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Conventional approaches like Agrobacterium-mediated transformation, viral transduction, biolistic particle bombardment, and polyethylene glycol (PEG)-facilitated delivery methods have been optimized for transporting specific genes to various plant cells. These conventional approaches in genetically modified crops are dependent on several factors like plant types, cell types, and genotype requirements, as well as numerous disadvantages such as time-consuming, untargeted distribution of genes, and high cost of cultivation. Therefore, it is suggested to develop novel techniques for the transportation of genes in crop plants using tailored nanoparticles (NPs) of manipulative and controlled high-performance features synthesized using green and chemical routes. It is observed that site-specific delivery of genes exhibits high efficacy in species-independent circumstances which leads to an increased level of productivity. Therefore, to achieve these outcomes, NPs can be utilized as gene nano-carriers for excellent delivery inside crops (i.e., cotton, tobacco, rice, wheat, okra, and maize) for desired genetic engineering modifications. As outcomes, this review provides an outline of the conventional techniques and current application of numerous nano-enabled gene delivery needed for crop gene manipulation, the benefits, and drawbacks associated with state-of-the-art techniques, which serve as a roadmap for the possible applicability of nanomaterials in plant genomic engineering as well as crop improvement in the future. [ABSTRACT FROM AUTHOR]

Plant viruses have evolved different viral suppressors of RNA silencing (VSRs) to counteract RNA silencing which is a small RNA-mediated sequence-specific RNA degradation mechanism. Previous studies have already shown that the coat protein (CP) of cucumber mosaic virus (CMV) reduced RNA silencing suppression (RSS) activity of the VSR of CMV, the 2b protein. To demonstrate the universality of this CP-VSR interference, our study included three different viruses: CMV and peanut stunt virus (PSV) from the Bromoviridae, and plum pox virus (PPV) from the Potyviridae family. The RSS activity of the three VSRs (CMV 2b, PSV 2b, and PPV HC-Pro) was compared using Agrobacterium-mediated transient expression in Nicotiana benthamiana and the effect of CMV CP, PSV CP and PPV CP was validated on the RSS activity of their cognate and heterologous VSRs as well. Furthermore, the VSRs were also evaluated in PTGS suppressor-deficient CMV mutant (CMV NVE/10–12/AAA) virus-infected plants. The joint presence of CPs and VSRs resulted in decreased RSS activity in each combination, regardless of the origin of the two proteins, suggesting a universal role of the viral CPs in fine tuning of RSS. Interestingly the PSV CP elicited the strongest negative effect on the RSS activity of all three VSRs. [ABSTRACT FROM AUTHOR]

Crown gall is one widespread grapevine disease worldwide, caused by Allorhizobium vitis (syn. Agrobacterium vitis) and Agrobacterium tumefaciens (syn. Rhizobium radiobacter). All. Vitis, is the predominant species and primary cause of the disease. This study aimed to identify and characterize molecularly the agrobacteria in plants with crown gall symptoms in Mendoza vineyards. Diseased plants with trunk-based galls were sampled from different areas of Mendoza. Two multiplex PCRs were performed for bacterial identification and characterization, demonstrating that 91.6% of the strains obtained were agrobacteria (77% A. tumefaciens and 23% All. vitis). Fifty percent of All. vitis and 16% of A. tumefaciens identified strains were pathogenic. Pathogenicity tests were also conducted on Kalanchoe daigremontiana, with resulting tumorigenic symptoms. [ABSTRACT FROM AUTHOR]

Milk thistle is a valuable medicinal plant, and obtaining silymarin (SLM) is the main reason for the cultivation of this plant. Due to the non-agricultural properties of milk thistle, its cultivation and harvesting are accompanied by difficulties. In vitro producing SLM by hairy root culture can overcome these problems. Various factors such as plant genotype, Agrobacterium rhizogenes strain used for infection, type and concentration of elicitor and, etc., can significantly enhance SLM production. The impact of various genotypes on SLM production in milk thistle hairy root culture has yet to be investigated. In this study examined the effect of five different genotypes of milk thistle and methyl jasmonate (MeJA) on the amount of SLM in milk thistle hairy roots based on completely randomized design. HPLC was used to quantify the amount of SLM in the hairy root samples. Based on the results, SLM production showed a considerable difference among ecotypes, with levels ranging from 109.8 to 648.7 ppm. MeJA increased the amount of SLM in all genotypes except for Mashhad ecotype. Due to the approximately 5.9-fold difference in SLM levels among different genotypes and the opposite responses of genotypes to MeJA indicate a high variation in this trait among different milk thistle ecotypes. Based on these results, screening milk thistle germplasm to find ecotypes with the highest levels of SLM in hairy root culture and also considering the strong mutual effect between elicitor and ecotype can be fruitful for increasing SLM content. [ABSTRACT FROM AUTHOR]

Bacteria associated with plants, whether rhizospheric, epiphytic, or endophytic, play a crucial role in plant productivity and health by promoting growth through complex mechanisms known as plant growth promoters. This study aimed to isolate, characterize, identify, and evaluate the potential of endophytic bacteria from the resurrection plant Selaginella lepidophylla in enhancing plant growth, using Arabidopsis thaliana ecotype Col. 0 as a model system. Plant growth-promotion parameters were assessed on the bacterial isolates; this assessment included the quantification of indole-3-acetic acid, phosphate solubilization, and biological nitrogen fixation, a trehalose quantification, and the siderophore production from 163 endophytic bacteria isolated from S. lepidophylla. The bacterial genera identified included Agrobacterium, Burkholderia, Curtobacterium, Enterobacter, Erwinia, Pantoea, Pseudomonas, and Rhizobium. The plant growth promotion in A. thaliana was evaluated both in Murashige Skoog medium, agar-water, and direct seed inoculation. The results showed that the bacterial isolates enhanced primary root elongation and lateral root and root hair development, and increased the fresh and dry biomass. Notably, three isolates promoted early flowering in A. thaliana. Based on these findings, we propose the S. lepidophylla bacterial isolates as ideal candidates for promoting growth in other agriculturally important plants. [ABSTRACT FROM AUTHOR]

