Bialaphos Resistance Research Articles

Zinc oxide nanoparticles enhance plasmid transfer among growth-promoting endophytes in Arabidopsis thaliana

Nanoparticles (NPs) hold great promise for agricultural applications, yet their potential impact on exogenous gene transfer within plant remains poorly understood. In this study, we utilized the non-conjugative plasmid pCAMBIA1300, harboring the bialaphos resistance (bar) gene expressed in plant and the kanamycin resistance (kanR) gene as selectable marker in bacteria. Our results revealed a significant increase in the transfer of plasmid (via carrier Escherichia coli DH5α), both intra- and inter-species within the endophyte, when Arabidopsis thaliana was exposed to environmentally relevant level of zinc oxide (ZnO) NPs at a concentration of 0.7 μg/mL throughout its lifespan. Intriguingly, the plasmid exhibited selective transfer to growth-promoting endophytes, such as Enterobacter, Serratia, and Achromobacter, with the presence of ZnO NPs expanding the pool of potential recipients. This result is due to the facilitation of an endophytic and mutualistic lifestyle of invasive E. coli DH5α and the enrichment of beneficial bacteria aided by ZnO NPs. The plant's descendant generations did not express the bar gene, and the endophytes carrying the exogenous plasmid did not transmit it to sub sequent generation. This research provides crucial insights for assessing the potential risks associated with gene contamination and ensuring the safe and sustainable use of NPs in agriculture.

Identification and Expression Analysis of EPSPS and BAR Families in Cotton.

Weeds seriously affect the yield and quality of crops. Because manual weeding is time-consuming and laborious, the use of herbicides becomes an effective way to solve the harm caused by weeds in fields. Both 5-enolpyruvyl shikimate-3-phosphate synthetase (EPSPS) and acetyltransferase genes (bialaphos resistance, BAR) are widely used to improve crop resistance to herbicides. However, cotton, as the most important natural fiber crop, is not tolerant to herbicides in China, and the EPSPS and BAR family genes have not yet been characterized in cotton. Therefore, we explore the genes of these two families to provide candidate genes for the study of herbicide resistance mechanisms. In this study, 8, 8, 4, and 5 EPSPS genes and 6, 6, 5, and 5 BAR genes were identified in allotetraploid Gossypium hirsutum and Gossypium barbadense, diploid Gossypium arboreum and Gossypium raimondii, respectively. Members of the EPSPS and BAR families were classified into three subgroups based on the distribution of phylogenetic trees, conserved motifs, and gene structures. In addition, the promoter sequences of EPSPS and BAR family members included growth and development, stress, and hormone-related cis-elements. Based on the expression analysis, the family members showed tissue-specific expression and differed significantly in response to abiotic stresses. Finally, qRT-PCR analysis revealed that the expression levels of GhEPSPS3, GhEPSPS4, and GhBAR1 were significantly upregulated after exogenous spraying of herbicides. Overall, we characterized the EPSPS and BAR gene families of cotton at the genome-wide level, which will provide a basis for further studying the functions of EPSPS and BAR genes during growth and development and herbicide stress.

Establishment of a transformation system in close relatives of wheat under the assistance of TaWOX5

Species closely related to wheat are important genetic resources for agricultural production, functional genomics studies and wheat improvement. In this study, a wheat gene related to regeneration, TaWOX5, was applied to establish the Agrobacterium-mediated transformation systems of Triticum monococcum, hexaploid triticale, and rye (Secale cereale L.) using their immature embryos. Transgenic plants were efficiently generated. During the transformation process, the Agrobacterium infection efficiency was assessed by histochemical staining for β-glucuronidase (GUS). Finally, the transgenic nature of regenerated plants was verified by polymerase chain reaction (PCR)-based genotyping for the presence of the GUS and bialaphos resistance (bar) genes, histochemical staining for GUS protein, and the QuickStix strip assay for bar protein. The transformation efficiency of T. monococcum genotype PI428182 was 94.4%; the efficiencies of four hexaploid triticale genotypes Lin456, ZS3297, ZS1257, and ZS3224 were 52.1, 41.2, 19.4, and 16.0%, respectively; and the transformation efficiency of rye cultivar Lanzhou Heimai was 7.8%. Fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) analyses indicated that the GUS transgenes were integrated into the distal or near centromere (proximal) regions of the chromosomes in transgenic T. monococcum and hexaploid triticale plants. In the transgenic hexaploid triticale plants, the foreign DNA fragment was randomly integrated into the AABB and RR genomes. Furthermore, the transgene was almost stably inherited in the next generation by Mendel’s law. The findings in this study will promote the genetic improvement of the three plant species for grain or forage production and the improvement of cereal species including wheat for functional genomics studies.

