Efficient Transformation Method Research Articles

Establishment and Validation of an Efficient Agrobacterium Tumefaciens-Mediated Transient Transformation System for Salix Psammophila

Salix psammophila, C. Wang & Chang Y. Yang, a desert-adapted shrub, is recognized for its exceptional drought tolerance and plays a vital role in ecosystem maintenance. However, research on S. psammophila has been limited due to the lack of an efficient and reliable genetic transformation method, including gene functional studies. The Agrobacterium-mediated transient overexpression assay is a rapid and powerful tool for analyzing gene function in plant vivo. In this study, tissue culture seedlings of S. psammophila were utilized as the recipient materials, and the plant expression vector pCAMBIA1301, containing the GUS reporter gene, was transferred into the seedlings via an Agrobacterium-mediated method. To enhance the efficiency of the system, the effects of secondary culture time, Agrobacterium concentration, infection time, and co-culture duration on the transient transformation efficiency of S. psammophila were explored. The optimal combination for the instantaneous transformation of S. psammophila tissue culture seedlings mediated by Agrobacterium was determined as follows: a secondary culture time of 30 d, a value of OD600 of 0.8, an infection time of 3 h, and a co-culture duration of 48 h. Subsequently, the effectiveness of the transformation system was validated using the S. psammophila drought response gene SpPP2C80. To further confirm the accuracy of the system, SpPP2C80-overexpressing Arabidopsis was constructed and drought resistance analysis was performed. The results were consistent with the transient overexpression of SpPP2C80 in S. psammophila tissue culture seedlings, indicating that this system can be effectively employed for studying gene function in S. psammophila. These findings provide essential information for investigating gene function in non-model plants and pave the way for advancements in molecular biology research in S. psammophila.

A Rapid Method for Obtaining the Transgenic Roots of Crassulaceae Plants.

Crassulaceae plants are valued for their horticultural, ecological, and economic significance, but their genetic improvement is hindered by the absence of efficient and stable genetic transformation methods. Therefore, the development of a tailored genetic transformation method is crucial for enhancing the progress of the genetic improvement of Crassulaceae plants. The results indicate that, in the transformation experiments conducted on Kalanchoe tetraphylla, the K599 strain exhibited the highest transformation efficiency (76.67%), while C58C1 was least efficient (21.43%). An acetosyringone concentration of 100 μM was optimal for the hairy root transformation, and the immersion method yielded the highest efficiency. Additionally, the Silwet L-77 concentration significantly influenced the transformation efficiency, with 0.05% leading to a decrease. Upon four Crassulaceae species, notable differences were observed, with K. tetraphylla exhibiting the highest efficiency of 100%, and Sedum alfredii displaying the lowest efficiency of 5%. The RUBY reporter gene offers a more distinct advantage over GFP in observing the transformation effects. This study developed a simple, feasible, and cost-effective method for obtaining transgenic roots from leaves of Crassulaceae. The methodology provides technical support for the genetic improvement and gene function research of Crassulaceae plants.

Exploring an economic and highly efficient genetic transformation and genome-editing system for radish through developmental regulators and visible reporter.

Radish (Raphanus sativus L.) is one of the most important root vegetable crops worldwide. However, gene function exploration and germplasm innovation still face tremendous challenges due to its extremely low transformation efficiency. Here, an economic and highly efficient genetic transformation method for radish was explored by Agrobacterium rhizogenes-mediated transformation with the help of combining special developmental regulator (DR) genes and the visual identification reporter. Firstly, the RUBY gene, a betalain biosynthesis system, could result in a visual red-violet color used as a convenient and effective reporter for monitoring transgenic hairy roots screening of radish. However, the hairy roots-to-shoots conversion system of radish still stands as a barrier to the obtainment of whole transgenic plants, although different hormone combinations and various culture conditions were tried. Following, two DR genes including Wuschel2 (Wus2) and isopentenyl transferase (ipt), as well as their combination Wus2-ipt were introduced for the shoot regeneration capacity improvement. The results showed that the transgenic shoots could be directly generated without externally supplying any hormones in the presence of a Wus2-ipt combination. Then, Wus2-ipt along with the RUBY reporter was employed to establish an efficient genetic transformation system of radish. Moreover, this system was applied in generating gene-edited radish plants and the phytoene desaturase (RsPDS) gene was effectively knockout through albino phenotype observation and sequencing analysis. These findings have the potential to be widely applied in genetic transformation and genome-editing genetic improvement of other vegetable species.

