T1 Plants Research Articles

Physiological, biochemical and economic characteristics of transgenic winter wheat plants with RNA suppressor of the proline dehydrogenase gene

Aim. To analyze the physiological, biochemical and economic characteristics of genetically modified plants of new promising genotypes of winter bread wheat of seed generation T2 with a double-stranded RNA suppressor of the proline dehydrogenase gene. Methods. Agrobacterium-mediated transformation in vitro; biochemical determination of proline dehydrogenase enzyme activity and free proline content; morphometric indicators and elements of crop structure; mathematical statistics. Results. It is shown that transgenic plants, in contrast to control, grow on a medium with mannitol more intensely, retaining a green color. It was found that both under normal conditions and under conditions of water deficiency, plants of seed generation T2 have an increased level of free Proline in the leaves compared to control genotypes. It was found that transformants are characterized by reduced activity of the enzyme proline dehydrogenase, which is manifested by changes in normal – stress – normal conditions. Transgenic T2 plants had a higher tolerance to water deficiency compared to the original, which was reflected in the nature of their growth. In conditions of soil moisture deficiency, the yield of most transformed lines was higher compared to untransformed plants. Conclusions. The results suggest that the use of a vector construct with a double-stranded RNA suppressor of the ProDH gene is effective for creating transgenic winter bread wheat plants with increased tolerance to water deficiency.

Conversion of sheath blight susceptible indica and japonica rice cultivars into moderately resistant through expression of antifungal β-1,3-glucanase transgene from Trichoderma spp.

Rice is an important food crop for three billion people worldwide. The crop is vulnerable to several diseases. Sheath blight caused by fungal pathogen Rhizoctonia solani is a significant threat to rice cultivation accounting for up to 50% yield losses. The pathogen penetrates leaf blades and sheaths, leading to plant necrosis; and major disease resistance gene against the pathogen is not available. This study describes development of sheath blight resistant transgenic indica and japonica rice cultivars through introduction of antifungal β-1,3-glucanase transgene cloned from Trichoderma. The transgene integration and expression in transformed T0 rice plants was examined by PCR, RT-PCR, qRT-PCR demonstrating up to 5-fold higher expression as compared to non-transgenic plants. The bioassay of T0, T1 and homozygous T2 progeny plants with virulent R. solani isolate revealed that plants carrying high level of β-1,3-glucanase expression displayed moderately resistant reaction to the pathogen. The optical micrographs of leaf sheath cells from moderately resistant plant after pathogen inoculation displayed presence of a few hyphae with sparse branching; on the contrary, pathogen hyphae in susceptible non-transgenic plant cells were present in abundance with profuse hyphal branching and forming prominent infection cushions. The disease severity in T2 progeny plants was significantly less as compared to non-transgenic plants confirming role of β-1,3-glucanase in imparting resistance.

Life cycle assessment of ceramic roof tiles: A temporal perspective

The production of ceramic roof tiles is energy intensive, leading to high carbon dioxide emissions intensity. The aim of this study is to conduct a life cycle assessment study to evaluate the environmental profile of ceramic roof tiles produced in two Portuguese ceramic plants (T1 and T2) that use different technologies. This assessment is performed on a temporal basis (over a 10-year period) to evaluate the effect that ceramic processual changes have on environmental impacts. System boundaries include the whole life cycle—from extraction and acquisition of raw materials up to tile end of life. Environmental impacts were calculated for eight impact categories. The impact results show an overall improvement in the environmental performance of roof tiles produced in T1 and T2 for all impact categories over the 10-year period as a result of the continued introduction of technical and energy saving measures during the tile manufacturing. Overall, roof tile 2 shows the worst environmental profile for all impact categories, with the exceptions of acidification and photochemical oxidant formation, due to the use of refractory material in the firing process. These exceptions can be explained by the existence of a dry flue gas cleaning adsorber with a filter coupled at kilns in T2. • Ceramic roof tile LCA study with inventory data over a 10-year period enables an integrated timeframe perspective of roof tiles environmental burdens. • Overall, improvement in the environmental performance of the T1 and T2 plants for all impact categories over the 10-year period was observed. • Changes to production technology affect the potential environmental impacts of ceramic roof tiles. • Overall, the production stage (modules A1–A3) is the hotspot for all impact categories.