Three novel alleles of OsGS1 developed by base-editing-mediated artificial evolution confer glufosinate tolerance in rice

Only few glufosinate-tolerant genes, such as phosphinothricin acetyltransferase (PAT) and bialaphos resistance (bar) identified from Streptomyces, are currently available for developing genetically modified rice in agricultural application. Following the rapid development of genome editing technology, generation of novel glufosinate-tolerant gene resources through artificial evolution of endogenous genes is more promising and highly desirable in rice molecular breeding program. In this study, the endogenous Glutamine synthetase1 (OsGS1) was artificially evolved by base-editing-mediated gene evolution (BEMGE) in rice cells to create novel alleles conferring glufosinate tolerance in rice germplasms. Two novel glufosinate-tolerant OsGS1 alleles (OsGS1-AVPS and OsGS1-+AF) and one reported tolerant allele (OsGS1-SGTA) were successfully identified from approximately 4200 independent hygromycin-tolerant calli. Germination assays and spray tests revealed that these three OsGS1 alleles confer glufosinate tolerance in rice. Furthermore, OsGS1-AVPS and OsGS1-SGTA were quickly deployed into the elite rice cultivar Nangeng 46 through precise base editing. Overall, our results demonstrate the feasibility of developing glufosinate-tolerant rice by editing an endogenous rice gene in molecular breeding programs.

Efficient and genotype independent maize transformation using pollen transfected by DNA-coated magnetic nanoparticles.

Current gene delivery methods for maize are limited to specific genotypes and depend on time-consuming and labor-intensive tissue culture techniques. Here, we report a new method to transfect maize that is culture-free and genotype independent. To enhance efficiency of DNA entry and maintain high pollen viability of 32%-55%, transfection was performed at cool temperature using pollen pretreated to open the germination aperture (40%-55%). Magnetic nanoparticles (MNPs) coated with DNA encoding either red fluorescent protein (RFP), β-glucuronidase gene (GUS), enhanced green fluorescent protein (EGFP) or bialaphos resistance (bar) was delivered into pollen grains, and female florets of maize inbred lines were pollinated. Red fluorescence was detected in 22% transfected pollen grains, and GUS stained 55% embryos at 18 d after pollination. Green fluorescence was detected in both silk filaments and immature kernels. The T1 generation of six inbred lines showed considerable EGFP or GUS transcripts (29%-74%) quantitated by polymerase chain reaction, and 5%-16% of the T1 seedlings showed immunologically active EGFP or GUS protein. Moreover, 1.41% of the bar transfected T1 plants were glufosinate resistant, and heritable bar gene was integrated into the maize genome effectively as verified by DNA hybridization. These results demonstrate that exogenous DNA could be delivered efficiently into elite maize inbred lines recalcitrant to tissue culture-mediated transformation and expressed normally through our genotype-independent pollen transfection system.

Optimized Transformation and Gene Editing of the B104 Public Maize Inbred by Improved Tissue Culture and Use of Morphogenic Regulators.

Plant transformation is a bottleneck for the application of gene editing in plants. In Zea mays (maize), a breakthrough was made using co-transformation of the morphogenic transcription factors BABY BOOM (BBM) and WUSCHEL (WUS) to induce somatic embryogenesis. Together with adapted tissue culture media, this was shown to increase transformation efficiency significantly. However, use of the method has not been reported widely, despite a clear need for increased transformation capacity in academic settings. Here, we explore use of the method for the public maize inbred B104 that is widely used for transformation by the research community. We find that only modifying tissue culture media already boosts transformation efficiency significantly and can reduce the time in tissue culture by 1 month. On average, production of independent transgenic plants per starting embryo increased from 1 to 4% using BIALAPHOS RESISTANCE (BAR) as a selection marker. In addition, we reconstructed the BBM-WUS morphogenic gene cassette and evaluated its functionality in B104. Expression of the morphogenic genes under tissue- and development stage-specific promoters led to direct somatic embryo formation on the scutellum of zygotic embryos. However, eight out of ten resulting transgenic plants showed pleiotropic developmental defects and were not fertile. This undesirable phenotype was positively correlated with the copy number of the morphogenic gene cassette. Use of constructs in which morphogenic genes are flanked by a developmentally controlled Cre/LoxP recombination system led to reduced T-DNA copy number and fertile T0 plants, while increasing transformation efficiency from 1 to 5% using HIGHLY-RESISTANT ACETOLACTATE SYNTHASE as a selection marker. Addition of a CRISPR/Cas9 module confirmed functionality for gene editing applications, as exemplified by editing the gene VIRESCENT YELLOW-LIKE (VYL) that can act as a visual marker for gene editing in maize. The constructs, methods, and insights produced in this work will be valuable to translate the use of BBM-WUS and other emerging morphogenic regulators (MRs) to other genotypes and crops.