Map-based cloning of LPD, a major gene positively regulates leaf prickle development in eggplant.

A critical gene for leaf prickle development (LPD) in eggplant was mapped on chromosome E06 and was confirmed to be SmARF10B through RNA interference using a new genetic transformation technique called SACI developed in this study Prickles on eggplant pose challenges for agriculture and are undesirable in cultivated varieties. This study aimed to uncover the genetic mechanisms behind prickle formation in eggplant. Using the F2 and F2:3 populations derived from a cross between the prickly wild eggplant, YQ, and the prickle-free cultivated variety, YZQ, we identified a key genetic locus (LPD, leaf prickle development) on chromosome E06 associated with leaf prickle development through BSA-seq and QTL mapping. An auxin response factor gene, SmARF10B, was predicted as the candidate gene as it exhibited high expression in YQ's mature leaves, while being significantly low in YZQ. Downregulating SmARF10B in YQ through RNAi using a simple and efficient Agrobacterium-mediated genetic transformation method named Seedling Apical Cut Infection (SACI) developed in this study substantially reduced the size and density of leaf prickles, confirming the role of this gene in prickle development. Besides, an effective SNP was identified in SmARF10B, resulting in an amino acid change between YQ and YZQ. However, this SNP did not consistently correlate with prickle formation in eight other eggplant materials examined. This study sheds light on the pivotal role of SmARF10B in eggplant prickle development and introduces a new genetic transformation method for eggplant, paving the way for future research in this field.

A simple and efficient non-tissue culture method for genetic transformation of grape immature zygotic embryos via VvBBM overexpression

The process of genetic transformation of grapevine has long been hampered by the inefficiency and complexity of tissue culture-based methods, severely limiting cultivar improvement and functional genome research. In this study, we aimed to develop a novel, non-tissue culture-based method for the genetic transformation of grape immature zygotic embryos without relying on tissue culture. This approach involves the overexpression of the Vitis vinifera BABY BOOM (VvBBM) gene, a key member of the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor family, which is known to enhance plant regeneration and transformation efficiency. Using ‘Cabernet Sauvignon’ immature zygotic embryos, we achieved a transformation efficiency of 42.85 % under non-tissue culture conditions, significantly reducing the cycle duration to approximately two months compared to traditional genetic transformation methods. Phenotypic and molecular analyses of the transformed grapevine plants confirmed the integration and overexpression of the VvBBM gene, resulting in improved growth metrics, including increased plant height, root length, and total root number, in a genotype-independent manner. Our results highlight the potential of VvBBM overexpression to circumvent the limitations imposed by tissue culture dependence, providing a rapid, efficient, and scalable alternative to conventional methods. Our study not only provides a practical approach to overcoming the barriers to genetic transformation in grapevine, but also opens new avenues for the application of this technology in the breeding of other economically important crops.

Micropropagation and genetic transformation of Byblis liniflora

Byblis, a small genus of carnivorous plants predominantly found in Australia, is characterized by its passive trapping mechanism and unique floral features. The chemical composition of Byblis, including identified phenylethanoid glycosides, particularly acteoside, highlights its pharmacological potential with various biological activities. In vitro culture techniques have been established for propagation, with micropropagation protocols developed for different Byblis species. However, information on genetic transformation, vital for trait modification and enhanced pharmacological interest, remains limited. This study focuses on optimizing micropropagation, adventitious regeneration, and genetic transformation methods for Byblis liniflora. Adventitious regeneration rates were highest in medium with reduced Murashige and Skoog salts (MS/10) and sucrose (3 gL−1) concentrations. Zeatin supplementation (1 mgL−1) further improved regeneration rates and bud development with 100% of regenerated root explants and 8.8 shoots per explant. Liquid MB3 medium supplemented with indole-3-acetic acid (IAA) 5 mgL−1 facilitated efficient rooting and acclimatization. The establishment of an efficient Rhizobium-mediated genetic transformation method yielded transgenic plants expressing green fluorescent protein (GFP). Molecular analysis confirmed transgene integration, marking the first successful genetic transformation in the Byblis genus. These advancements pave the way for exploring gene function and enhancing pharmacological properties, thereby broadening our understanding and utilization of carnivorous plants like Byblis.

