Explore the vast range of titles available.

Background Genome editing by CRISPR/Cas9 has become a popular approach to induce targeted mutations for crop trait improvement. Soybean ( Glycine max L. Merr.) is an economically important crop worldwide. Although gene editing has been demonstrated in soybean, its utilization in stably transformed plants through whole plant regeneration is still not widespread, largely due to difficulties with transformation or low mutation efficiencies. Results We sought to establish a simple, efficient, and specific CRISPR/Cas9 system to induce heritable mutations in soybean through stable transformation. We targeted phytoene desaturase (PDS) genes due to the distinctive dwarf and albino phenotypes of the loss of function mutant. To evaluate gene editing efficiency and specificity, three constructs targeting each of the two homologous soybean PDS genes specifically, as well as two constructs targeting both simultaneously with one guide RNA were created. Instead of using cotyledonary nodes from germinated seedlings, we used ‘half-seed’ explants derived from imbibed seeds for Agrobacterium -mediated transformation of cultivar Williams 82. Transformed plants for all five constructs were recovered. Dwarf and albino phenotypes were observed in transgenic plants harboring the constructs targeting both PDS genes. Gene editing at the desired loci was detected in the majority of T0 transgenic plants, with 75–100% mutation efficiencies. Indel frequencies varied widely among plants (3–100%), with those exhibiting visible mutant phenotypes showing higher frequencies (27–100%). Deletion was the predominant mutation type, although 1-nucleotide insertion was also observed. Constructs designed to target only one PDS gene did not induce mutation in the other homologous counterpart; and no mutation at several potential off-target loci was detected, indicating high editing specificity. Modifications in both PDS genes were transmitted to T1 progenies, including plants that were negative for transgene detection. Strong mutant phenotypes were also observed in T1 plants. Conclusions Using simple constructs containing one guide RNA, we demonstrated efficient and specific CRISPR/Cas9-mediated mutagenesis in stably transformed soybean plants, and showed that the mutations could be inherited in progenies, even in plants that lost transgenes through segregation. The established system can be employed to edit other genes for soybean trait improvement.

Background The type II clustered, regularly interspaced, short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 (Cas9) system is a novel molecular tool for site-specific genome modification. The CRISPR-Cas9 system was recently introduced into plants by transient or stable transformation. Findings Here, we report gene targeting in rice via the Agrobacterium tumefaciens -mediated CRISPR-Cas9 system. Three 20-nt CRISPR RNAs were designed to pair with diverse sites followed by the protospacer adjacent motif (PAM) of the rice herbicide resistance gene BEL . After integrating the single-guide RNA (sgRNA) and Cas9 cassette in a single binary vector, transgenic rice plants harboring sgRNA:Cas9 were generated by A. tumefaciens -mediated stable transformation. By analyzing the targeting site on the genome of corresponding transgenic plants, the mutations were determined. The mutagenesis efficiency was varied from ~2% to ~16%. Furthermore, phenotypic analysis revealed that the biallelic mutated transgenic plant was sensitive to bentazon. Conclusions Our results indicate that the agricultural trait could be purposely modified by sgRNA:Cas9-induced gene targeting. CRISPR-Cas9 system could be exploited as a powerful tool for trait improvements in crop breeding.

This manuscript describes a unique protocol for the rapid transformation of Medicago truncatula A17 cell suspension cultures mediated by Agrobacterium tumefaciens. Medicago cells were collected on day 7 of the growth curve, which corresponded to the beginning of the exponential phase. They were then co-cultured with Agrobacterium for 3 days before being spread onto a petri dish with appropriate antibiotic selection. The Receptor Binding Domain of the Spike protein of SARS-CoV-2 was used as a model to develop this protocol. The presence of the transgene was assessed using PCR, and the integrity of the product was evaluated by SDS-PAGE and Western-blotting.Key messageWe have developed an effective and simple protocol for the transformation of Medicago truncatula A17. The success of this protocol relies on proper handling of the cultures and is based solely on co-cultivating plant cells with Agrobacterium. This method does not require experienced professionals or specialized equipment, making it faster, easier, and more affordable than other available protocols. Additionally, there is no need for protoplast preparation or vacuum application.

