr60 transcripts were transiently upregulated 1 d post-inoculation with Pgt, but not in mock-inoculated plants. The upregulation of Sr60 was associated with stable upregulation of several pathogenesis-related genes. The Sr60-resistant haplotype found in T. monococcum was not found in polyploid wheat, suggesting an opportunity to introduce a novel resistance gene. Sr60 was successfully introgressed into hexaploid wheat, and we developed a diagnostic molecular marker to accelerate its deployment and pyramiding with other resistance genes. The cloned Sr60 also can be a useful component of transgenic cassettes including other resistance genes with complementary resistance profiles.

Use of non-integrating Zm-Wus2 vectors to enhance maize transformation

Abstract: The use of Baby boom (Bbm) and Wuschel2 (Wus2) has made maize transformation more efficient across an increasingly wide range of inbreds. However, the benefits have come with the requirement of excising these transformation helper components to enable plant regeneration, which adds size to the T-DNA, and complexity to the transformation system. A new system with the advantages of smaller size and simplicity for the selectable marker gene-containing T-DNA is described. First, expression of Zm-Wus2 alone driven by the maize Pltp promoter (Zm-Pltppro), was determined to be sufficient to induce rapid somatic embryo formation from the scutella of maize immature embryos. It was also demonstrated that co-infecting with two strains of Agrobacterium, one with a Wus2 expression cassette, and the other with a combination of both selectable and visual marker cassettes, produced transformed T0 plants that contained only a single copy of the selectable marker T-DNA, without the integration of Wus2. Furthermore, the process was optimized by varying the ratio of the two Agrobacterium strains, and by modulating Wus2 expression to enable high-frequency recovery of selectable marker-containing T0 plants that did not contain Wus2. Several factors may have contributed to this outcome. Wus2 expression in localized cell(s) appeared to stimulate somatic embryogenesis in neighboring cells, including those that had integrated the selectable marker. In addition, in cells in which the Wus2 T-DNA did not integrate but the selectable marker T-DNA did, transient Wus2 expression stimulated somatic embryo formation and regeneration of stable T0 plants that contained the selectable marker. In addition, augmenting the Pltp promoter with three viral enhancer elements to increase Wus2 expression stimulated embryogenesis while precluding their regeneration. The phenomenon has now been designated as “altruistic transformation.”

Overexpression of the transcription factor GROWTH-REGULATING FACTOR5 improves transformation of dicot and monocot species

Abstract: 1 Abstract Successful regeneration of genetically modified plants from cell culture is highly dependent on the species, genotype, and tissue-type being targeted for transformation. Studies in some plant species have shown that when expression is altered, some genes regulating developmental processes are capable of triggering plant regeneration in a variety of plant cells and tissue-types previously identified as being recalcitrant to regeneration. In the present research, we report that developmental genes encoding GROWTH-REGULATING FACTORS positively enhance regeneration and transformation in both monocot and dicot species. In sugar beet (Beta vulgaris ssp. vulgaris), ectopic expression of Arabidopsis GRF5 (AtGRF5) in callus cells accelerates shoot formation and dramatically increases transformation efficiency. More importantly, overexpression of AtGRF5 enables the production of stable transformants in recalcitrant sugar beet varieties. The introduction of AtGRF5 and GRF5 orthologs into canola (Brassica napus L.), soybean (Glycine max L.), and sunflower (Helianthus annuus L.) results in significant increases in genetic transformation of the explant tissue. A positive effect on proliferation of transgenic callus cells in canola was observed upon overexpression of GRF5 genes and AtGRF6 and AtGRF9. In soybean and sunflower, the overexpression of GRF5 genes seems to increase the proliferation of transformed cells, promoting transgenic shoot formation. In addition, the transformation of two putative AtGRF5 orthologs in maize (Zea mays L.) significantly boosts transformation efficiency and resulted in fully fertile transgenic plants. Overall, the results suggest that overexpression of GRF genes render cells and tissues more competent to regeneration across a wide variety of crop species and regeneration processes. This sets GRFs apart from other developmental regulators and, therefore, they can potentially be applied to improve transformation of monocot and dicot plant species.

A common wild rice-derived BOC1 allele reduces callus browning in indica rice transformation.