Response of Potted Citrus Trees Subjected to Water Deficit Irrigation with the Application of Superabsorbent Polyacrylamide Polymers

Searching for new strategies to mitigate the effects of low water availability for citrus production, a study was carried out on potted mandarin cv. W. Murcott, with the objective of evaluating the physiological and growth response of the plants to polyacrylamide gel application in the substrate in water restriction conditions. The following treatments were evaluated, T0 (control) with 100% ETc water replenishment, T1 with 50% ETc water replenishment, and T2 with 50% ETc water replenishment plus the application of polyacrylamide polymers to the substrate. Temperature and water volumetric content (ϴ: m3 m−3) were evaluated in the substrate. Plant water-status parameters such as stem water potential (SWP), stomatal conductance (gs), and chlorophyll fluorescence (Fv/Fm), as well as biomass, nutrients levels, and proline biosynthesis were measured in the plants in response to the treatments. The results showed that the substrate moisture for T2 was kept significantly higher than T0 and T1, despite receiving the same irrigation rate as T1 and a half of T0; however, this higher moisture availability in the substrate of T2 was not reflected in the plant’s water status or growth. On the contrary, the T2 plants showed responses such as lower total biomass, lower vegetative development, and lower root biomass, as well as a higher concentration of proline in the root. According to these results, it is concluded that polymers such as polyacrylamide sodium allow the retention of water in the substrate, but do not necessarily release that water for plants, probably because that moisture is kept in the hydrogel and not released to the substrate media or the roots, or if released, in this case, this occurs with an increase in the concentration of sodium available to the plants, which could lead the citrus crop to a worse situation of water and/or osmotic stress.

Increasing the level of resistant starch in 'Presidio' rice through multiplex CRISPR-Cas9 gene editing of starch branching enzyme genes.

Rice (Oryza sativa L.) is an excellent source of starch, which is composed of amylopectin and amylose. Resistant starch (RS) is a starch product that is not easily digestible and absorbed in the stomach or small intestine and instead is passed on directly to the large intestine. Cereals high in RS may be beneficial to improve human health and reduce the risk of diet-related chronic diseases. It has been reported through chemical mutagenesis and RNA interference studies that starch branching enzymes (SBEs) play a major role in contributing to higher levels of RS in cereal crops. In this study, we used multiplex clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated protein 9 (Cas9) genome editing to simultaneously target all four SBE genes in rice using the endogenous transfer RNA (tRNA)-processing system for expressing the single-guide RNAs (sgRNAs) targeting these genes. The CRISPR-Cas9 vector construct with four SBE gene sgRNAs was transformed into the U.S. rice cultivar Presidio using Agrobacterium-mediated transformation. Knockout mutations were identified at all four SBE genes across eight transgene-positive T0 plants. Transgene-free T1 lines with different combinations of disrupted SBE genes were identified, with several SBE-edited lines showing significantly increased RS content up to 15% higher than the wild-type (WT) cultivar Presidio. Although further efforts are needed to fix all of the mutant alleles as homozygous, our study demonstrated the potential of multiplex genome editing to develop high-RS lines.

CRISPR/Cas9-mediated saturated mutagenesis of the cotton MIR482 family for dissecting the functionality of individual members in disease response.

Many genes encoding nucleotide‐binding leucine‐rich repeat receptors (NLRs) are regulated and fine‐tuned by miR482 to balance the trade‐off between disease resistance and growth. Dicotyledonous plants, including cotton, usually have multiple miR482 isoforms. Each miR482 isoform can regulate several NLRs that in turn can be regulated by several different miR482 isoforms. Dissecting the functionality of individual miR482 isoforms in disease response and in balancing the disease resistance and growth trade‐off demands a collection of mutants mutated in individual miR482 members (single or multiple). In this study, we generated such a collection of cotton miR482 mutants using CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) genome editing and transformation of pooled guide RNAs (gRNAs). In total, 84 T0 plants representing 40 independent transgenic events and harboring mutation in each of the 10 miR482 isoforms were generated. The average editing efficiency of the 18 transformed gRNAs is 75%, ranging from 0 (3 gRNAs) to 100% (8 gRNAs). Most miR482 isoforms have a diverse range of mutations, including small indels (1–44 bp) and substitutions, which are expected to impair biogenesis of miR482. All nine mutant populations used in Verticillium dahliae infection experiments showed a disease index lower than the control, with four being significantly lower. The disease assay also suggests a different role of different miR482 isoforms in disease response and a potential dosage effect of miR482l. The study demonstrates the feasibility of saturation mutagenesis of plant miRNA families with dozens of genetic loci using CRISPR/Cas9 and provides the cotton community a valuable resource for uncovering the miR482‐NLR module(s) underlying the interaction between cotton and different pathogens.