The study we presented here demonstrates an efficient regeneration and transformation protocol for Eclipta prostrata (L.) L (Syn.: Eclipta alba (L.) Hassk). The establishment of regeneration protocol starts with collection of germplasm from the wild. Seeds collected from morphologically healthy plants were used as the source for in-vitro seedlings production. The optimized Murashige and Skoog (MS) medium with 4.44 μM Benzyl aminopurine (BAP) and 3% sucrose demonstrated the successful approach for generating the number of shoots (36–44 shoots/node) during the regeneration process. The altered shoots were efficiently rooted using hormone-free half MS media (HMS 20.7 g/L). The specific Agrobacterium tumefaciens strain GV3101, carrying pBI121-CFP gene (modified cyan fluorescent protein) in binary vector with kanamycin resistance, was used for plant transformation. The transformation process involved inoculating nodal explants with A. tumefaciens cells at an optical density of 0.5 and allowing an infection period of 45 min and co-cultivation for 48 h. Kanamycin, at a concentration of 35 mg/L and 45 min of infection time, was employed as a selectable condition to screen and identify successfully transformed plants. PCR analysis (T1 and T2 transgenic lines) and fluorescent microscopy (T2 transgenic lines) were utilized to confirm the transgenic nature of the stable regenerated plants, ensuring successful integration of foreign genetic material. We have established a simple and stable transformation system for E. prostrata using a regeneration protocol that has promising implications. Using this protocol researchers may enhance the production of specific bioactive compounds, thereby increasing the medicinal and pharmaceutical value of E. prostrata. [ABSTRACT FROM AUTHOR]

Eremosparton songoricum (Litv.) Vass. is a desert legume exhibiting extreme drought tolerance and the ability to withstand various harsh environments, making it a good candidate for investigating stress tolerance mechanisms and exploring valuable stress-resistant genes. However, the absence of a genetic transformation system for E. songoricum poses significant limitations for functionally validating these stress-resistant genes in situ. In this study, we developed an Agrobacterium-mediated transient transformation system for E. songoricum utilizing the β-glucuronidase (GUS) gene as a reporter. We investigated three types of explants (seedlings, assimilated branches and callus) and the effects of different Agrobacterium strains, seedling ages, OD600 values, acetosyringone (AS) concentrations, sucrose concentrations and infection times on the transformation efficiency. The results reveal that the optimal transformation system was infecting one-month-old regenerating assimilated branches with the Agrobacterium strain C58C1. The infection solution comprised 1/2 MS medium with 3% sucrose and 200 μM AS at an OD600 of 0.8, infection for 3 h and then followed by 2 days of dark cultivation, which achieving a maximum transformation rate of 97%. The maximum transformation rates of the seedlings and calluses were 57.17% and 39.51%, respectively. Moreover, we successfully utilized the assimilated branch transient transformation system to confirm the role of the previously reported transcription factor EsDREB2B in E. songoricum. The overexpression of EsDREB2B enhanced drought tolerance by increasing the plant's reactive oxygen species (ROS) scavenging capacity in situ. This study established the first transient transformation system for a desert legume woody plant, E. songoricum. This efficient system can be readily applied to investigate gene functions in E. songoricum. It will expedite the exploration of genetic resources and stress tolerance mechanisms in this species, offering valuable insights and serving as a reference for the transformation of other desert plants and woody legumes. [ABSTRACT FROM AUTHOR]

Tropical grassland soils, especially those with alkaline properties, often exhibit limited phosphorus availability due to its precipitation in insoluble forms. Phosphate-solubilizing bacteria (PSB) and rhizobia have demonstrated their potential to enhance the availability of this nutrient and promote the growth of forage legumes. This study, conducted under controlled conditions in a mesh house, evaluated the effect of co-inoculation with PSB, including Micrococcus sp. Sfcm-14-01, Agrobacterium sp. Sfl-043-09, and Enterobacter sp. Sfcm-014-02 and Sfcm-054-06, along with rhizobia (Ensifer terangae R1-012-02 and Bradyrhizobium glycinis Rcm-025-01), under different levels of phosphorus fertilization on the legumes Leucaena leucocephala and Centrosema macrocarpum. The results indicate significant increases in various growth parameters, such as chlorophyll levels (SPAD), biomass (dry weight of roots and aerial parts) (mg), the foliar phosphorus concentration (ppm), and the concentration of available phosphorus in the soil, particularly under low-phosphorus fertilization conditions. The highest level of available phosphorus in the soil was achieved with 75% of the recommended fertilization dose, resulting in concentrations of 13.73 ppm for L. leucocephala and 7.69 ppm for C. macrocarpum, representing increases in phosphorus availability of 170.81% and 240.27%, respectively, compared with no fertilization or inoculation. These findings suggest that the co-inoculation of PSB and native rhizobia is a promising strategy to enhance the biomass productivity and mineral content of forage in tropical grazing systems, especially under phosphorus-limited conditions. [ABSTRACT FROM AUTHOR]

The ability to efficiently genetically modify plant species is crucial, driving the need for innovative technologies in plant biotechnology. Existing plant genetic transformation systems include Agrobacterium-mediated transformation, biolistics, protoplast-based methods, and nanoparticle techniques. Despite these diverse methods, many species exhibit resistance to transformation, limiting the applicability of most published methods to specific species or genotypes. Tissue culture remains a significant barrier for most species, although other barriers exist. These include the infection and regeneration stages in Agrobacterium, cell death and genomic instability in biolistics, the creation and regeneration of protoplasts for protoplast-based methods, and the difficulty of achieving stable transformation with nanoparticles. To develop species-independent transformation methods, it is essential to address these transformation bottlenecks. This review examines recent advancements in plant biotechnology, highlighting both new and existing techniques that have improved the success rates of plant transformations. Additionally, several newly emerged plant model systems that have benefited from these technological advancements are also discussed. [ABSTRACT FROM AUTHOR]

Agrobacterium-mediated transformation of plants often results in the integration of multiple copies of T-DNA and backbone DNA from binary vectors into the host genome. However, the interplay between T-DNA and backbone DNA remains elusive. In this study, 70.8% of T1Arabidopsis transformants exhibited integration of both T-DNA and backbone DNA, and no cases of only backbone integration were observed. To elucidate the integration patterns, we employed bulk-genome resequencing in Arabidopsis and identified 20 integration sites across 10 T1 transgenic plants, most of which were flanked by left borders of T-DNA at both ends. On average, each integration site contained 6.3 copies of T-DNA and 2.65 copies of backbone DNA. The junction structures between T-DNA and the backbone were highly variable, revealing a previously underappreciated frequency of readthrough at both the left and right borders. Transient expression studies in Nicotiana benthamiana leaves demonstrated that T-DNA and backbone DNA were simultaneously transferred into transformed cells, although the backbone DNA had lower copy numbers than T-DNA. These findings suggest a close relationship between T-DNA and backbone DNA during their transfer and integration, thus offering new insights into the mechanism underlying Agrobacterium-mediated transformation. [ABSTRACT FROM AUTHOR]