Abstract: Callus browning, a common trait derived from the indica rice cultivar (Oryza sativa L.), is a challenge to transformation regeneration. Here, we report the map-based cloning of BROWNING OF CALLUS1 (BOC1) using a population derived from crossing Teqing, an elite indica subspecies exhibiting callus browning, and Yuanjiang, a common wild rice accession (Oryza rufipogon Griff.) that is less susceptible to callus browning. We show that BOC1 encodes a SIMILAR TO RADICAL-INDUCED CELL DEATH ONE (SRO) protein. Callus browning can be reduced by appropriate upregulation of BOC1, which consequently improves the genetic transformation efficiency. The presence of a Tourist-like miniature inverted-repeat transposable element (Tourist MITE) specific to wild rice in the promoter of BOC1 increases the expression of BOC1 in callus. BOC1 may decrease cell senescence and death caused by oxidative stress. Our study provides a gene target for improving tissue culturability and genetic transformation. Callus browning heavily affects indica rice transformation regeneration. Here, the authors show transposon insertion in the promoter of BOC1 gene, encoding a SIMILAR TO RADICAL-INDUCED CELL DEATH ONE protein, can upregulate its expression and decrease callus browning in cultivated rice by releasing oxidative stress.

Generation of transgene-free PDS mutants in potato by Agrobacterium-mediated transformation.

Abstract: Gene editing using the CRISPR/Cas9 system has become a routinely applied method in several plant species. The most convenient gene delivery system is Agrobacterium-mediated gene transfer with antibiotic selection and stable genomic integration of transgenes, including Cas9. For elimination of transgenes in the segregating progeny, selfing is applied in many plant species. This approach, however, cannot be widely employed in potato because most of the commercial potato cultivars are self-incompatible. In this study, the efficiency of a transient Cas9 expression system with positive/negative selection based on codA-nptII fusion was tested. The PHYTOENE DESATURASE (PDS) gene involved in carotenoid biosynthesis was targeted. A new vector designated PROGED::gPDS carrying only the right border of T-DNA was constructed. Using only the positive selection function of PROGED::gPDS and the restriction enzyme site loss method in PCR of genomic DNA after digestion with the appropriate restriction enzyme, it was demonstrated that the new vector is as efficient in gene editing as a traditional binary vector with right- and left-border sequences. Nevertheless, 2 weeks of positive selection followed by negative selection did not result in the isolation of PDS mutants. In contrast, we found that with 3-day positive selection, PDS mutants appear in the regenerating population with a minimum frequency of 2–10%. Interestingly, while large deletions (> 100 bp) were generated by continuous positive selection, the 3-day selection resulted in deletions and substitutions of only a few bp. Two albinos and three chimaeras with white and green leaf areas were found among the PDS mutants, while all the other PDS mutant plants were green. Based on DNA sequence analysis some of the green plants were also chimaeras. Upon vegetative propagation from stem segments in vitro, the phenotype of the plants obtained even by positive selection did not change, suggesting that the expression of Cas9 and gPDS is silenced or that the DNA repair system is highly active during the vegetative growth phase in potato. Gene-edited plants can be obtained from potatoes by Agrobacterium-mediated transformation with 3-day antibiotic selection with a frequency high enough to identify the mutants in the regenerating plant population using PCR.

Learning Generalized Transformation Equivariant Representations via Autoencoding Transformations.

Abstract: Transformation Equivariant Representations (TERs) aim to capture the intrinsic visual structures that equivary to various transformations by expanding the notion of translation equivariance underlying the success of Convolutional Neural Networks (CNNs). For this purpose, we present both deterministic AutoEncoding Transformations (AET) and probabilistic AutoEncoding Variational Transformations (AVT) models to learn visual representations from generic groups of transformations. While the AET is trained by directly decoding the transformations from the learned representations, the AVT is trained by maximizing the joint mutual information between the learned representation and transformations. This results in Generalized TERs (GTERs) equivariant against transformations in a more general fashion by capturing complex patterns of visual structures beyond the conventional linear equivariance under a transformation group. The presented approach can be extended to (semi-)supervised models by jointly maximizing the mutual information of the learned representation with both labels and transformations. Experiments demonstrate the proposed models outperform the state-of-the-art models in both unsupervised and (semi-)supervised tasks. Moreover, we show that the unsupervised representation can even surpass the fully supervised representation pretrained on ImageNet when they are fine-tuned for the object detection task.