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.

CRISPR/Cas9-mediated knockout of a prolyl-4-hydroxylase subfamily in Nicotiana benthamiana using DsRed2 for plant selection.

The properties of host plants used for molecular farming can be modified by CRISPR/Cas9 genome editing to improve the quality and yield of recombinant proteins. However, it is often necessary to target multiple genes simultaneously, particularly when using host plants with large and complex genomes. This is the case for Nicotiana benthamiana, an allotetraploid relative of tobacco frequently used for transient protein expression. A multiplex genome editing system incorporating the DsRed2 fluorescent marker for the identification and selection of transgenic plants was established. As proof of principle, NbP4H4 was targeted encoding a prolyl-4-hydroxylase involved in protein O-linked glycosylation. Using preselected gRNAs with efficiencies confirmed by transient expression, transgenic plant lines with knockout mutations in all four NbP4H4 genes were obtained. Leaf fluorescence was then used to screen for the absence of the SpCas9 transgene in T1 plants, and transgene-free lines with homozygous or biallelic mutations were identified. The analysis of plant-produced recombinant IgA1 as a reporter protein revealed changes in the numberof peptides containing hydroxyproline residues and pentoses in the knockout plants. The selection of efficient gRNAs combined with the DsRed2 marker reduces the effort needed to generate N. benthamiana mutants and simplifies the screening processes to obtain transgene-free progeny.

The Rice Qa-SNAREs in SYP13 Subfamily Are Involved in Regulating Arbuscular Mycorrhizal Symbiosis and Seed Fertility.

Qa-SNARE gene SYP132 (isoform α) was previously reported to affect arbuscular mycorrhizal (AM) symbiosis in the legume species Medicago truncatula. In non-legumes especially monocots, it remains unknown whether certain SNARE genes are also involved in AM symbiosis. In this work, we studied a rice orthologous gene OsSYP132, which showed induced expression in mycorrhizal roots and two paralogous genes OsSYP131a and OsSYP131b, which were not induced by the AM fungus Rhizophagus irregularis. After employing CRISPR/Cas9 technique to generate their mutants, the Ossyp131a homozygous mutant T0 plants exhibited a dwarf phenotype and produced no fertile seeds, indicating a required role of this gene in seed fertility. Unlike the case in legume, the Ossyp132 mutants exhibited normal mycorrhizal phenotype, so did the Ossyp131b mutants. In the Ossyp131b Ossyp132 double mutants, however, the colonization rate and arbuscule abundance level decreased markedly, indicating an impaired fungal proliferation ability in rice roots. Such a defect was further confirmed by the reduced expression levels of AM marker genes. Our results in rice therefore demonstrated that while SYP13II members showed evolutionary and induction patterns specific to symbiosis, AM symbiosis is in fact controlled by the combined action of both SYP13I and SYP13II clades, revealing a functional redundancy among SYNTAXIN genes in mutualism.

Targeted base editing in the mitochondrial genome of Arabidopsis thaliana

Beyond their well-known role in respiration, mitochondria of land plants contain biologically essential and/or agriculturally important genes whose function and regulation are not fully understood. Until recently, it has been difficult to analyze these genes or, in the case of crops, to improve their functions, due to a lack of methods for stably modifying plant mitochondrial genomes. In rice, rapeseed, and Arabidopsis thaliana, mitochondria-targeting transcription activator-like effector nucleases (mitoTALENs) have recently been used to disrupt targeted genes in an inheritable and stable manner. However, this technique can also induce large deletions around the targeted sites, as well as cause ectopic homologous recombinations, which can change the sequences and gene order of mitochondrial genomes. Here, we used mitochondria-targeting TALEN-based cytidine deaminase to successfully substitute targeted C:G pairs with T:A pairs in the mitochondrial genomes of plantlets of A. thaliana without causing deletions or changes in genome structure. Expression vectors of the base editor genes were stably introduced into the nuclear genome by the easy-to-use floral dipping method. Some T1 plants had apparent homoplasmic substitutions that were stably inherited by seed progenies, independently of the inheritance of nuclear-introduced genes. As a demonstration of the method, we used it to restore the growth of an organelle transcript processing 87 (otp87) mutant that is defective in the editing of RNA transcripts of the mitochondrial atp1 gene and to identify bases in atp1 that affect the efficiency of RNA editing by OTP87.