Abstract Virus-Induced Gene Silencing (VIGS) is a versatile tool in plant science, yet its application to non-model species like sunflower demands extensive optimization due to transformation challenges. In this study, we aimed to elucidate the factors that significantly affect the efficiency of Agrobacterium-VIGS in sunflowers. After testing a number of approaches, we concluded that the seed vacuum technique followed by 6 h of co-cultivation produced the most efficient VIGS results. Genotype-dependency analysis revealed varying infection percentages (62–91%) and silencing symptom spreading in different sunflower genotypes. Additionally, we explored the mobility of tobacco rattle virus (TRV) and phenotypic silencing manifestation (photo-bleaching) across different tissues and regions of VIGS-infected sunflower plants. We showed the presence of TRV is not necessarily limited to tissues with observable silencing events. Finally, time-lapse observation demonstrated a more active spreading of the photo-bleached spots in young tissues compared to mature ones. This study not only offers a robust VIGS protocol for sunflowers but also provides valuable insights into genotype-dependent responses and the dynamic nature of silencing events, shedding light on TRV mobility across different plant tissues.

Genetic transformation and regeneration procedures in Saintpaulia are dependent on different parameters, which limit the genetic manipulation efficiency. We focused on introducing an efficient model for gene transformation and regeneration after that, via RSM. Two commercial cultivars were transformed by the GUS reporter gene via Agrobacterium-mediated method. Results showed that variables infection time, BA and IAA concentrations, and cultivar type significantly (p ≤ .05) affected gene transformation. According to the highest desirability (0.94) of the obtained model, the highest transformation rate was achieved (68.7%) by 15 s dip in bacterial suspension followed by regeneration steps in the medium supplemented with 100 µg/l BA + 1000 µg/l IAA. Subsequently, the given model validity was evaluated by a target gene (etr1-1). The highest rate of etr1-1 transformation (76.25%) was achieved by 30 s infection time in the proposed medium containing 100 µg/l BA + 1000 µg/l IAA, according to the model. Genomic and reverse transcription PCRs confirmed that GUS, nptII and etr1-1 genes were successfully integrated and expressed in both cultivars at each transformation. Our results indicate that optimising the time of infection and media using the RSM design could greatly improve the accuracy and feasibility of a model for achieving a high rate of genetic transformation. [ABSTRACT FROM AUTHOR]

Gingerols are phenolic biomedical compounds found in ginger (Zingiber officinale) whose low aqueous solubility limits their medical application. To improve their solubility and produce novel glucosides, an α -glucosidase (glycoside hydrolase) from Agrobacterium radiobacter DSM 30147 (Ar G) was subcloned, expressed, purified, and then confirmed to have additional α -glycosyltransferase activity. After optimization, the Ar G could glycosylate gingerols into three mono-glucosides based on the length of their acyl side chains. Compound 1 yielded 63.0 %, compound 2 yielded 26.9 %, and compound 3 yielded 4.37 %. The production yield of the gingerol glucosides optimally increased in 50 mM phosphate buffer (pH 6) with 50 % (w/v) maltose and 1000 mM Li+ at 40 °C for an 24-h incubation. The structures of purified compound 1 and compound 2 were determined as 6-gingerol-5- O - α -glucoside (1) and novel 8-gingerol-5- O - α -glucoside (2), respectively, using nucleic magnetic resonance and mass spectral analyses. The aqueous solubility of the gingerol glucosides was greatly improved. Further assays showed that, unusually, 6-gingerol-5- O - α -glucoside had 10-fold higher anti-inflammatory activity (IC 50 value of 15.3 ± 0.5 μM) than 6-gingerol, while the novel 8-gingerol-5- O - α -glucoside retained 42.7 % activity (IC 50 value of 106 ± 4 μM) compared with 8-gingerol. The new α -glucosidase (Ar G) was confirmed to have acidic α -glycosyltransferase activity and could be applied in the production of α -glycosyl derivatives. The 6-gingerol-5- O - α -glucoside can be applied as a clinical drug for anti-inflammatory activity. [Display omitted] [ABSTRACT FROM AUTHOR]

Agrobacterium sp. are notable for their ability to produce substantial amounts of exopolysaccharides. Our study identified an exopolysaccharide (Galacan, 4982.327 kDa) from Agrobacterium sp. FN01. Galacan is a heteropolysaccharide primarily composed of glucose and galactose at a molar ratio of 25:1. The FT-IR results suggested that Galacan had typical absorption peaks of polysaccharide. The results of periodate oxidation, Smith degradation, and NMR confirmed the presence of structural units, such as β-D-Galp(→, →3)β-D-Galp(1→, →2,3)β-D-Glcp(1→, β-D-Glcp(1→, and →2)β-D-Glcp(1→. Galacan demonstrated significant biological activities. In experiments conducted with zebrafish, it facilitated the proliferation of Lactobacillus brevis in the intestinal tract, suggesting potential prebiotic properties. Moreover, in vivo studies revealed its antihyperglycemic effects, as evidenced by significant reductions in blood glucose levels and enhanced fluorescence intensity of pancreatic β cells in a streptozotocin (STZ)-induced hyperglycemic zebrafish model. Additionally, antiaging assays demonstrated Galacan's ability to inhibit β-galactosidase activity and enhance telomerase activity in a hydrogen peroxide (HP)-induced aging zebrafish model. These findings emphasized the potential of Galacan as a natural prebiotic with promising applications in diabetes prevention and antiaging interventions. [ABSTRACT FROM AUTHOR]

Virus-Induced Gene Silencing (VIGS) is a versatile tool in plant science, yet its application to non-model species like sunflower demands extensive optimization due to transformation challenges. In this study, we aimed to elucidate the factors that significantly affect the efficiency of Agrobacterium-VIGS in sunflowers. After testing a number of approaches, we concluded that the seed vacuum technique followed by 6 h of co-cultivation produced the most efficient VIGS results. Genotype-dependency analysis revealed varying infection percentages (62–91%) and silencing symptom spreading in different sunflower genotypes. Additionally, we explored the mobility of tobacco rattle virus (TRV) and phenotypic silencing manifestation (photo-bleaching) across different tissues and regions of VIGS-infected sunflower plants. We showed the presence of TRV is not necessarily limited to tissues with observable silencing events. Finally, time-lapse observation demonstrated a more active spreading of the photo-bleached spots in young tissues compared to mature ones. This study not only offers a robust VIGS protocol for sunflowers but also provides valuable insights into genotype-dependent responses and the dynamic nature of silencing events, shedding light on TRV mobility across different plant tissues. [ABSTRACT FROM AUTHOR]