An efficient Agrobacterium-mediated transformation method using hypocotyl as explants for Brassica napus

Abstract: Rapeseed (Brassica napus) is an important oil crop that supplies a considerable amount of global vegetable oil production. Genetic transformation system is important to gene functional analysis and molecular breeding. Here, an efficient Agrobacterium-mediated transformation protocol using hypocotyl of rapeseed as explants is described. To develop this protocol, we compared several essential factors that would affect the transformation efficiency, such as Agrobacterium strains, selection marker genes, and genotypes of rapeseed. Comparison of different Agrobacterium strains showed that the GV3101 had higher transformation efficiency than that of C58C1 and EHA105. HPTII, NPTII, and RePAT were used as selection marker genes in tissue culture. The results showed that the transformation efficiency was 3.7–4.8%, 2.2–22.5%, and 1.6–5.9% when the hypocotyl of Westar was infected by GV3101 and screened under hygromycin, kanamycin, and basta, respectively. The transformation efficiency of Westar was the highest and ZS11 was the lowest when five different genotypes of rapeseed (Westar, ZS9, ZS11, GY284, and WH3417) were infected by GV3101. Using this protocol, it will take 8–10 weeks to obtain transgenic plants. This protocol has been used to study gene function in several genotypes of rapeseed in our laboratory. These results indicate that it is efficient to obtain transgenic plant of rapeseed using this protocol.

First-generation genome editing in potato using hairy root transformation

Abstract: Genome editing and cis-gene breeding have rapidly accelerated crop improvement efforts, but their impacts are limited by the number of species capable of being genetically transformed. Many dicot species, including some vital potato relatives being used to accelerate breeding and genetics efforts, remain recalcitrant to standard Agrobacterium tumefaciens-based transformation. Hairy root transformation using Agrobacterium rhizogenes (A. rhizogenes) provides an accelerated approach to generating transgenic material but has been limited to analysis of hairy root clones. In this study, strains of A. rhizogenes were tested in the wild diploid potato relative Solanum chacoense, which is recalcitrant to infection by Agrobacterium tumefaciens. One strain of A. rhizogenes MSU440 emerged as being capable of delivering a T-DNA carrying the GUS marker and generating transgenic hairy root clones capable of GUS expression and regeneration to whole plants. CRISPR/Cas9 reagents targeting the potato PHYTOENE DESATURASE (StPDS) gene were expressed in hairy root clones and regenerated. We found that 64%-98% of transgenic hairy root clones expressing CRISPR/Cas9 reagents carried targeted mutations, while only 14%-30% of mutations were chimeric. The mutations were maintained in regenerated lines as stable mutations at rates averaging at 38% and were capable of germ-line transmission to progeny. This novel approach broadens the numbers of genotypes amenable to Agrobacterium-mediated transformation while reducing chimerism in primary events and accelerating the generation of edited materials.

Pseudomonas aeruginosa is capable of natural transformation in biofilms.

Abstract: Natural transformation is a mechanism that enables competent bacteria to acquire naked, exogenous DNA from the environment. It is a key process that facilitates the dissemination of antibiotic resistance and virulence determinants throughout bacterial populations. Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that produces large quantities of extracellular DNA (eDNA) that is required for biofilm formation. P. aeruginosa has a remarkable level of genome plasticity and diversity that suggests a high degree of horizontal gene transfer and recombination but is thought to be incapable of natural transformation. Here we show that P. aeruginosa possesses homologues of all proteins known to be involved in natural transformation in other bacterial species. We found that P. aeruginosa in biofilms is competent for natural transformation of both genomic and plasmid DNA. Furthermore, we demonstrate that type-IV pili (T4P) facilitate but are not absolutely essential for natural transformation in P. aeruginosa.

An efficient Agrobacterium-mediated genetic transformation method for foxtail millet (Setaria italica L.).