Production of Conjoined Transgenic and Edited Barley and Wheat Plants for Nud Genes Using the CRISPR/SpCas9 System.

The Nudum (Nud) gene controls the caryopsis type of cereal crops by regulating lipid biosynthetic pathways. Based on the HvNud sequence and its homologous gene sequences in wheat, a conserved sgRNA was designed to obtain the mutants from the barley variety “Vlamingh” and the wheat variety “Fielder” via Agrobacterium-mediated transformation. A total of 19 and 118 transgenic plants were obtained, and 11 and 61 mutant plants were identified in T0 transgenic plants in barley and wheat after PCR-RE detection, and the editing efficiencies of the targeted gene were 57.9 and 51.7% in barley and wheat, respectively. The grain shape of the barley mutants was naked. Five different combinations of mutations for wheat TaNud genes were identified in the T0 generation, and their homozygous-edited plants were obtained in the T1 generation. Interestingly, the conjoined plants in which one plant has different genotypes were first identified. The different tillers in an individual T0 plant showed independent transgenic or mutant events in both barley and wheat, and the different genotypes can stably inherit into T1 generation, indicating that the T0 transgenic plants were the conjoined type. In addition, we did not find any off-target mutations in both barley and wheat. A candidate method for detecting putative-edited wheat plants was suggested to avoid losing mutations in this investigation. This study provides not only materials for studying the function of the Nud gene in barley and wheat but also a system for detecting the mutants in wheat.

An Improved Agrobacterium-Mediated Transformation and Genome-Editing Method for Maize Inbred B104 Using a Ternary Vector System and Immature Embryos.

For maize genome-editing and bioengineering, genetic transformation of inbred genotypes is most desired due to the uniformity of genetic background in their progenies. However, most maize inbred lines are recalcitrant to tissue culture and transformation. A public, transformable maize inbred B104 has been widely used for genome editing in recent years. This is primarily due to its high degree of genetic similarity shared with B73, an inbred of the reference genome and parent of many breeding populations. Conventional B104 maize transformation protocol requires 16–22 weeks to produce rooted transgenic plants with an average of 4% transformation frequency (number of T0 plants per 100 infected embryos). In this Method paper, we describe an advanced B104 transformation protocol that requires only 7–10 weeks to generate transgenic plants with an average of 6.4% transformation frequency. Over 66% of transgenic plants carried CRISPR/Cas9-induced indel mutations on the target gene, demonstrating that this protocol can be used for genome editing applications. Following the detailed and stepwise procedure described here, this quick and simplified method using the Agrobacterium ternary vector system consisting of a T-DNA binary vector and a compatible helper plasmid can be readily transferable to interested researchers.

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.

Identification and Characterization of the MIKC-Type MADS-Box Gene Family in Brassica napus and Its Role in Floral Transition.

Increasing rapeseed yield has always been a primary goal of rapeseed research and breeding. However, flowering time is a prerequisite for stable rapeseed yield and determines its adaptability to ecological regions. MIKC-type MADS-box (MICK) genes are a class of transcription factors that are involved in various physiological and developmental processes in plants. To understand their role in floral transition-related pathways, a genome-wide screening was conducted with Brassica napus (B. napus), which revealed 172 members. Using previous data from a genome-wide association analysis of flowering traits, BnaSVP and BnaSEP1 were identified as candidate flowering genes. Therefore, we used the CRISPR/Cas9 system to verify the function of BnaSVP and BnaSEP1 in B. napus. T0 plants were edited efficiently at the BnaSVP and BnaSEP1 target sites to generate homozygous and heterozygous mutants with most mutations stably inherited by the next generation. Notably, the mutant only showed the early flowering phenotype when all homologous copies of BnaSVP were edited, indicating functional redundancy between homologous copies. However, no changes in flowering were observed in the BnaSEP1 mutant. Quantitative analysis of the pathway-related genes in the BnaSVP mutant revealed the upregulation of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and FLOWERING LOCUS T (FT) genes, which promoted early flowering in the mutant. In summary, our study created early flowering mutants, which provided valuable resources for early maturing breeding, and provided a new method for improving polyploid crops.