The article titled "Correction" in the Plant Journal provides updates and corrections to figures in a previous article titled "Modulation of drought resistance by the abscisic acid receptor PYL5 through inhibition of clade A PP2Cs." The corrections include the removal of accidental duplications, correction of gel splicing errors, and the inclusion of additional data. The corrected figures show the interaction of HAB1 with PYL5, PYL8, and PYL6, the subcellular localization of PYL5 and its interaction with HAB1, and the ABA-hypersensitive inhibition of germination and root growth in PYL5-overexpressing lines. The authors apologize for the errors. [Extracted from the article]

Agrobacteria are a diverse, polyphyletic group of prokaryotes with multipartite genomes capable of transferring DNA into the genomes of host plants, making them an essential tool in plant biotechnology. Despite their utility in plant transformation, genome-wide transcriptional regulation is not well understood across the three main lineages of agrobacteria. Transcription start sites (TSSs) are a necessary component of gene expression and regulation. In this study, we used differential RNA-seq and a TSS identification algorithm optimized on manually annotated TSS, then validated with existing TSS to identify thousands of TSS with nucleotide resolution for representatives of each lineage. We extend upon the 356 TSSs previously reported in Agrobacterium fabrum C58 by identifying 1,916 TSSs. In addition, we completed genomes and phenotyping of Rhizobium rhizogenes C16/80 and Allorhizobium vitis T60/94, identifying 2,650 and 2,432 TSSs, respectively. Parameter optimization was crucial for an accurate, high-resolution view of genome and transcriptional dynamics, highlighting the importance of algorithm optimization in genome-wide TSS identification and genomics at large. The optimized algorithm reduced the number of TSSs identified internal and antisense to the coding sequence on average by 90.5% and 91.9%, respectively. Comparison of TSS conservation between orthologs of the three lineages revealed differences in cell cycle regulation of ctrA as well as divergence of transcriptional regulation of chemotaxis-related genes when grown in conditions that simulate the plant environment. These results provide a framework to elucidate the mechanistic basis and evolution of pathology across the three main lineages of agrobacteria. IMPORTANCE Transcription start sites (TSSs) are fundamental for understanding gene expression and regulation. Agrobacteria, a group of prokaryotes with the ability to transfer DNA into the genomes of host plants, are widely used in plant biotechnology. However, the genome-wide transcriptional regulation of agrobacteria is not well understood, especially in less-studied lineages. Differential RNA-seq and an optimized algorithm enabled identification of thousands of TSSs with nucleotide resolution for representatives of each lineage. The results of this study provide a framework for elucidating the mechanistic basis and evolution of pathology across the three main lineages of agrobacteria. The optimized algorithm also highlights the importance of parameter optimization in genome-wide TSS identification and genomics at large.

Crown gall is one widespread grapevine disease worldwide, caused by Allorhizobium vitis (syn. Agrobacterium vitis) and Agrobacterium tumefaciens (syn. Rhizobium radiobacter). All. Vitis, is the predominant species and primary cause of the disease. This study aimed to identify and characterize molecularly the agrobacteria in plants with crown gall symptoms in Mendoza vineyards. Diseased plants with trunk-based galls were sampled from different areas of Mendoza. Two multiplex PCRs were performed for bacterial identification and characterization, demonstrating that 91.6% of the strains obtained were agrobacteria (77% A. tumefaciens and 23% All. vitis). Fifty percent of All. vitis and 16% of A. tumefaciens identified strains were pathogenic. Pathogenicity tests were also conducted on Kalanchoe daigremontiana, with resulting tumorigenic symptoms. Highlights: • Crown gall is one widespread grapevine disease in the world, caused by Allorhizobium vitis (syn. Agrobacterium vitis) and Agrobacterium tumefaciens (syn. Rhizobium radiobacter). • This study aimed to identify and characterize molecularly the causal agent of crown gall in Mendoza vineyards. • One hundred and forty-eight symptomatic plants were collected from various vine-growing areas of Mendoza. • Allorhizobium vitis and Agrobacterium tumefaciens strains were molecularly identified as pathogenic and octopine/nopaline type. • All vitis is the predominant pathogenic species in grapevines studied in Mendoza.

Agrobacterium-mediated plant transformation is widely used in plant genetic engineering. However, its efficiency is limited by plant immunity against Agrobacterium. Chili pepper (Capsicum annuum L.) is an important vegetable that is recalcitrant to Agrobacterium-mediated transformation. In this work, Agrobacterium was found to induce a strong immune response in pepper, which might be the reason for T-DNA being difficult to express in pepper. An Agrobacterium mutant screen was conducted and a point mutation in the hisI gene was identified due to a weak immune response and enhanced transient expression mediated by this Agrobacterium mutant in pepper leaves. Further genetic analysis revealed that histidine biosynthesis deficiency caused by mutations in many genes of this pathway led to reduced pepper cell death, presumably due to reduced bacterial growth. However, mutation analysis of threonine and tryptophan biosynthesis genes showed that the biosynthesis of different amino acids may play different roles in Agrobacterium growth and stimulating the pepper immune response. The possible application of Agrobacterium amino acid biosynthesis mutations in plant biology was discussed.

Key messageGRF-GIF chimeric proteins from multiple source species enhance in vitro regeneration in both wild and cultivated lettuce. In addition, they enhance regeneration in multiple types of lettuce including butterheads, romaines, and crispheads. The ability of plants to regenerate in vitro has been exploited for use in tissue culture systems for plant propagation, plant transformation, and genome editing. The success of in vitro regeneration is often genotype dependent and continues to be a bottleneck for Agrobacterium-mediated transformation and its deployment for improvement of some crop species. Manipulation of transcription factors that play key roles in plant development such as BABY BOOM, WUSCHEL, and GROWTH-REGULATING FACTORs (GRFs) has improved regeneration and transformation efficiencies in several plant species. Here, we compare the efficacy of GRF-GIF gene fusions from multiple species to boost regeneration efficiency and shooting frequency in four genotypes of wild and cultivated lettuce (Lactuca spp. L.). In addition, we show that GRF-GIFs with mutated miRNA 396 binding sites increase regeneration efficiency and shooting frequency when compared to controls. We also present a co-transformation strategy for increased transformation efficiency and recovery of transgenic plants harboring a gene of interest. This strategy will enhance the recovery of transgenic plants of other lettuce genotypes and likely other crops in the Compositae family.