Transformation of Teosinte (Zea mays ssp. parviglumis) via Biolistic Bombardment of Seedling-Derived Callus Tissues.

Modern maize exhibits a significantly different phenotype than its wild progenitor teosinte despite many genetic similarities. Of the many subspecies of Zea mays identified as teosinte, Zea mays ssp. parviglumis is the most closely related to domesticated maize. Understanding teosinte genes and their regulations can provide great insights into the maize domestication process and facilitate breeding for future crop improvement. However, a protocol of genetic transformation, which is essential for gene functional analyses, is not available in teosinte. In this study, we report the establishment of a robust callus induction and regeneration protocol using whorl segments of seedlings germinated from mature seeds of Zea parviglumis. We also report, for the first time, the production of fertile, transgenic teosinte plants using the particle bombardment. Using herbicide resistance genes such as mutant acetolactate synthase (Als) or bialaphos resistance (bar) as selectable markers, we achieved an average transformation frequency of 4.17% (percentage of independent transgenic events in total bombarded explants that produced callus). Expression of visual marker genes of red fluorescent protein tdTomato and β-glucuronidase (gus) could be detected in bombarded callus culture and in T1 and T2 progeny plants. The protocol established in this work provides a major enabling technology for research toward the understanding of this important plant in crop domestication.

Convergence of Bar and Cry1Ac Mutant Genes in Soybean Confers Synergistic Resistance to Herbicide and Lepidopteran Insects.

Soybean is a globally important crop species, which is subject to pressure by insects and weeds causing severe substantially reduce yield and quality. Despite the success of transgenic soybean in terms of Bacillus thuringiensis (Bt) and herbicide tolerance, unforeseen mitigated performances have still been inspected due to climate changes that favor the emergence of insect resistance. Therefore, there is a need to develop a biotech soybean with elaborated gene stacking to improve insect and herbicide tolerance in the field. In this study, new gene stacking soybean events, such as bialaphos resistance (bar) and pesticidal crystal protein (cry)1Ac mutant 2 (M#2), are being developed in Vietnamese soybean under field condition. Five transgenic plants were extensively studied in the herbicide effects, gene expression patterns, and insect mortality across generations. The increase in the expression of the bar gene by 100% in the leaves of putative transgenic plants was a determinant of herbicide tolerance. In an insect bioassay, the cry1Ac-M#2 protein tested yielded higher than expected larval mortality (86%), reflecting larval weight gain and weight of leaf consumed were less in the T1 generation. Similarly, in the field tests, the expression of cry1Ac-M#2 in the transgenic soybean lines was relatively stable from T0 to T3 generations that corresponded to a large reduction in the rate of leaves and pods damage caused by Lamprosema indicata and Helicoverpa armigera. The transgenic lines converged two genes, producing a soybean phenotype that was resistant to herbicide and lepidopteran insects. Furthermore, the expression of cry1Ac-M#2 was dominant in the T1 generation leading to the exhibit of better phenotypic traits. These results underscored the great potential of combining bar and cry1Ac mutation genes in transgenic soybean as pursuant of ensuring resistance to herbicide and lepidopteran insects.