Overexpression of Choline Oxidase Gene in Three Filial Generations of Rice Transgenic Lines.

Glycinebetaine (GB) accumulation in many halophytic plants, animals, and microorganisms confers abiotic stress tolerance to salinity, drought, and extreme temperatures. Although there are a few genetic and biochemical pathways to synthesize GB, but isolation of a single gene Choline Oxidase (codA) from Arthrobacter spp. have opened a new hatch to engineer the susceptible plants. The effects of overexpressed codA gene, through multiple copy insertion and GB accumulation on salinity tolerance in rice were studied. Seed-derived embryogenic calli of 'Tarom Molaie' cultivar were targeted with two plasmids pChlCOD and pCytCOD both harboring the codA gene using the biolistic mediated transformation. The regenerated T0 plants were screened by PCR analysis. A line containing three copies of codA gene and harboring pChlCOD and pCytCOD was identified by Southern blot analysis. The expression of codA gene in this transgenic line was then confirmed by RT-PCR. The Mendelian segregation pattern of the inserted sequences was accomplished by the progeny test using PCR. The effects of overexpression of codA on salinity tolerance were evaluated at germination and seedling stage using T2-pChl transgenic line and control seeds in the presence of 0, 100, 200, and 300 mM NaCl. Finally, leaf growth dynamics of T2-pChlCOD transgenic line and control line under hydroponic conditions in the presence of 0, 40, 80, and 120 mM NaCl were assessed. The seed germination experiment results showed that the transformed seeds had a higher germination rate than the controls under all salinity treatments. But also, the leaf growth dynamics showed that the control plants had a more favorable leaf growth dynamic in all of the treatments. Although, the transgenic lines (T0, T1 and T2) exhibited lower performance than the wild type, the transgenic line varied for GB and choline contents and increasing codA gene copy number led to increased GB content. In a salinity sensitive crop such as rice, GB may not significantly contribute to the plant protection against salt stress. Also, insufficiency of choline resources as GB precursor might have affected the overall growth ability of the transgenic line and resulted in decreased leaf growth dynamics.

Targeted generation of Null Mutants in ZmGDIα confers resistance against maize rough dwarf disease without agronomic penalty.

Targeted generation of Null Mutants in ZmGDIα confers resistance against maize rough dwarf disease without agronomic penalty.

Applications of CRISPR/Cas9 as New Strategies for Short Breeding to Drought Gene in Rice.

Recent unpredictable climate change is the main reason for the decline in rice yield. In particular, drought stress is a major constraint in reducing yield and quality for rice at rainfed agriculture areas, such as Asia and South America. CRISPR/Cas9 provides an effective solution for gene function study and molecular breeding due to specific editing of targeted genome sequences. In addition, CRISPR/Cas9 application can significantly reduce the time required to develop new cultivars with improved traits compared to conventional complex and time-consuming breeding. Here, drought-induced gene Oryza sativa Senescence-associated protein (OsSAP) was edited by CRISPR/Cas9. To investigate the possible role of OsSAP in drought stress, genome-editing plants were subjected to drought stress until the soil moisture content reached 20%, and the reactive oxygen species (ROS) scavenging efficiency of genome-editing plants were decreased. When the genome-editing plants were subjected to drought stress, survival rate, shoot length, root length, content of chlorophyll number of tiller, and 1,000-grain weight decreased, and more H2O2 and O2− were detected in leaves. In addition, expression levels of several critical stress-related transcription factors were decreased in the OsSAP genome-editing plant. These results suggest that OsSAP function as a positive regulator during drought stress response in rice. We analyzed the expression of OsSAP and Cas9 in T0 and T1 plants as well as T2 seeds. As the course of generation advancement progressed, Cas9 expression remained stable or weakened but the OsSAP expression was continuously removed from the T0 plant. The coefficient of variation (CV) in both T1 plants and T2 seeds was lower than 5%. Overall, our results suggest that CRISPR/Cas9 could be a novel and important tool for efficiently generating specific and inheritable targeted genome editing in rice, with short breeding cycles.