Abstract: A simple and robust Agrobacterium-mediated gene expression system in the C4 panicoid model crop, foxtail millet has been developed with up to 27 % transformation efficiency. Foxtail millet (Setaria italica L.) is a model crop to study C4 photosynthesis, abiotic stress tolerance, and bioenergy traits. Advances in molecular genetics and genomics had identified several potential genes in this crop that would serve as candidates for imparting climate-resilient traits in related millets, cereals, and biofuel crops. However, the lack of an efficient genetic transformation system has been impeding the functional characterization of these genes in foxtail millet per se. Given this, an easy and efficient regeneration and transformation protocol was optimized using mature seeds as a choicest explant. The suitability of secondary embryogenic calli over primary calli is underlined due to their high competence. The use of perfect combinations of plant growth regulators together with the ionic strength of organic and inorganics salts was found to influence regeneration and genetic transformation. We studied and optimized various crucial factors that affect the genetic transformation of foxtail millet calli using Agrobacterium tumefaciens-mediated approach. Secondary embryogenic calli and LBA44404 strain were found to be the best targets for transformation. The use of high sucrose and glucose, together with freshly prepared tobacco leaves extract, Silwet L-77 and acetosyringone, improved the efficiency of the genetic transformation of foxtail millet. Moreover, the use of an in vitro regeneration system with 84% callusing efficiency and 70–74% regeneration frequency led to a high recovery of transformants. Altogether, the present study reports a highly efficient (~ 27%) transformation system in foxtail millet that will expedite forward and reverse genetic studies in this important crop.

A protein-independent fluorescent RNA aptamer reporter system for plant genetic engineering

Abstract: Reporter systems are routinely used in plant genetic engineering and functional genomics research. Most such plant reporter systems cause accumulation of foreign proteins. Here, we demonstrate a protein-independent reporter system, 3WJ-4 × Bro, based on a fluorescent RNA aptamer. Via transient expression assays in both Escherichia coli and Nicotiana benthamiana, we show that 3WJ-4 × Bro is suitable for transgene identification and as an mRNA reporter for expression pattern analysis. Following stable transformation in Arabidopsis thaliana, 3WJ-4 × Bro co-segregates and co-expresses with target transcripts and is stably inherited through multiple generations. Further, 3WJ-4 × Bro can be used to visualize virus-mediated RNA delivery in plants. This study demonstrates a protein-independent reporter system that can be used for transgene identification and in vivo dynamic analysis of mRNA.

Adaptive Fuzzy Asymptotic Tracking Control of State-Constrained High-Order Nonlinear Time-Delay Systems and Its Applications.

Abstract: This article discusses the adaptive fuzzy asymptotic tracking control for high-order nonlinear time-delay systems with full-state constraints. Fuzzy-logic systems and a separation principle are utilized to relax growth assumptions imposed on unknown nonlinearities. The adverse effect caused by unknown time delays is eliminated by choosing appropriate Lyapunov-Krasovskii functionals. By integrating nonlinear-transformed functions with a key coordinate transformation into the control design and constructing a specific compact set on the initial values of system states, the desired trajectory and parameter estimates, it is rigorously proved that all closed-loop signals are semiglobally bounded, the fuzzy approximation is valid, the full-state constraints are not violated without feasibility conditions on virtual controllers, and asymptotic tracking is achieved. The effectiveness and advantages of this control scheme are confirmed by two examples including a single-link robotic system.

Ni–Fe Catalysts Supported on γ-Al2O3/HZSM-5 for Transformation of Palmitic Acid into Hydrocarbon Fuel

Abstract: Ni–Fe catalysts supported on γ-Al2O3 and HZSM-5 were proved to be active for fatty acids conversion into hydrocarbon fuel. The strong interaction between Ni and γ-Al2O3 produced NiAl2O4 species wit...

Learning Poisson systems and trajectories of autonomous systems via Poisson neural networks.

Abstract: We propose the Poisson neural networks (PNNs) to learn Poisson systems and trajectories of autonomous systems from data. Based on the Darboux-Lie theorem, the phase flow of a Poisson system can be written as the composition of (1) a coordinate transformation, (2) an extended symplectic map and (3) the inverse of the transformation. In this work, we extend this result to the unknotted trajectories of autonomous systems. We employ structured neural networks with physical priors to approximate the three aforementioned maps. We demonstrate through several simulations that PNNs are capable of handling very accurately several challenging tasks, including the motion of a particle in the electromagnetic potential, the nonlinear Schr{\"o}dinger equation, and pixel observations of the two-body problem.