Many economically important crops and vegetables belonging to the cruciferous family are heavily endangered by clubroot disease caused by Plasmodiophora brassicae infection. Breeding of clubroot resistant cultivars based on mapping and cloning of resistant genes is commonly regarded as the most cost-effective and efficient way to fight against this disease. The traditional way of R gene functional validation requires stable transformation that is both time- and labor-consuming. In this study, a rapid and efficient hairy-root transgenic protocol mediated by Agrobacterium rhizogenes was developed. The transformation positive rate was over 80% in Brassica napus showed by GUS reporter gene and this transformation only took 1/6 of the time compared with stable transformation. The system was applicable to different B. napus varieties and other cruciferous crops including Brassica rapa and Brassica oleracea. In particular, two known CR genes, CRA3.7.1 and CRA8.2.4 were used respectively, as example to show that the system works well for CR gene study combined with subsequent P. brassicae infection in B. napus. Most importantly, it works both in over-expression that led to disease resistance, as well as in RNAi which led to disease susceptible phenotype. Therefore, this system can be used in batch-wise identification of CR genes, and also offered the possibility of manipulating key genes within the P. brassicae genome that could improve our knowledge on hostepathogen interaction. [ABSTRACT FROM AUTHOR]

The qualified presumption of safety (QPS) process was developed to provide a safety assessment approach for microorganisms intended for use in food or feed chains. In the period covered by this statement, no new information was found that would change the status of previously recommended QPS TUs. The TUs in the QPS list were updated based on a verification, against their respective authoritative databases, of the correctness of the names and completeness of synonyms. A new procedure has been established to ensure the TUs are kept up to date in relation to recent taxonomical insights. Of 81 microorganisms notified to EFSA between October 2023 and March 2024 (45 as feed additives, 25 as food enzymes or additives, 11 as novel foods), 75 were not evaluated because: 15 were filamentous fungi, 1 was Enterococcus faecium, 10 were Escherichia coli, 1 was a Streptomyces (all excluded from the QPS evaluation) and 46 were TUs that already have a QPS status. Two of the other eight notifications were already evaluated for a possible QPS status in the previous Panel Statement: Heyndrickxia faecalis (previously Weizmannia faecalis) and Serratia marcescens. One was notified at genus level so could not be assessed for QPS status. The other five notifications belonging to five TUs were assessed for possible QPS status. Akkermansia muciniphila and Actinomadura roseirufa were still not recommended for QPS status due to safety concerns. Rhizobium radiobacter can be recommended for QPS status with the qualification for production purposes. Microbacterium arborescens and Burkholderia stagnalis cannot be included in the QPS list due to a lack of body of knowledge for its use in the food and feed chain and for B. stagnalis also due to safety concerns. A. roseirufa and B. stagnalis have been excluded from further QPS assessment. [ABSTRACT FROM AUTHOR]

SUMMARY: Uncovering the function of phytopathogen effectors is crucial for understanding mechanisms of pathogen pathogenicity and for improving our ability to protect plants from diseases. An increasing number of effectors have been predicted in various plant pathogens. Functional characterization of these effectors has become a major focus in the study of plant–pathogen interactions. In this study, we designed a novel screening system that combines the TMV (tobacco mosaic virus)‐GFP vector and Agrobacterium‐mediated transient expression in the model plant Nicotiana benthamiana. This system enables the rapid identification of effectors that interfere with plant immunity. The biological function of these effectors can be easily evaluated by observing the GFP fluorescence signal using a UV lamp within just a few days. To evaluate the TMV‐GFP system, we initially tested it with well‐described virulence and avirulence type III effectors from the bacterial pathogen Ralstonia solanacearum. After proving the accuracy and efficiency of the TMV‐GFP system, we successfully screened a novel virulence effector, RipS1, using this approach. Furthermore, using the TMV‐GFP system, we reproduced consistent results with previously known cytoplasmic effectors from a diverse array of pathogens. Additionally, we demonstrated the effectiveness of the TMV‐GFP system in identifying apoplastic effectors. The easy operation, time‐saving nature, broad effectiveness, and low technical requirements of the TMV‐GFP system make it a promising approach for high‐throughput screening of effectors with immune interference activity from various pathogens. Significance Statement: The development of a robust high‐throughput functional screening assay for pathogen effectors will facilitate the study in plant‐pathogen interactions. We established a novel screening system using the TMV‐GFP vector, enabling the rapid identification of effectors that interfere with plant immunity by observing GFP fluorescence under a UV lamp within a few days. The TMV‐GFP system is a promising approach for high‐throughput screening of effectors from diverse pathogens. [ABSTRACT FROM AUTHOR]

Genetic transformation is helpful in enhancing crops, utilising promoters that can be constitutive, inducible, or tissue-specific. However, the use of constitutive promoters may hinder plant growth due to energy consumption during cellular processes. To optimise transgene effects, tissue-specific promoters like root-specific ones prove valuable in addressing root-related issues and enhancing productivity. Yet, identified root-specific promoters in crop are limited. To address this gap, the expression pattern of the root-specific SlREO promoter was examined across various crops. Sequencing confirmed its identity and high homology (99%) with the NCBI database, distinct from other plants tested. Using the PLACE database, six motifs associated with root expression were identified, along with several other important elements. The 2.4 kb SlREO promoter was linked to a ß-glucuronidase (GUS) reporter gene alongside the CaMV35S promoter in pRI 201-AN- GUS vectors to study its expression. Histochemistry revealed strong root-specific expression in tomato (Solanum lycopersicum) root tissues and limited expression in stems. However, the SlREO promoter did not consistently maintain its root-specific expression in other plants. Conversely, the CaMV35S promoter exhibited constitutive expression across all tissues in various plants. This study underscores the potential of the SlREO promoter as a root-specific regulatory element, offering avenues for improving crops, particularly against environmental stresses. The improvement of tomato (Solanum lycopersicum) for various biotic and abiotic traits that affect the roots functions is vital, and root-specific promoters are tools to improve the gene expression of transgenes. SlREO is one tissue-specific promoters in tomato. This study cloned and characterised SlREO in Indian tomato cultivar, and evaluated its activity in various crops. Transgenic studies suggest that SlREO is a crop-dependent tissue-specific promoter that cannot be used to improve the expression of genes in other crops. This article belongs to the Collection Functional Genomics for Developing Climate Resilient Crops. [ABSTRACT FROM AUTHOR]