Development of marker-free transgenic pigeon pea (Cajanus cajan) expressing a pod borer insecticidal protein

Pigeon pea, a grain legume of the semiarid tropics, is a rich source of high-quality protein. The productivity of this pulse is seriously affected by lepidopteron insect pests. To generate a sustainable insect-resistant plant, synthetically prepared bioactive key constituents of a crystal protein (Syn Cry1Ab) of Bacillus thuringiensis were expressed in pigeon pea under the guidance of a tissue-specific promoter of the RuBP carboxylase/oxygenase small subunit (rbcS) gene. Regenerated transgenic plants with the cry1Ab expression cassette (cry1Ab-lox-bar-lox) showed the optimum insect motility rate (90%) in an in vitro insect bioassay with second instar larvae, signifying the insecticidal potency of Syn Cry1Ab. In parallel, another plant line was also generated with a chimaeric vector harbouring a cre recombinase gene under the control of the CaMV 2 × 35S promoter. Crossing between T1 plants with a single insertion of cry1Ab-lox-bar-lox T-DNA and T1 plants with moderate expression of a cre gene with a linked hygromycin resistance (hptII) gene was performed to exclude the bialaphos resistance (bar) marker gene. Excision of the bar gene was achieved in T1F1 hybrids, with up to 35.71% recombination frequency. Insect-resistant pigeon pea plants devoid of selectable marker genes (syn Cry1Ab- bar and cre-hptII) were established in a consecutive generation (T1F2) through genetic segregation.

TALEN-mediated bar-knockout Rice Production and Transcriptome Profiling

Gene editing technologies such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) systems have been developed to create targeted DNA mutagenesis in many crop plants. This report describes application of the TALEN system to generate bialaphos resistance (bar)-knockout null segregants in herbicide-tolerant rice (Ba15) and microarray analysis on transcriptome changes of mutated lines, to identify unexpected effects resulting from off-targets. We generated 41 T0 plants and identified TALEN-mediated bar sequence mutations in 14 of them. Non-target site single nucleotide polymorphisms (SNPs) and small insertion/deletions (InDels) accounted for a large proportion of the mutations. Segregations of phosphinothricin acetyltransferase (PAT) protein expression levels were observed in T1 generations of two lines, R6 and R9. In addition, most T1 offspring harbored the TALE-R expression cassette and acquired some de novo mutations that were not inherited from their T0 parents. Three bar-knockout T1 lines were tested for PAT protein expression in progeny seedlings, and their T2 plants possessed inactive bar. We selected three bar-knockout T2 plants that were TALE-DNA-free for microarray analysis, aiming to understand the transcriptome differences between mutated null segregants and their recipient line. Only 31 differentially expressed genes (DEGs) were identified in the bar-knockout rice lines, possibly resulting from somaclonal variations from the in vitro cell culture process. Taken together, TALEN-mediated bar mutations have little effect on the whole transcriptome profile of rice. We believe our results will be helpful to study unexpected consequences in gene-edited crops.

Virus-Mediated Transient Expression Techniques Enable Gene Function Studies in Black-Grass.

Even though considerable progress has been made in weed ecology, weed molecular biology has been hindered by an inability to genetically manipulate weeds. Genetic manipulation is essential to demonstrate a causative relationship between genotype and phenotype. Herein we demonstrate that virus-mediated transient expression techniques developed for other monocots can be used in black-grass (Alopecurus myosuroides) for loss- and gain-of-function studies. We not only use virus induced gene silencing (VIGS) to create the black-grass exhibiting reduced PHYTOENE DESATURASE expression and virus-mediated overexpression (VOX) to drive GREEN FLUORESCENT PROTEIN, we demonstrate these techniques are applicable to testing hypotheses related to herbicide resistance in black-grass. We use VIGS to demonstrate that AmGSTF1 is necessary for the resistant biotype Peldon to survive fenoxaprop application and show the heterologous expression of the bialaphos resistance gene with VOX is sufficient to confer resistance to an otherwise lethal dose of glufosinate. Black-grass is the most problematic weed for winter-cereal farmers in the UK and Western Europe as it has rapidly evolved adaptions that allow it to effectively avoid current integrated weed management practices. Black-grass also reduces yields and therefore directly threatens food security and productivity. Novel disruptive technologies which mitigate resistance evolution and enable better control over this pernicious weed are therefore required. These virus-mediated protocols offer a step change in our ability to alter genes of interest under controlled laboratory conditions and therefore to gain a molecular-level understanding of how black-grass can survive in the agri-environment.

Induction of competent cells for Agrobacterium tumefaciens-mediated stable transformation of common bean (Phaseolus vulgaris L.).