The NAC Transcription Factor ANAC087 Induces Aerial Rosette Development and Leaf Senescence in Arabidopsis.

CmNACP1 mRNA has been shown to move long distance through the phloem in Cucurbita maxima (pumpkin) and through a graft junction. Whereas the phloem transport of several different mRNAs has been documented in other systems as well, its function remains, for most of these RNAs, largely unknown. To gain insight into the possible role of these RNAs, we searched for the closest homologs of CmNACP1 in Arabidopsis, a model plant much more amenable for analysis. A phylogenetic approach using the predicted NAC domain indicated that ANAC059, ANAC092, ANAC079, ANAC100, ANAC046, and ANAC087 form a single clade with CmNACP1. In the present work, we analyzed the possible function of the ANAC087 gene in more detail. The promoter region of this gene directed expression in the vasculature, and also in trichomes, stem, apexes, and developing flowers which supports the notion that ANAC087 and CmNACP1 are orthologs. Overexpression of the ANAC087 gene induced increased branching in inflorescence stem, and also development of ectopic or aerial rosettes in T1 and T2 plants. Furthermore, overexpression of ANAC087 leads to accelerated leaf senescence in 44 days post-germination (dpg). Interestingly, a similar phenotype was observed in plants expressing the ANAC087 gene upstream region, also showing an increase in ANAC087 transcript levels. Finally, the results shown in this work indicate a role for ANAC087 in leaf senescence and also in rosette development.

Efficient gene targeting in soybean using Ochrobactrum haywardense-mediated delivery of a marker-free donor template.

Gene targeting (GT) for precise gene insertion or swap into pre-defined genomic location has been a bottleneck for expedited soybean precision breeding. We report a robust selectable marker-free GT system in soybean, one of the most economically important crops. An efficient Oh H1-8 (Ochrobactrum haywardense H1-8)-mediated embryonic axis transformation method was used for the delivery of CRISPR-Cas9 components and donor template to regenerate T0 plants 6–8 weeks after transformation. This approach generated up to 3.4% targeted insertion of the donor sequence into the target locus in T0 plants, with ∼ 90% mutation rate observed at the genomic target site. The GT was demonstrated in two genomic sites using two different donor DNA templates without the need for a selectable marker within the template. High-resolution Southern-by-Sequencing analysis identified T1 plants with precise targeted insertion and without unintended plasmid DNA. Unlike previous low-frequency GT reports in soybean that involved particle bombardment–mediated delivery and extensive selection, the method described here is fast, efficient, reproducible, does not require a selectable marker within the donor DNA, and generates nonchimeric plants with heritable GT.

CRISPR/Cas9 gene editing and natural variation analysis demonstrate the potential for HvARE1 in improvement of nitrogen use efficiency in barley.

Nitrogen is a major determinant of grain yield and quality. As excessive use of nitrogen fertilizer leads to environmental pollution and high production costs, improving nitrogen use efficiency (NUE) is fundamental for a sustainable agriculture. Here, we dissected the role of the barley abnormal cytokinin response1 repressor 1 (HvARE1) gene, a candidate for involvement in NUE previously identified in a genome-wide association study, through natural variation analysis and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing. HvARE1 was predominantly expressed in leaves and shoots, with very low expression in roots under low nitrogen conditions. Agrobacterium-mediated genetic transformation of immature embryos (cv. Golden Promise) with single guide RNAs targeting HvARE1 generated 22 T0 plants, from which four T1 lines harbored missense and/or frameshift mutations based on genotyping. Mutant are1 lines exhibited an increase in plant height, tiller number, grain protein content, and yield. Moreover, we observed a 1.5- to 2.8-fold increase in total chlorophyll content in the flag leaf at the grain filling stage. Delayed senescence by 10-14 d was also observed in mutant lines. Barley are1 mutants had high nitrogen content in shoots under low nitrogen conditions. These findings demonstrate the potential of ARE1 in NUE improvement in barley.