represents five fast-growing tropical tree species in the Cannabaceae and is the only plant lineage besides legumes that can establish nitrogen-fixing nodules with rhizobium. Comparative analyses between legumes and allows identification of conserved genetic networks controlling this symbiosis. However, such studies are hampered due to the absence of powerful reverse genetic tools for . Here, we present a fast and efficient protocol for -mediated transformation and CRISPR/Cas9 mutagenesis of . Using this protocol, knockout mutants are obtained within 3 months. Due to efficient micro-propagation, bi-allelic mutants can be studied in the T generation, allowing phenotypic evaluation within 6 months after transformation. We mutated four genes - , and - that control cytokinin, ethylene, or strigolactone hormonal networks and that in legumes commit essential symbiotic functions. Knockout mutants in and displayed developmental phenotypes, namely reduced procambium activity in and disturbed sex differentiation in mutants. The symbiotic phenotypes of and mutant lines differ from those in legumes. In contrast, and are essential for nodule formation, a phenotype similar as reported for legumes. This indicates a conserved role for these GRAS-type transcriptional regulators in rhizobium symbiosis, illustrating the value of trees as a research model for reverse genetic studies.

Carnations ( Dianthus caryophyllus L.) are amongst the three most commercially valuable cut flowers worldwide. However, traditional breeding methods are often time-consuming and labor-intensive. Although genome editing is used as an alternative method for creating new varieties, the high heterozygosity of carnations inhibits the ability to maintain varietal characteristics in null segregants except for target-derived traits. The use of homozygous lines is a possible solution. Therefore, this study aimed to establish regeneration, transformation, and genome editing methods using seed-carnation varieties. The effects of four auxins (indole-3-butyric acid, IBA; a-naphthaleneacetic acid, NAA; 2,4-dichlorophenoxyacetic acid, 2,4-D; and 3-indoleacetic acid, IAA) and five cytokinins (6-benzyladenine, BA; thidiazuron, TDZ; kinetin, KT; zeatin, ZT; and N 6 -2-isopentenyl adenine, 2IP) on callus and shoot induction were evaluated. The combination of 0.05 mg/l 2,4-D and 4 mg/l TDZ had the highest shoot formation rate at 28%. In addition, shoot hyperhydricity was reduced by increasing the size of culture vessels. Sucrose, agar, and AgNO 3 concentrations, as well as pH, were optimized to facilitate regeneration. Hygromycin at 12.5 mg/l was subsequently used as the selection agent after Agrobacterium -mediated transformation. Finally, the phytoene desaturase gene was knocked out using the CRISPR/Cas9 system. The obtained albino shoot had a one-base deletion or two-base insertion in the genome sequence. To our knowledge, this is the first study to establish a system for genome editing of callus-derived shoots from a homozygous seed-propagated carnation, which may contribute to the rapid breeding of the new varieties. Key message This is the first report to establish regeneration, transformation, and genome editing methods using homozygous seed-carnation cultivars to maintain varietal characteristics in null segregants.

Key message This study mined a gene, HaNAC146 , holds promise as a valuable candidate gene for developing crops with improved stress tolerance and high production potential. NAC (NAM/ATAF/CUC) is one of the largest transcription factor families. They play important roles in regulating plant development, aging, morphogenesis, as well as biotic and abiotic stress. There is a delicate balance between stress resistance and plant growth and development. To date, few genes have been identified in crops that can simultaneously enhance resistance and increase production. Sunflower, as a pioneering crop in saline-alkali soils, exhibit a certain level of tolerance to drought, barren, and saline-alkali stress. In this study, we identified a transcription factor gene, HaNAC146 , which can improve both the growth and abiotic stress tolerance in transgenic Arabidopsis thaliana . Our main findings indicated that HaNAC146 is induced in sunflower by various abiotic stress and some plant hormones. It is localized in the nucleus and has transcriptional activation activity. HaNAC146 can promote growth, and increase seed production by enhancing photosynthesis in transgenic Arabidopsis . Utilizing a transient transformation system in sunflower and a stable transformation platform in Arabidopsis , we demonstrated that HaNAC146 can enhance the resistance of both sunflower seedlings and Arabidopsis to salt and drought stress. This enhancement is achieved through multiple pathways, including increasing antioxidant capacity, accumulating osmotic modulating substances, improving photosynthetic efficiency, activating the expression of downstream stress-responsive genes and promoting stomatal closure with plant sensitivity to abscisic acid (ABA). These results also indicated that robust growth is a key factor in plant resistance to abiotic stress. This unique stress-responsive transcription factor, HaNAC146 , holds promise as a valuable candidate gene for developing crops with improved stress tolerance and high production potential.