Uptake, translocation and transformation of three pharmaceuticals in green pea plants

Abstract: Abstract Treated water from wastewater treatment plants that is increasingly used for irrigation may contain pharmaceuticals and, thus, contaminate soils. Therefore, this study focused on the impact of soil conditions on the root uptake of selected pharmaceuticals and their transformation in a chosen soil–plant system. Green pea plants were planted in 3 soils. Plants were initially irrigated with tap water. Next, they were irrigated for 20 days with a solution of either atenolol (ATE), sulfamethoxazole (SUL), carbamazepine (CAR), or all of these three compounds. The concentrations of pharmaceuticals and their metabolites [atenolol acid (AAC), N1-acetyl sulfamethoxazole (N1AS), N4-acetyl sulfamethoxazole (N4AS), carbamazepine 10,11-epoxide (EPC), 10,11-dihydrocarbamazepine (DHC), trans-10,11-dihydro-10,11-dihydroxy carbamazepine (RTC), and oxcarbazepine (OXC)] in soils and plant tissues were evaluated after harvest. The study confirmed high (CAR), moderate (ATE, AAC, SUL), and minor (N4AC) root uptake of the studied compounds by the green pea plants, nonrestricted transfer of the CAR species into the different plant tissues, and a very high efficiency in metabolizing CAR in the stems and leaves. The results showed neither a synergic nor competitive influence of the application of all compounds in the solution on their uptake by plants. The statistical analysis proved the negative relationships between the CAR sorption coefficients and the concentrations of CAR, EPC, and OXC in the roots (R = –0.916, –0.932, and –0.925, respectively) and stems (R = –0.837, –0.844, and –0.847, respectively).

Ranking essential bacterial processes by speed of mutant death

Abstract: Mutant phenotype analysis of bacteria has been revolutionized by genome-scale screening procedures, but essential genes have been left out of such studies because mutants are missing from the libraries analyzed. Since essential genes control the most fundamental processes of bacterial life, this is a glaring deficiency. To address this limitation, we developed a procedure for transposon insertion mutant sequencing that includes essential genes. The method, called transformation transposon insertion mutant sequencing (TFNseq), employs saturation-level libraries of bacterial mutants generated by natural transformation with chromosomal DNA mutagenized heavily by in vitro transposition. The efficient mutagenesis makes it possible to detect large numbers of insertions in essential genes immediately after transformation and to follow their loss during subsequent growth. It was possible to order 45 essential processes based on how rapidly their inactivation inhibited growth. Inactivating ATP production, deoxyribonucleotide synthesis, or ribosome production blocked growth the fastest, whereas inactivating cell division or outer membrane protein synthesis blocked it the slowest. Individual mutants deleted of essential loci formed microcolonies of nongrowing cells whose sizes were generally consistent with the TFNseq ordering. The sensitivity of essential functions to genetic inactivation provides a metric for ranking their relative importance for bacterial replication and growth. Highly sensitive functions could represent attractive antibiotic targets since even partial inhibition should reduce growth.

Gene transfer to plants by electroporation: methods and applications

Abstract: Developing gene transfer technologies enables the genetic manipulation of the living organisms more efficiently. The methods used for gene transfer fall into two main categories; natural and artificial transformation. The natural methods include the conjugation, transposition, bacterial transformation as well as phage and retroviral transductions, contain the physical methods whereas the artificial methods can physically alter and transfer genes from one to another organisms' cell using, for instance, biolistic transformation, micro- and macroinjection, and protoplast fusion etc. The artificial gene transformation can also be conducted through chemical methods which include calcium phosphate-mediated, polyethylene glycol-mediated, DEAE-Dextran, and liposome-mediated transfers. Electrical methods are also artificial ways to transfer genes that can be done by electroporation and electrofusion. Comparatively, among all the above-mentioned methods, electroporation is being widely used owing to its high efficiency and broader applicability. Electroporation is an electrical transformation method by which transient electropores are produced in the cell membranes. Based on the applications, process can be either reversible where electropores in membrane are resealable and cells preserve the vitality or irreversible where membrane is not able to reseal, and cell eventually dies. This problem can be minimized by developing numerical models to iteratively optimize the field homogeneity considering the cell size, shape, number, and electrode positions supplemented by real-time measurements. In modern biotechnology, numerical methods have been used in electrotransformation, electroporation-based inactivation, electroextraction, and electroporative biomass drying. Moreover, current applications of electroporation also point to some other uncovered potentials for various exploitations in future.