Stable transformation of common bean (Phaseolus vulgaris L.) has been successful, to date, only using biolistic-mediated transformation and shoot regeneration from meristem-containing embryo axes. In this study, using precultured embryo axes, and optimal co-cultivation conditions resulted in a successful transformation of the common bean cultivar Olathe using Agrobacterium tumefaciens strain EHA105. Plant regeneration through somatic embryogenesis was attained through the preculture of embryo axes for 12 weeks using induced competent cells for A. tumefaciens-mediated gene delivery. Using A. tumefaciens at a low optical density (OD) of 0.1 at a wavelength of 600 nm for infection and 4-day co-cultivation, compared to OD600 of 0.5, increased the survival rate of the inoculated explants from 23% to 45%. Selection using 0.5 mg L-1 glufosinate (GS) was effective to identify transformed cells when the bialaphos resistance (bar) gene under the constitutive 35S promoter was used as a selectable marker. After an 18-week selection period, 1.5% -2.5% inoculated explants, in three experiments with a total of 600 explants, produced GS-resistant plants through somatic embryogenesis. The expression of bar was confirmed in first- and second-generation seedlings of the two lines through reverse polymerase chain reaction. Presence of the bar gene was verified through genome sequencing of two selected transgenic lines. The induction of regenerable, competent cells is key for the successful transformation, and the protocols described may be useful for future transformation of additional Phaseolus germplasm.

Effects of Nonstarch Genetic Modifications on Starch Structure and Properties.

This paper examines if, in maize, starch structure and starch-dependent properties might be altered by pleiotropic effects arising from genetic modifications that are not directly related to starch synthesis. The molecular structure, specifically the starch chain-length distributions (CLDs), of two maize lines transformed with Bar (bialaphos resistance) and Cry1c genes (an artificial gene, encoding proteinaceous insecticidal δ-endotoxins) were compared to those of their control lines. The two transgenes are responsible for herbicidal resistance and insect tolerance, respectively. The starch CLDs were measured by enzymatic debranching and measuring the molecular weight distributions of the resulting linear chains. It was found that although all the lines had similar amylose contents, the CLDs of both amylopectin and amylose for Cry1c were noticeably different from the others, having more short amylopectin and long amylose chains. These CLDs are known to affect functional properties, and indeed it was found that the Cry1c transgenic lines showed a lower gelatinization temperature and faster digestion rate than the control or Bar lines. However, a slower digestion rate is nutritionally desirable. Thus, pleiotropic effects from genetic modifications can indirectly but significantly affect the starch synthesis pathway and thus change functional properties of significance for human health.

Genetic Transformation of Common Wheat (Triticum aestivum L.) Using Biolistics.

The following protocol describes the genetic transformation of wheat using the BioRad PDS/1000-He particle delivery system. Immature embryos are isolated 12-16days post-anthesis, the embryonic axis is removed, and the immature scutella are precultured for 1-2days prior to particle bombardment. Gold particles are coated with plasmid DNA containing the gene(s) of interest plus a selectable marker gene, in this instance bar (bialaphos resistance), and are fired into the cells to deliver the DNA. Subsequent tissue culture and regeneration steps allow recovery of plantlets, assisted by the inclusion of PPT (phosphinothricin tripeptide), the active ingredient of glufosinate-ammonium containing herbicides, to help select transformants. This updated method introduces selection earlier in the regeneration process which provides a shortened protocol while maintaining high transformation efficiencies.

Protein expression and gene editing in monocots using foxtail mosaic virus vectors.

Plant viruses can be engineered to carry sequences that direct silencing of target host genes, expression of heterologous proteins, or editing of host genes. A set of foxtail mosaic virus (FoMV) vectors was developed that can be used for transient gene expression and single guide RNA delivery for Cas9‐mediated gene editing in maize, Setaria viridis, and Nicotiana benthamiana. This was accomplished by duplicating the FoMV capsid protein subgenomic promoter, abolishing the unnecessary open reading frame 5A, and inserting a cloning site containing unique restriction endonuclease cleavage sites immediately after the duplicated promoter. The modified FoMV vectors transiently expressed green fluorescent protein (GFP) and bialaphos resistance (BAR) protein in leaves of systemically infected maize seedlings. GFP was detected in epidermal and mesophyll cells by epifluorescence microscopy, and expression was confirmed by Western blot analyses. Plants infected with FoMV carrying the bar gene were temporarily protected from a glufosinate herbicide, and expression was confirmed using a rapid antibody‐based BAR strip test. Expression of these proteins was stabilized by nucleotide substitutions in the sequence of the duplicated promoter region. Single guide RNAs expressed from the duplicated promoter mediated edits in the N. benthamiana Phytoene desaturase gene, the S. viridis Carbonic anhydrase 2 gene, and the maize HKT1 gene encoding a potassium transporter. The efficiency of editing was enhanced in the presence of synergistic viruses and a viral silencing suppressor. This work expands the utility of FoMV for virus‐induced gene silencing (VIGS), virus‐mediated overexpression (VOX), and virus‐enabled gene editing (VEdGE) in monocots.