Ulva prolifera is a fast-growing green seaweed that has garnered considerable interest in both fundamental and applied research. Here, we established a molecular tool by employing a selectable marker gene that allowed the isolation of U. prolifera cells integrating exogenous DNA. We developed a modular plasmid for expressing exogenous genes in U. prolifera based on the bacterial antibiotic-resistance marker, aminoglycoside phosphotransferase gene ( aph7” ). Integration of aph7” in macroalgae can generate transformants resistant to hygromycin B. In addition, we characterized the promoter region of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase gene (pUpRbcS) to drive the expression of aph7” . The transcripts were consistently confirmed from antibiotic-selected transformants, stably retaining the exogenous gene in the succeeding generations. Subsequently, a CRISPR-based knock-in system was established, facilitating the integration of aph7” cassette in the endogenous selection gene encoding for adenine phosphoribosyltransferase ( UpAPT ). APT gene can serve as an endogenous marker in algae that exhibits a lethal phenotype under cultivation with 2-fluoroadenine. The resulting knock-in mutants could resist the co-selection of the antibiotic hygromycin B and 2-fluoroadenine. Our results advance U. prolifera as a genetic platform, enabling functional research to elucidate Ulva biology, and to bring forth biotechnological utilization of algal resources.

Premise Stable transformation is the biggest barrier to studying gene function in plants. In most species, transformation requires tissue culture and regeneration methods that may be arduous and cause undesirable genetic changes. Floral dip methods bypass these challenges by directly transforming ovules of developing flowers, but these methods have limited success outside of the Brassicaceae. Methods and Results We demonstrate that floral dip methods are effective for some genotypes of the yellow monkeyflower, Mimulus guttatus, an ecological and evolutionary model system. In genotypes where floral dip failed, we developed an effective floral bud injection method that allows plants to be infiltrated multiple times and reduces floral abscission and male sterility. Conclusions Through a combination of floral dip and injection methods, we transformed both coastal perennial and inland annual genotypes of M. guttatus, setting the stage for understanding the molecular genetic underpinnings of local adaptation to the divergent habitats occupied by these distinctive ecotypes.

Key message This study described the biosynthesis of 4-hydroxydihydrocinnamaldehyde sharing with monolignol pathway and supplemented the biosynthesis of colchicine in G. superba , 4-hydroxydihydrocinnamaldehyde produced in tobacco BY2 cells provided an important stepstone. The precursor, 4-hydroxydihydrocinnamaldehyde (4-HDCA), participates in the biosynthesis of the carbon skeleton of colchicine, which is derived from L-phenylalanine. However, one hypothesis proposed that 4-HDCA is synthesized by sharing the early part of the monolignol pathway in G. superba . In this study, we validated this prediction and identified the enzymatic functions involved in this pathway. Gs DBR1 is a crucial enzyme to illustrate 4-HDCA diverging from monolignol pathway, we first confirmed its reductase activity on 4-coumaraldehyde, an important intermediate compound in monolignol biosynthesis. Then, the biochemical function of recombinant enzymes belonging to the other four families were verified to elucidate the entire process of 4-HDCA biosynthesis from L-phenylalanine . After reconstruction, the 4-HDCA was 78.4 ng/g with fresh weight (FW) of transgenic tobacco cells, and the yield increased to 168.22 ng/g·FW after improved treatment with methyl jasmonate (MeJA). The elucidation of 4-HDCA biosynthesis sharing the monolignol pathway supplemented the biosynthesis of colchicine in G. superba, and the production of 4-HDCA in tobacco cells provides an important step in the development of plant cell cultures as heterologous bio-factories for secondary metabolite production.