A Highly Efficient Agrobacterium rhizogenes-Mediated Hairy Root Transformation Method of Idesia polycarpa and the Generation of Transgenic Plants.

Idesia polycarpa is a promising woody oilseed species because of its high oil yield. However, its use is greatly limited due to the lack of varieties with good qualities; additionally, gene function has been less studied in this plant because an efficient transformation method has not been established yet. In this study, we established a rapid and efficient hairy root transformation method by infecting the whole seedling, the rootless seedling, and the leaf petiole with Agrobacterium rhizogenes using different infection methods. Among these transformation methods, a higher transformation efficiency was obtained using the whole seedling, which could reach up to 71.91%. Furthermore, we found that the seedling age significantly affected the transformation efficiency, either using whole or rootless seedlings. Additionally, we found that the transgenic roots could regenerate transgenic shoots. Taken together, our study lays the foundation for future study and for genetically modifying wood traits in the future.

Transcription Factor McHB7 Improves Ice Plant Drought Tolerance through ABA Signaling Pathway.

As global climate change continues, drought episodes have become increasingly frequent. Studying plant stress tolerance is urgently needed to ensure food security. The common ice plant is one of the model halophyte plants for plant stress biology research. This study aimed to investigate the functions of a newly discovered transcription factor, Homeobox 7 (HB7), from the ice plant in response to drought stress. An efficient Agrobacterium-mediated transformation method was established in the ice plant, where ectopic McHB7 expression may be sustained for four weeks. The McHB7 overexpression (OE) plants displayed drought tolerance, and the activities of redox enzymes and chlorophyll content in the OE plants were higher than the wild type. Quantitative proteomics revealed 1910 and 495 proteins significantly changed in the OE leaves compared to the wild type under the control and drought conditions, respectively. Most increased proteins were involved in the tricarboxylic acid cycle, photosynthesis, glycolysis, pyruvate metabolism, and oxidative phosphorylation pathways. Some were found to participate in abscisic acid signaling or response. Furthermore, the abscisic acid levels increased in the OE compared with the wild type. McHB7 was revealed to bind to the promoter motifs of Early Responsive to Dehydration genes and abscisic acid-responsive genes, and protein-protein interaction analysis revealed candidate proteins responsive to stresses and hormones (e.g., abscisic acid). To conclude, McHB7 may contribute to enhance plant drought tolerance through abscisic acid signaling.

Impact of weak nonlocal nonlinearity on modulational instability and chirp-free soliton trains in parabolic law optical fiber

This study considers the generation and evolution of chirp-free soliton trains in a focusing cubic-quintic nonlinear optical fiber media (by utilizing experimental parameters), under the influence of weak nonlocal nonlinearity. Analysis of modulational instability (MI) reveals that the MI gain exist only in the anomalous dispersion regime, with the quintic nonlinear term increasing the maximum gain and sideband frequencies. The nonlocal nonlinearity parameter generally suppresses the impact of MI, because the maximum gain is greatly reduced with increase in nonlocality. By using the subsidiary ordinary differential equation method, stationary periodic soliton trains are obtained; with the intensity of the optical beam greatly reduced and frequency increased as nonlocality is gradually stepped up. However, an increase in nonlocality instead reduces the frequency of the traveling optical signals; derived via an efficient transformation method. Our results strongly suggest that nonlocal nonlinearity has potential applications in the control of laser beams; as nonlinear periodic optical signals can be easily transformed to very weak quasi-plane waves.

TMSCl Promoted Direct Conversion of Cyclic Anhydrides to (Un)Symmetric-Diesters/Amide Esters.