One-step generation of composite soybean plants with transgenic roots by Agrobacterium rhizogenes -mediated transformation

Abstract: Agrobacterium rhizogenes-mediated (ARM) transformation is a highly efficient technique for generating composite plants composed of transgenic roots and wild-type shoot, providing a powerful tool for studying root biology. The ARM transformation has been established in many plant species, including soybean. However, traditional transformation of soybean, transformation efficiency is low. Additionally, the hairy roots were induced in a medium, and then the generated composite plants were transplanted into another medium for growth. This two-step operation is not only time-consuming, but aggravates contamination risk in the study of plant-microbe interactions. Here, we report a one-step ARM transformation method with higher transformation efficiency for generating composite soybean plants. Both the induction of hairy roots and continuous growth of the composite plants were conducted in a single growth medium. The primary root of a 7-day-old seedling was decapitated with a slanted cut, the residual hypocotyl (maintained 0.7-1 cm apical portion) was inoculated with A. rhizogenes harboring the gene construct of interest. Subsequently, the infected seedling was planted into a pot with wet sterile vermiculite. Almost 100% of the infected seedlings could produce transgenic positive roots 16 days post-inoculation in 7 tested genotypes. Importantly, the transgenic hairy roots in each composite plant are about three times more than those of the traditional ARM transformation, indicating that the one-step method is simpler in operation and higher efficiency in transformation. The reliability of the one-step method was verified by CRISPR/Cas9 system to knockout the soybean Rfg1, which restricts nodulation in Williams 82 (Nod-) by Sinorhizobium fredii USDA193. Furthermore, we applied this method to analyze the function of Arabidopsis YAO promoter in soybean. The activity of YAO promoter was detected in whole roots and stronger in the root tips. We also extended the protocol to tomato. We established a one-step ARM transformation method, which is more convenient in operation and higher efficiency (almost 100%) in transformation for generating composite soybean plants. This method has been validated in promoter functional analysis and rhizobia-legume interactions. We anticipate a broad application of this method to analyze root-related events in tomato and other plant species besides soybean.

System Transformation-Based Neural Control for Full-State-Constrained Pure-Feedback Systems via Disturbance Observer.

Abstract: In this article, a novel disturbance observer-based adaptive neural control (ANC) scheme is proposed for full-state-constrained pure-feedback nonlinear systems using a new system transformation method. A nonlinear transformation function in a uniformed design framework is constructed to convert the original states with constrained bounds into the ones without any constraints. By combining an auxiliary first-order filter, an augmented nonlinear system without any state constraint is derived to circumvent the difficulty of the controller design caused by the nonaffine input signal. Based on the augmented nonlinear system, a nonlinear disturbance observer (NDO) is designed to enhance the disturbance rejection ability. Subsequently, the NDO-based ANC scheme is presented by combining the second-order filters with backstepping. The proposed scheme confines all states within the predefined bounds, eliminates the condition on both the known sign and bounds of control gains, improves the robustness of the closed-loop system, and alleviates the computational burden. Two simulation examples are performed to show the validity of the presented scheme.

Agrobacterium-Mediated Genetic Transformation of Wild Oryza Species Using Immature Embryos.