Seed endophytes played a crucial role on host plants stress tolerance and heavy metal (HM) accumulation. Dysphania ambrosioides is a hyperaccumulator and showed strong tolerance and extraordinary accumulation capacities of multiple HMs. However, little is known about its seed endophytes response to field HM-contamination, and its role on host plants HM tolerance and accumulation. In this study, the seed endophytic community of D. ambrosioides from HM-contaminated area (H) and non-contaminated area (N) were investigated by both culture-dependent and independent methods. Moreover, Cd tolerance and the plant growth promoting (PGP) traits of dominant endophytes from site H and N were evaluated. The results showed that in both studies, HM-contamination reduced the diversity and richness of endophytic community and changed the most dominant endophyte, but increased resistant species abundance. By functional trait assessments, a great number of dominant endophytes displayed multiple PGP traits and Cd tolerance. Interestingly, soil HM-contamination significantly increased the percentage of Cd tolerance isolates of Agrobacterium and Epicoccum, but significantly decreased the ration of Agrobacterium with the siderophore production ability. However, the other PGP traits of isolates from site H and N showed no significant difference. Therefore, it was suggested that D. ambrosioides might improve its HM tolerance and accumulation through harboring more HM-resistant endophytes rather than PGP endophytes, but to prove this, more work need to be conducted in the future. [ABSTRACT FROM AUTHOR]

Vitex negundo L. is an aromatic, woody, blooming shrub in the Lamiaceae family which can grow into a small tree. Traditionally, V. negundo root has been used to cure diabetes, colic, boils, leprosy, and rheumatism. Keeping the importance of its roots in mind, an attempt has been taken for development of a protocol for efficient hairy root proliferation system. The Agrobacterium rhizogenes strains (MTCC 532 and MTCC 2364) were used for hairy root induction. For A. rhizogenes infection, both in vitro and in vivo leaves as well as internodes were used as explant. In vitro leaves and internodal explants were obtained by the inoculation of matured nodal segments on the optimum medium [MS + 2.0 mg/L N6-Benzylaminopurine (BAP)] with c.a. 91.6% shoot regeneration and an average of 8.1 shoots per explants. In vitro leaf showed best hairy root induction followed by in vitro internode on ½ MS medium augmented with acetosyringone. Highest transformation efficiency was achieved using MTCC 2364 strain, while no transformation was observed in MTCC 532 strain. Different factors affecting transformation including co-cultivation period, infection time and optical density (O.D.) value were standardized. The highest efficacy, 88.8% hairy root induction was observed in in vitro leaves infected by MTCC 2364 for 60 minutes infection time with an O.D. value of 0.29 maintained over a 44-48 hours of co-cultivation period. The prescribed protocol may be used as a reference for development of industrial scale hairy root production for bioactive compound located in root of V. negundo. [ABSTRACT FROM AUTHOR]

Simple Summary: Commensal bacteria in the vaginal canal are vital to reproductive health. Infertility or severe urinary tract infections may result from vaginal dysbiosis. This research examined the aerobic bacterial flora in vaginal samples from bitches during antepartum, postpartum, and Lactatio sine graviditate. The 100 vaginal samples analyzed yielded 82% positive microbiological results and 18% negative. Microbiologic profile revealed 17 genera. Micrococcaceae, Staphylococcaceae, Morganellaceae, Bacillaceae, and Rhizobiaceae were the most isolated bacteria. The first strains of Agrobacterium radiobacter, Ochrobactrum anthropi, Chromobacterium violaceum, Burkholderia mallei, Bacillus pumllus, and Staphylococcus xylosus were recovered from vaginal secretions. Microbiological studies show that breastfeeding bitches' vaginal discharge is variable and may be impacted by coitus, sampling season, age, and reproductive status. The vaginal tract comprises commensal microorganisms, which play an essential role in the health of the reproductive tract. Any dysbiosis in the vaginal microenvironment may lead to severe urinary tract infections or even infertility. This study aimed to evaluate the aerobic bacterial flora isolated from vaginal samples from 100 lactating bitches in the antepartum period (n = 3), postpartum period (n = 80), and with Lactatio sine graviditate (n = 17). Before vaginal swabs, all the bitches went through a gynecology consult, along with milk and blood sampling. Standard microbiological techniques were used for bacterial isolation. Among the 100 vaginal samples analyzed, 82% had a positive microbiological outcome, while 18% were negative. The microbiologic profile listed 17 different genera. The main isolated bacterial families were Micrococcaceae, Staphylococcaceae, Morganellaceae, Bacillaceae, and Rhizobiaceae. At the same time, strains like Agrobacterium radiobacter, Ochrobactrum anthropi, Chromobacterium violaceum, Burkholderia mallei, Bacillus pumllus, or Staphylococcus xylosus were isolated for the first time from the vaginal secretion of lactating bitches. The microbiological data demonstrates that lactating bitches' vaginal discharge is heterogeneous and may be affected by coitus, sampling season, age, and reproductive status. [ABSTRACT FROM AUTHOR]

Betalain has received increased attention because of its high nutritional value and crucial physiological functions. Based on the elucidation of its core biosynthetic pathway, betalain can be produced in additional plants by metabolic engineering. Synthesis of betalain in carrot (Daucus carota L.) can improve its nutritional quality and economic value by extracting betalain from the fleshy root, non-edible part, and processing residue of carrot. In this study, two different constructs, namely, pYB:mCD (AomelOS, BvCYP76AD1S, and BvDODA1S) and pYB:CDD (BvCYP76AD1S, BvDODA1S, and MjcDOPA5GTS), were introduced into carrot for betanin synthesis by Agrobacterium-mediated transformation. Betanin can be synthetized in both transgenic calli, and pYB:mCD-transgenic callus can be used to produce betacyanin by suspension culture. However, pYB:mCD-transgenic seedlings can synthetize betanin only by tyrosine feeding. The pYB:CDD-transgenic lines can synthetize betanin in whole plants. The betanin content in fleshy root of pYB:CDD-transgenic carrot was (63.4 ± 9) μg.g-1 fresh weight according to quantitative analysis. These betanin-producing carrot plant materials can be used to synthesize betanin for industrial application or consumption as dietary sources. [ABSTRACT FROM AUTHOR]