We present a mild, efficient, and one-pot method for the silyl-promoted transformation of cyclic anhydrides into homo- and hetero-dicarboxylic acid diesters and amide esters. This versatile reaction operates under ambient conditions, on a gram scale, and accommodates a wide range of alcohols, amines, and cyclic anhydrides. The one-pot process involves a two-step sequence, starting with the nucleophilic opening of anhydride by an amine or alcohol, followed by esterification. TMSCl serves a dual role, acting as a sacrificial reagent to remove in situ water and as a Lewis acid to promote the anhydride opening. The reaction proceeds successfully in the absence and presence of a base, as confirmed by NMR and crossover experiments, which validated the formation of dicarboxylic acid monoester and alkyl silyl mixed diester respectively. Controlled experiments have shown that the one-pot process yields higher efficiencies when compared to the same reaction conducted using a two-step process. This is the first comprehensive study demonstrating a broad substrate scope for the conversion of cyclic anhydride into diesters and amide esters. The method finds application in the synthesis of various commercial plasticizers.

Development of an efficient Agrobacterium-mediated transformation method and its application in tryptophan pathway modification in Catharanthus roseus.

The biosynthetic pathway of Catharanthus roseus vinca alkaloids has a long research history, including not only identification of metabolic intermediates but also the mechanisms of inter-cellular transport and accumulation of biosynthesized components. Vinca alkaloids pathway begins with strictosidine, which is biosynthesized by condensing tryptamine from the tryptophan pathway and secologanin from the isoprenoid pathway. Therefore, increasing the supply of precursor tryptophan may enhance vinca alkaloid content or their metabolic intermediates. Many reports on the genetic modification of C. roseus use cultured cells or hairy roots, but few reports cover the production of transgenic plants. In this study, we first investigated a method for stably producing transgenic plants of C. roseus, then, using this technique, we modified the tryptophan metabolism system to produce transgenic plants with increased tryptophan content. Transformed plants were obtained by infecting cotyledons two weeks after sowing with Agrobacterium strain A13 containing a plant expression vector, then selecting with 1/2 B5 medium supplemented with 50 mg l-1 kanamycin and 20 mg l-1 meropenem. Sixty-eight regenerated plants were obtained from 4,200 cotyledons infected with Agrobacterium, after which genomic PCR analysis using NPTII-specific primers confirmed gene presence in 24 plants with a transformation rate of 0.6%. Furthermore, we performed transformation into C. roseus using an expression vector to join trpE8 and aroG4 genes, which are feedback-resistant mutant genes derived from Escherichia coli. The resulting transformed plants showed exactly the same morphology as the wild-type, albeit with a marked increase in tryptophan and alkaloids content, especially catharanthine in leaves.

Rapid formation of denitrification granules for nitrite accumulation by increasing nitrogen loading rates and resistance to industrial wastewater

The nitrite (NO2–) accumulation in partial denitrification (PD) offers the possibility of widespread application of anammox process. In this study, the rapid establishment of PD granular system was achieved by increasing nitrogen loading rates (NLR) from 0.9 to 4.8 kg N/(m3·d), with the nitrate-to-nitrite transforming ratio (NTR) increasing rapidly to 87.0 % within 18 days. Growth evidence indicated that the functional genus Thauera was significantly enriched (12.5 %→76.4 %), with nitrate (NO3–) reduction rates (SNO3) improving by 5.4 times from 13.0 to 70.7 mg N/(g VSS·h). Importantly, the rapid aggregation of PD biomass as granules ensured robustness and resistance of PD feeding with the electroplating tail wastewater (NO3–-N of 103.0 ± 5.0 mg/L), obtaining stable NTR above 91.5 %. This study demonstrated the achievability of the fast development of PD granules and the adaptability and robustness of treating nitrate-containing industrial wastewater, which provided a promising method for efficient nitrogen transformation in industrial applications.

An efficient method for hairy root transformation and transgenic plant regeneration of Melia azedarach

An efficient method for hairy root transformation and transgenic plant regeneration of Melia azedarach

Tracking organelle activities through efficient and stable root genetic transformation system in woody plants.