Abstract: Genetic transformation is one of the most important technologies for revealing or modulating gene function. It is used widely in both functional genomics and molecular breeding of rice. Demands on its use in wild Oryza species is increasing because of their high genetic diversity. Given the difficulties in genetic crosses between distantly related species, genetic transformation offers a way to alter or transfer genetic traits in wild rice accessions. However, transformation of wild Oryza accessions by conventional methods using calli induced from scutellum tissue of embryos in mature seeds often fails. Here, we report methods using immature embryos for the genetic transformation of a broad range of Oryza species. First, we investigated the ability of callus induction and regeneration from immature embryos of 192 accessions in 20 species under several culture conditions. We regenerated plants from immature embryos of 90 accessions in 16 species. Next, we optimized the conditions of Agrobacterium infection using a vector carrying the GFP gene driven by the maize ubiquitin promoter. GFP signals were observed in 51 accessions in 11 species. We analyzed the growth and seed set of transgenic plants of O. barthii, O. glumaepatula, O. rufipogon, and O. brachyantha. The plants grew to maturity and set seeds normally. Southern blot analyses using DNA from T0 plants showed that all GFP plants were derived from independent transformation events. We confirmed that the T-DNAs were transmitted to the next generation through the segregation of GFP signals in the T1 generation. These results show that many Oryza species can be transformed by using modified immature-embryo methods. This will accelerate the use of wild Oryza accessions in molecular genetic analyses and molecular breeding.

Transgenic Cotton (Gossypium hirsutum L.) to Combat Weed Vagaries: Utility of an Apical Meristem-Targeted in planta Transformation Strategy to Introgress a Modified CP4-EPSPS Gene for Glyphosate Tolerance.

Abstract: Weeds burden plant growth as they compete for space, sunlight, and soil nutrients leading to 25-80% yield losses. Glyphosate [N-(phosphonomethyl)glycine] is a widely used broad spectrum non-selective herbicide that controls weeds by inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme and interfering with the shikimate biosynthesis pathway. Cotton (Gossypium hirsutum L.) is one of the most important commercial crops grown worldwide for its fiber. We have developed herbicide tolerant transgenic cotton (cv. P8-6) by introgression of a codon-optimized and modified EPSPS gene (CP4-EPSPS) possessing an N-terminal chloroplast targeting peptide from Petunia hybrida. Because of the recalcitrant nature of cotton, a genotype-independent non-tissue culture-based apical meristem-targeted in planta transformation approach was used to develop transformants. Although in planta transformation methodologies are advantageous in developing a large number of transgenic plants, effective screening strategies are essential for initial identification of transformants. In the present study, the use of a two-level rigorous screening strategy identified 2.27% of T1 generation plants as tolerant to 800 and 1,500 mg/L of commercially available glyphosate (Roundup). Precise molecular characterization revealed stable integration, expression, and inheritance of CP4-EPSPS in advanced generations of the promising transgenic events. Further, superiority of selected transgenic plants in tolerating increasing levels of glyphosate (500-4,000 mg/L) was ascertained through reduced accumulation of shikimate. This report is the first of its kind where cotton transformants tolerating high levels of glyphosate (up to 4,000 mg/L) and accumulating low levels of shikimate have been identified. This study not only reiterated the genotype-independent nature of the transformation strategy but also reiterated the translational utility of the CP4-EPSPS gene in management of weeds.

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes.

Abstract: Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) and Wuschel2 (Wus2). Bbm is regulated by the maize phospholipid transferase gene (Pltp) promoter, and Wus2 is under the control of a maize auxin-inducible (Axig1) promoter. An Agrobacterium strain carrying these morphogenic genes on transfer DNA (T-DNA) and extra copies of Agrobacterium virulence (vir) genes are used to infect maize immature embryo explants. Somatic embryos form on the scutella of infected embryos and can be selected by herbicide resistance and germinated into plants. A heat-activated cre/loxP recombination system built into the DNA construct allows for removal of morphogenic genes from the maize genome during an early stage of the transformation process. Transformation frequencies of approximately 14%, 4%, and 4% (numbers of independent transgenic events per 100 infected embryos) can be achieved for W22, B73, and Mo17, respectively, using this protocol.

A detached leaf assay for testing transient gene expression and gene editing in cowpea (Vigna unguiculata [L.] Walp.).