Phytochromes are photoreceptor proteins with a bilin chromophore that undergo photoconversion between two spectrally different forms, Pr and Pfr. Three domains, termed PAS, GAF, and PHY domains, constitute the N‐terminal photosensory chromophore module (PCM); the C‐terminus is often a histidine kinase module. In the Agrobacterium fabrum phytochrome Agp1, the autophosphorylation activity of the histidine kinase is high in the Pr and low in the Pfr form. Crystal structure analyses of PCMs suggest flexibility around position 308 in the Pr but not in the Pfr form. Here, we performed time‐resolved fluorescence anisotropy measurements with different Agp1 mutants, each with a single cysteine residue at various positions. The fluorophore label Atto‐488 was attached to each mutant, and time‐resolved fluorescence anisotropy was measured in the Pr and Pfr forms. Fluorescence anisotropy curves were fitted with biexponential functions. Differences in the amplitude A2 of the second component between the PCM and the full‐length variant indicate a mechanical coupling between position 362 and the histidine kinase. Pr‐to‐Pfr photoconversion induced no significant changes in the time constant t2 at any position. An intermediate t2 value at position 295, which is located in a compact environment, suggests flexibility around the nearby position 308 in Pr and in Pfr. [ABSTRACT FROM AUTHOR]

A key goal in current research on industrial curdlan production is the expansion of carbon sources for fermentation. In this study, a recombinant bacterial strain, sp-AmyAXCC, capable of fermenting and synthesizing curdlan using dextrin as a carbon source, was produced via heterologous expression of IPTG-inducible α-amylase from Xanthomonas campestris NRRL B-1459 in Agrobacterium tumefaciens ATCC31749. External expression of the enzyme was confirmed by western blotting, and the expression levels of exogenous proteins during the fermentation process were monitored. Additionally, the properties of the curdlan product were characterized using attenuated total reflectance-Fourier transform infrared spectroscopy and X-ray diffraction. The recombinant strain produced curdlan at a titer of 30.40 ± 0.14 g/L, gel strength of 703.5 ± 34.2 g/cm2, and a molecular weight of 3.58 × 106 Da, which is 33% greater than the molecular weight of native curdlan (2.69 × 106 Da). In the batch fermentation of sp-AmyAXCC with 12% dextrin as a carbon source, the titer of curdlan was 66.7 g/L with a yield of 0.56 g/g, and a productivity rate of 0.62 g/L/h at 108 h. The results of this study expand the substrate spectrum for Agrobacterium fermentation in curdlan production and provides guidance for further industrialization of curdlan production. [ABSTRACT FROM AUTHOR]

Rhododendron delavayi is an evergreen tree species, as a woody plant belonging to Ericaceae characterized by its long lifecycle and high ornamental value. Genetic engineering has made remarkable progress in improving various plant species. However, applying these advancements to Rhododendron remains a complex endeavor, primarily due to challenges associated with genetic transformation technology. In this study, the optimum transformation conditions for Agrobacterium infection of R. delavayi leaf disc were established as follows: the pre-culture time was 0 d, Agrobacterium liquid concentration OD600 = 0.7, infection time was 60 min, Acetosyringone concentration 20 mg/L, and co-culture time was 6 d. After removal of bacteria, first screening culture was 30 days with medium containing Kanamycin (Kan) at 30 mg/L and Cefotaxime (Cef) at 500 mg/L. Subsequent culture was for 60 days with medium supplemented with Kan at 30 mg/L and Cef at 400 mg/Ls. Third screening culture was for 90 days with medium containing Kan at 40 mg/L and Cef at 300 mg/L. Positive transgenic materials were identified after 120 days culture. Gene cloning and transformation proceeded with two MYB genes, RdMYB8 and RdMYB24, which had been isolated from petals of R. delavayi. These genes were individually inserted into overexpression vectors and then successfully transformed into Agrobacterium GV3101. Leaf discs of R. delavayi were inoculated with Agrobacterium carrying the respective genes, resulting in the successful acquisition of positive transgenic materials. The transgenic callus exhibited a red coloration and showed an increase in the total anthocyanin content compared to the control group. The study suggests that RdMYB8 and RdMYB24 are involved in anthocyanin synthesis in R. delavayi, and this Agrobacterium-mediated transformation system holds promise as a fundamental tool for advancing genetic engineering not only in R. delavayi.Key message: Leaf discs from Rhododendron delavayi are the optimal recipient material for Agrobacterium-mediated transformation. The transgenic callus overexpressing RdMYB8 exhibits enhanced anthocyanin biosynthesis. [ABSTRACT FROM AUTHOR]

Most legumes are able to develop a root nodule symbiosis in association with proteobacteria collectively called rhizobia. Among them, the tropical species Aeschynomene evenia has the remarkable property of being nodulated by photosynthetic Rhizobia without the intervention of Nod Factors (NodF). Thereby, A. evenia has emerged as a working model for investigating the NodF-independent symbiosis. Despite the availability of numerous resources and tools to study the molecular basis of this atypical symbiosis, the lack of a transformation system based on Agrobacterium tumefaciens significantly limits the range of functional approaches. In this report, we present the development of a stable genetic transformation procedure for A. evenia. We first assessed its regeneration capability and found that a combination of two growth regulators, NAA (= Naphthalene Acetic Acid) and BAP (= 6-BenzylAminoPurine) allows the induction of budding calli from epicotyls, hypocotyls and cotyledons with a high efficiency in media containing 0,5 μM NAA (up to 100% of calli with continuous stem proliferation). To optimize the generation of transgenic lines, we employed A. tumefaciens strain EHA105 harboring a binary vector carrying the hygromycin resistance gene and the mCherry fluorescent marker. Epicotyls and hypocotyls were used as the starting material for this process. We have found that one growth medium containing a combination of NAA (0,5 μM) and BAP (2,2 μM) was sufficient to induce callogenesis and A. tumefaciens strain EHA105 was sufficiently virulent to yield a high number of transformed calli. This simple and efficient method constitutes a valuable tool that will greatly facilitate the functional studies in NodF-independent symbiosis. [ABSTRACT FROM AUTHOR]