Due to the protracted transgenic timeline and low efficiency in stable genetic transformation of woody plants, there has been limited exploration of real-time organelle imaging within stable transgenic woody plant cells. Here, we established an efficient in vivo genetic transformation system for woody plants using an Agrobacterium rhizogenes-mediated approach. This system was successfully validated in multiple perennial woody species. Using citrus as a model, we introduced organelle-targeted fluorescent reporters via genetic transformation and investigated their subcellular localization and dynamics using advanced imaging techniques, such as confocal microscopy and live-cell imaging. Moreover, we subjected transgenic MT-GFP-labeled mitochondria in root cells to stress conditions simulating agricultural adversities faced by fruit crops. The stress-induced experiments revealed notable alterations in mitochondrial morphology. Our study contributes novel insights into membrane trafficking processes, protein localization dynamics, and cellular physiology in woody plants, while also providing stable and efficient genetic transformation methods for perennial woody species.

Electrotransformation of Foodborne Pathogen Cronobacter sakazakii by a Simple Method.

Cronobacter sakazakii is an opportunistic foodborne pathogen that mainly infects infants and immunocompromised people, with a high mortality rate. However, the efficient transformation method of this bacterium has not been systematically reported. In this study, we developed a fast and efficient transformation method for C. sakazakii by cold sucrose treatment. Compared with CaCl2 or glycerol treatment, the transformation efficiency of this method is significantly high when bacteria were cultured overnight at 42°C before cold sucrose treatment. Furthermore, applying this method, we successfully knocked out the pppA gene by direct electroporation. Collectively, our study provides a simple, time-saving, and efficient method for competent cell preparation of C. sakazakii, which is conducive to the further research of C. sakazakii.

Agrobacterium rhizogenes-Mediated Genetic Transformation and Establishment of CRISPR/Cas9 Genome-Editing Technology in Limonium bicolor

Limonium bicolor is a perennial herbaceous plant belonging to the Plumbaginaceae family. It can be used as a dried flower or in cut flower arrangements and serves as a model recretohalophyte. Its genome sequencing has been recently completed. However, the research on L. bicolor is limited by the absence of a highly efficient genetic transformation system. In this study, we established a highly efficient Agrobacterium rhizogenes-mediated L. bicolor genetic transformation method. The transgenic hairy roots were induced from the hypocotyl of L. bicolor using A. rhizogenes strain K599 harboring pRdGa4Cas9 plasmid (which carries an expression cassette of 35S::DsRed2). The transgenic shoots were regenerated from hairy root segments (~0.1 cm diameter), and induction efficiency was achieved at 100%. The transgenic shoots with 4–5 rosette leaves were directly planted into the soil to induce the transgenic roots. Therefore, transgenic plantlets were produced. The DsRed2 can be used as a reliable reporter gene in screening transgenic plantlets. Furthermore, we also established a CRISPR/Cas9 system in L. bicolor employing the A. rhizogenes-mediated genetic transformation approach. The highly efficient transformation method and CRIPSP/Cas9 system established will provide a valuable tool for functional genomics investigation and trait improvement in L. bicolor.

Cotyledon peeling method for passion fruit protoplasts: a versatile cell system for transient gene expression in passion fruit (Passiflora edulis).

Passion fruit (Passiflora edulis) is a perennial evergreen vine that grows mainly in tropical and subtropical regions due to its nutritional, medicinal and ornamental values. However, the molecular biology study of passion fruit is extremely hindered by the lack of an easy and efficient method for transformation. The protoplast transformation system plays a vital role in plant regeneration, gene function analysis and genome editing. Here, we present a new method ('Cotyledon Peeling Method') for simple and efficient passion fruit protoplast isolation using cotyledon as the source tissue. A high yield (2.3 × 107 protoplasts per gram of fresh tissues) and viability (76%) of protoplasts were obtained upon incubation in the enzyme solution [1% (w/v) cellulase R10, 0.25% (w/v) macerozyme R10, 0.4 M mannitol, 10 mM CaCl2, 20 mM KCl, 20 mM MES and 0.1% (w/v) BSA, pH 5.7] for 2 hours. In addition, we achieved high transfection efficiency of 83% via the polyethylene glycol (PEG)-mediated transformation with a green fluorescent protein (GFP)-tagged plasmid upon optimization. The crucial factors affecting transformation efficiency were optimized as follows: 3 μg of plasmid DNA, 5 min transfection time, PEG concentration at 40% and protoplast density of 100 × 104 cells/ml. Furthermore, the established protoplast system was successfully applied for subcellular localization analysis of multiple fluorescent organelle markers and protein-protein interaction study. Taken together, we report a simple and efficient passion fruit protoplast isolation and transformation system, and demonstrate its usage in transient gene expression for the first time in passion fruit. The protoplast system would provide essential support for various passion fruit biology studies, including genome editing, gene function analysis and whole plant regeneration.