Marker-free lines of phytase-transgenic Brassica napus show enhanced ability to utilize phytate

Through regeneration of 30 selected Brassica napus cultivars a non-genotype-specific and highly efficient regeneration system was established using two-step tissue culture. Plant regeneration frequencies from explants of cotyledons with petioles were 75–100% in different cultivars. Co-transformation was used to transfer exogenous genes with two expression vectors. LBA4404 Agrobacterium strains harboring the pBINphy (containing the phytase gene) and pBINBar (containing the selectable bialaphos resistance marker gene bar) were mixed at an equal ratio (1:1) and used to coinfect B. napus. A total of 10 transgenic B. napus lines expressing the bar and phytase genes were obtained, with a phytase/bar cointegration frequency of 50% in T0 plants. B. napus doubled haploid (DH) lines were obtained by microspore culture. Among these lines, a total of 14 phytase-transgenic DH B. napus lines free of selectable markers were obtained based on PCR screening. Western blotting showed that the phytase protein was normally expressed in the transgenic B. napus seeds. Both hydroponic and medium experiments showed that phytase-transgenic B. napus, but not nontransgenic control, could grow normally by utilizing phytate as the sole phosphorus (P) source. The phytase activity in the seeds of transgenic B. napus was approximately 3–20-fold higher than that in negative control seeds. Furthermore, the inorganic P content in the seeds of transgenic B. napus was also 2–6-fold higher than that in nontransgenic B. napus recipient seeds. Thus, the phytase-transgenic B. napus lines were developed that can utilize organic P and exhibit improved P uptake and utilization. The selectable marker free lines of phytase transgenic B. napus which can express and secrete phytase, grow normally by utilizing phytate as the sole phosphorus (P) source.

A sugarcane mosaic virusvector for gene expression in maize.

Zea mays L. ssp. mays (maize) is an important crop plant as well as model system for genetics and plant biology. The ability to select among different virus‐based platforms for transient gene silencing or protein expression experiments is expected to facilitate studies of gene function in maize and complement experiments with stable transgenes. Here, we describe the development of a sugarcane mosaic virus (SCMV) vector for the purpose of protein expression in maize. An infectious SCMV cDNA clone was constructed, and heterologous genetic elements were placed between the protein 1 (P1) and helper component‐proteinase (HC‐Pro) cistrons in the SCMV genome. Recombinant SCMV clones engineered to express green fluorescent protein (GFP), β‐glucuronidase (GUS), or bialaphos resistance (BAR) protein were introduced into sweet corn (Golden × Bantam) plants. Documentation of developmental time courses spanning maize growth from seedling to tasseling showed that the SCMV genome tolerates insertion of foreign sequences of at least 1,809 nucleotides at the P1/HC‐Pro junction. Analysis of insert stability showed that the integrity of GFP and BAR coding sequences was maintained longer than that of the much larger GUS coding sequence. The SCMV isolate from which the expression vector is derived is able to infect several important maize inbred lines, suggesting that this SCMV vector has potential to be a valuable tool for gene functional analysis in a broad range of experimentally important maize genotypes.

Establishment of an efficient alfalfa regeneration system via organogenesis and the co-expression of Arabidopsis SOS genes improves salt tolerance in transgenic alfalfa (Medicago sativa L.)