Abstract: The legume cowpea (Vigna unguiculata L.) is extensively grown in sub-Saharan Africa. Cowpea, like many legumes has proved recalcitrant to plant transformation. A rapid transient leaf assay was developed for testing gene expression and editing constructs prior to stable cowpea transformation, to accelerate cowpea and legume crop improvement. Attempts to develop a transient protoplast system for cowpea were unsuccessful. Leaflets from plants 3–4 weeks post-germination were age selected to establish a rapid Agrobacterium (Agro) infiltration-mediated transient system for efficacy testing of gene expression and CRISPR/Cas9 gene editing constructs. In planta, Agro-infiltration of leaflets with fluorescent expression constructs, resulted in necrosis. By contrast, Agro-infiltration of detached leaflets with an Arabidopsis (At) ubiquitin3 promoter:ZsGreen construct, followed by culture on solid nutrient medium resulted in fluorescence in over 48% of leaf cells. Expression efficiency was leaf age-dependent. Three cowpea meiosis genes were identified for CRISPR/Cas9 gene-editing, with the forward aim of meiosis-knock out for asexual seed induction in cowpea. Constructs were designed and tested containing candidate gene-specific guide RNAs, expressed using either the cowpea or Arabidopsis U6 promoters with Cas9 expression directed by either the Arabidopsis 40S ribosomal protein or parsley ubiquitin4-2 promoters. Leaflets were infiltrated with test gene-editing constructs and analytical methods developed to identify gene-specific mutations. A construct that produced mutations predicted to induce functional knockout of in the VuSPO11-1 meiosis gene was tested for efficacy in primary transgenic cowpea plants using a previously established stable transformation protocol. Vuspo11-1 mutants were identified, that cytologically phenocopied spo11-1 mutants previously characterized in Arabidopsis, and rice. Importantly, a biallelic male and female sterile mutant was identified in primary transgenics, exhibiting the expected defects in 100% of examined male and female meiocytes. The transient, detached cowpea leaf assay, and supporting analytical methods developed, provide a rapid and reproducible means for testing gene expression constructs, and constructs for inducing mutagenesis in genes involved in both vegetative and reproductive developmental programs. The method and tested editing constructs and components have potential application for a range of crop legumes.

An efficient system for Agrobacterium-mediated transient transformation in Pinus tabuliformis

Abstract: Functional genomic studies using genetics approaches of conifers are hampered by the complex and enormous genome, long vegetative growth period, and exertion in genetic transformation. Thus, the research carried out on gene function in Pinus tabuliformis is typically performed by heterologous expression based on the model plant Arabidopsis. However, due to the evolutionary and vast diversification from non-flowering (gymnosperms) to flowering (angiosperms) plants, several key differences may alter the underlying genetic concerns and the analysis of variants. Therefore, it is essential to develop an efficient genetic transformation and gene function identification protocol for P. tabuliformis. In the present study we established a highly efficient transgene Agrobacterium-mediated transient expression system for P. tabuliformis. Using a β-glucuronidase gene (GUS) as a reporter gene expression, the highest transformation efficiency (70.1%) was obtained by co-cultivation with Agrobacterium strain GV3101 at an optical density at 600 nm of 0.8, with 150 μM acetosyringone for 30 min followed by 3 days in the dark at 23 ± 1 °C. This protocol would be applied to other conifers; GUS staining was observed 24 h post-infection. We report a simple, fast, and resilient system for transient Agrobacterium-mediated transformation high-level expression of target genes in P. tabuliformis, which will also improve transformation efficiency in other conifer species.

Adaptive Full-State-Constrained Control of Nonlinear Systems With Deferred Constraints Based on Nonbarrier Lyapunov Function Method

Abstract: In this article, the problem of tracking control is considered for a class of uncertain strict-feedback nonlinear systems with deferred asymmetric time-varying full-state constraints. A novel adaptive robust full-state-constrained control scheme is developed. First, by introducing a novel shifting function, the original constrained system with any initial values is modified to a new constrained system, and the initial values of the modified constrained system remain 0. Then, to remove the feasibility condition caused by the barrier Lyapunov functions, the modified constrained system is further transformed into a new unconstrained system by a brand new nonlinear transformation. Furthermore, the tracking error system of the unconstrained system is constructed by using a new coordinate transformation, and a novel adaptive full-state-constrained control scheme is designed based on this error system through the backstepping recursion method and first-order filters. Finally, the resulting closed-loop system proves to be stable and numerical simulations are conducted to demonstrate the effectiveness of the developed control strategy.