Highly Efficient CRISPR/Cas9 Mediated Gene Editing in Ocimum basilicum 'FT Italiko' to Induce Resistance to Peronospora belbahrii.

Ocimum basilicum (sweet basil) is an economically important aromatic herb; in Italy, approximately 1000 ha of "Genovese-type" basil are grown annually in greenhouses and open fields and are subjected to Downy Mildew (DM) disease, caused by Peronospora belbahrii, leading to huge crop losses. Mutation of the Susceptibility (S) gene DMR6 (Downy Mildew Resistant 6) has been proven to confer a broad-spectrum resistance to DM. In this work, an effective Genome Editing (GE) approach mediated by CRISPR/Cas9 in O. basilicum 'Italiko', the élite cultivar used to produce "Pesto Genovese D.O.P", was developed. A highly efficient genetic transformation method mediated by A. tumefaciens has been optimized from cotyledonary nodes, obtaining 82.2% of regenerated shoots, 84.6% of which resulted in Cas9+ plants. Eleven T0 lines presented different type of mutations in ObDMR6; 60% were indel frameshift mutations with knock-out of ObDMR6 of 'FT Italiko'. Analysis of six T1 transgene-free seedlings revealed that the mutations of T0 plants were inherited and segregated. Based on infection trials conducted on T0 plants, clone 22B showed a very low percentage of disease incidence after 14 days post infection. The aromatic profile of all in vitro edited plants was also reported; all of them showed oxygenated monoterpenes as the major fraction.

Efficient floral dip transformation method using Agrobacterium tumefaciens on Cosmos sulphureus Cav.

Yellow cosmos (Cosmos sulphureus Cav.) is a specific flowering plant and considered a suitable genetic engineering model. Agrobacterium-mediated plant transformation is commonly used for plant genetic engineering. Floral dip transformation is one of the plant genetic transformation methods, and it involves dipping flower buds into an Agrobacterium suspension. Studies on floral dip transformation of yellow cosmos have never been reported. Therefore, an efficient method in plant genetic engineering must be established. This study developed an effective and efficient floral dip transformation method for yellow cosmos.In this study, flower buds with sizes of 5–7 mm were used. Several parameters have been observed to optimize the floral dip method. These parameters included the optical density (OD600) of Agrobacterium culture, concentration of surfactant, and duration of flower bud dipping into the Agrobacterium suspension.The results showed that the floral dip method was most efficient when the flower buds were dipped into Agrobacterium suspension with OD600 = 0.8 and containing 5% sucrose and 0.1% Silwet L-77 for 30 s. This method enhanced the transformation efficiency at a rate of 12.78 ± 1.53%. The neomycin phosphotransferase II and green fluorescent protein genes with sizes of 550 and 736 bp, respectively, were confirmed by polymerase chain reaction. In addition, the transgenic plants were kanamycin resistant and fluorescent under ultraviolet light observation. This finding suggests that the proposed floral dip transformation provides new insights into efficient plant genetic engineering methods for yellow cosmos.

An efficient and reproducible Agrobacterium-mediated genetic transformation method for the ornamental monocotyledonous plant Ornithogalum dubium Houtt

Ornithogalum is a genus from the Hyacinthaceae (Asparagaceae) family that comprises about 200 species with remarkable white, yellow, or orange flowers that display exceptional vase life. These properties have made it a popular cut flower and pot plant. Forward genetics approaches may be advantageous to generate novel phenotypes, but the Agrobacterium-mediated transformation of plants from this genus remains challenging. Here, a stable and efficient Agrobacterium-mediated transformation system was established for O. dubium. We found that the timing of transformation with respect to light exposure of the tissue affected transformation rates more than other tested parameters. In the transgenic plants obtained, T-DNA integrations were confirmed by polymerase chain reactions and positive plants were established in the greenhouse and displayed weak transgene expression. This study exposed an efficient platform for gene function research and germplasm improvement in O. dubium plants. The present protocol is now available for the development of novel improved O. dubium varieties.