The Salt Overly Sensitive (SOS) signal transduction pathway is pivotal in Na+ efflux and facilitates ion transport and homeostasis for improved salt tolerance in plants. Ten alfalfa varieties were used as experimental materials and two alfalfa regeneration systems were established and optimized. Cotyledons and hypocotyls were initially used as explants to induce embryogenic callus via the indirect production of somatic embryos to establish a callus acceptor system. Cotyledonary nodes were used as explants to induce adventitious bud formation via direct organogenesis, thereby establishing an in vitro regeneration system that could be used for the genetic transformation. Agrobacterium-mediated transformation of the cotyledonary nodes of the alfalfa ‘Golden Empress b’ was used to generate 25 independent sources of transformed plants exhibiting herbicide tolerance. Four of the positive transgenic plants were randomly selected for southern blot analysis, and three hybridization signals with one or two copies were detected. Reverse transcription polymerase chain reaction showed that the Bialaphos resistance (Bar) and SOS1 genes were expressed in transgenic plants and that multiple exogenous salt-tolerant genes were integrated into the transgenic plant genome and expressed at the transcriptional level. The overexpression of Arabidopsis SOS genes in alfalfa conferred a high degree of salinity tolerance, enhanced plant growth, lowered the accumulation of Na+, increased the accumulation of K+ in the leaves, and altered physiological and biochemical parameters in response to salt stress.

Genetic transformation of einkorn (Triticum monococcum L. ssp. monococcum L.), a diploid cultivated wheat species

BackgroundDomesticated einkorn (Triticum monococcum L.) is one of the oldest cultivated cereal crops in the world. Its small genome size (~ 5.7 GB), low ploidy (2n = 2x = 14, AmAm) and high genetic polymorphism make this species very attractive for use as a diploid model for understanding the genomics and proteomics of Triticeae. Einkorn, however, is still a recalcitrant monocotyledonous species for the application of modern biotechnologies, including transgenesis. This paper reports the factors that may influence transgene delivery, integration, expression and inheritance in einkorn.ResultsIn this study, we report the successful genetic transformation of einkorn using biolistic-mediated DNA delivery. Immature embryo-derived tissues of spring einkorn were bombarded with a plasmid containing the reporter gene GFP (green fluorescent protein) driven by the rice actin promoter (act1) and the selectable bar gene (bialaphos resistance gene) driven by the maize ubiquitin promoter (ubi1). Adjustments to various parameters such as gas pressure, microcarrier size and developmental stage of target tissue were essential for successful transient and stable transformation. Bombarded einkorn tissues are recalcitrant to regenerating plants, but certain modifications of the culture medium have been shown to increase the production of transgenic events. In various experiments, independent transgenic plants were produced at frequencies ranging from 0.0 to 0.6%. Molecular analysis, marker gene expression and herbicide treatment demonstrated that gfp/bar genes were stably integrated into the einkorn genome and successfully inherited over several generations. The transgenes, as dominant loci, segregated in both Mendelian and non-Mendelian fashion due to multiple insertions. Fertile homozygous T1-T2 populations of transgenic einkorn that are resistant to herbicides were selected.ConclusionTo the best of our knowledge, this is the first report of the production of genetically modified einkorn plants. We believe that the results of our research could be a starting point for the application of the current biotechnological-based technologies, such as transgenesis and genome editing, to accelerate comparative functional genomics in einkorn and other cereals.

Non-specific activities of the major herbicide-resistance gene BAR.

Bialaphos resistance (BAR) and phosphinothricin acetyltransferase (PAT) genes, which convey resistance to the broad-spectrum herbicide phosphinothricin (also known as glufosinate) via N-acetylation, have been globally used in basic plant research and genetically engineered crops 1-4 . Although early in vitro enzyme assays showed that recombinant BAR and PAT exhibit substrate preference toward phosphinothricin over the 20 proteinogenic amino acids 1 , indirect effects of BAR-containing transgenes in planta, including modified amino acid levels, have been seen but without the identification of their direct causes 5,6 . Combining metabolomics, plant genetics and biochemical approaches, we show that transgenic BAR indeed converts two plant endogenous amino acids, aminoadipate and tryptophan, to their respective N-acetylated products in several plant species. We report the crystal structures of BAR, and further delineate structural basis for its substrate selectivity and catalytic mechanism. Through structure-guided protein engineering, we generated several BAR variants that display significantly reduced non-specific activities compared with its wild-type counterpart in vivo. The transgenic expression of enzymes can result in unintended off-target metabolism arising from enzyme promiscuity. Understanding such phenomena at the mechanistic level can facilitate the design of maximally insulated systems featuring heterologously expressed enzymes.