Under changing climatic scenarios, grassland conservation and development have become imperative to impart functional sustainability to their ecosystem services. These goals could be effectively and efficiently achieved with targeted genetic improvement of native grass species. To the best of our literature search, very scant research findings are available pertaining to gene editing of noncultivated grass species (switch grass, wild sugarcane, Prairie cordgrass, Bermuda grass, Chinese silver grass, etc.) prevalent in natural and semi-natural grasslands. Thus, to explore this novel research aspect, this study purposes that gene editing techniques employed for improvement of cultivated grasses especially sugarcane might be used for non-cultivated grasses as well. Our hypothesis behind suggesting sugarcane as a model crop for genetic improvement of noncultivated grasses is the intricacy of gene editing owing to polyploidy and aneuploidy compared to other cultivated grasses (rice, wheat, barley, maize, etc.). Another reason is that genome editing protocols in sugarcane (x = 10-13) have been developed and optimized, taking into consideration the high level of genetic redundancy. Thus, as per our knowledge, this review is the first study that objectively evaluates the concept and functioning of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 technique in sugarcane regarding high versatility, target specificity, efficiency, design simplicity, and multiplexing capacity in order to explore novel research perspectives for gene editing of non-cultivated grasses against biotic and abiotic stresses. Additionally, pronounced challenges confronting sugarcane gene editing have resulted in the development of different variants (Cas9, Cas12a, Cas12b, and SpRY) of the CRISPR tool, whose technicalities have also been critically assessed. Moreover, different limitations of this technique that could emerge during gene editing of non-cultivated grass species have also been highlighted. [ABSTRACT FROM AUTHOR]

Tomato (Solanum lycopersicum L.) is one of the best suited plants for genetic studies and is widely used for introduction of economically important and novel genes through genetic engineering. This research was conducted in Balochistan Agriculture College in 2021-2022. In this study comprehensive research was conducted in two phases in first phase an in-vitro regeneration protocol using four tomato genotypes was established and in second phase an agrobacterium mediated transformation protocol was established at Baluchistan Agriculture College Quetta. The desired results for surface sterilization were achieved when seeds were subjected to combination of surface sterilant; 70% ethanol 1 minute followed by + 5% sodium hypochlorite + 100 µL tween-20 for 30 minutes. The cotyledonary leaf explants were treated with plant growth hormones zeatin, IAA and BAP at 1, 2 and 3 mg/l concentrations each. The best response to morphogenesis was measured for zeatin 2 mg/l in all four genotypes; Matina 93%, Rio Grande 83%, VF-001 80% and VF-002 70%. Root regeneration was observed in the hormone free subculture media simultaneously along the shoot elongation. Healthy plantlets were transferred to pots for hardening and acclimatization. Afterwards normal fruits were harvested at maturity. In second phase the same four tomato genotypes were used for transformation via agrobacterium strain LBA4404 (containing hptII marker gene). Highest percentage of hygromycin positive explants was found at OD600 0.60, 25 minutes incubation period and 48 hours co-cultivation while minimum hygromycin positive percentage was observed at OD600 of 0.40. Screening for putative transgenic shoots was done at selection pressure of 4mg hygromycin/l and two of the four genotypes Rio Grande and Matina stood against this selection pressure for more than three weeks and were declared transformed shoots. Future recommendation: the transformation protocol established and best genotype found shall be used for tomato genetic enhancement. [ABSTRACT FROM AUTHOR]

• Agrobacterium sp. T3F4 oxidized Se(-II)/Se(0) into Se(IV) and As(III) into As(V). • Strain T3F4 increased Se bioavailability and immobilized As in seleniferous As-polluted soil. • Strain T3F4 promoted Se uptake and decreased As accumulation in Brassica rapa L. • Se(IV) alleviated As(V) toxicity via competitive uptake by B. rapa. Toxic arsenic (As) and trace element selenium (Se) are transformed by microorganisms but their complex interactions in soil-plant systems have not been fully understood. An As- and Se- oxidizing bacterium, Agrobacterium sp. T3F4, was applied to a native seleniferous As-polluted soil to investigate As/Se uptake by the vegetable Brassica rapa L. and As-Se interaction as mediated by strain T3F4. The Se content in the aboveground plants was significantly enhanced by 34.1%, but the As content was significantly decreased by 20.5% in the T3F4-inoculated pot culture compared to the control (P < 0.05). Similar result was shown in treatment with additional 5 mg/kg of Se(IV) in soil. In addition, the As contents in roots were significantly decreased by more than 35% under T3F4 or Se(IV) treatments (P<0.05). Analysis of As-Se-bacterium interaction in a soil simulation experiment showed that the bioavailability of Se significantly increased and As was immobilized with the addition of the T3F4 strain (P < 0.05). Furthermore, an As/Se co-exposure hydroponic experiment demonstrated that As uptake and accumulation in plants was reduced by increasing Se(IV) concentrations. The 50% growth inhibition concentration (IC50) values for As in plants were increased about one-fold and two-fold under co-exposure with 5 and 10 µmol/L Se(IV), respectively. In conclusion, strain T3F4 improves Se uptake but decreases As uptake by plants via oxidation of As and Se, resulting in decrease of soil As bioavailability and As/Se competitive absorption by plants. This provides a potential bioremediation strategy for Se biofortification and As immobilization in As-polluted soil. [Display omitted] [ABSTRACT FROM AUTHOR]

In August 2020, the unusual appearance of crown gall symptoms was observed on the tobacco plants (hybrid PVH2310) grown in fields in the Golubinci (Srem district, Serbia) locality. The causal agent isolated from galls located on tobacco roots formed circular, convex, and glistening light blue colonies, and then dark to olive-green-colored bacterial colonies on a semi-selective D1 medium. Molecular analysis based on multiplex PCR and multi-locus sequence analysis (MLSA) using concatenated sequences of the atpD, dnaK, glnA, and rpoB genes as well as 16S rRNA identified Serbian tobacco isolates such as Agrobacterium tumefaciens (biovar 1). Two duplex PCR methods confirmed the presence of the virD2 and virC genes in tobacco isolates. Pathogenicity tests performed on carrot discs and squash fruits resulted in tumor/gall formation after 12 to 16 days post inoculation, respectively. Pathogenicity was also confirmed on tobacco plants, where isolates caused tumor development 21−25 days after inoculation. API 50 CH generated results regarding the biochemical features of the Serbian tobacco isolates. As A. tumefaciens (biovar 1) as a cause of tobacco crown gall has previously been documented solely in Japan, there is presently no data on its wider occurrence. Therefore, this first detailed investigation of A. tumefaciens isolated from naturally infected tobacco in Serbia will contribute to a better understanding of it at the global level. [ABSTRACT FROM AUTHOR]

Phosphorus is one of the major biogenic elements. Its inflow facilitates eutrophication of lake water. In aquatic ecosystems, phosphorus is present mostly in organic compounds. Ability of aquatic microorganisms to assimilate phosphorus from organophosphorous compounds results from activity of alkaline phosphatases; activity of these enzymes may be an indicator of the state of the ecosystem, phosphate load, and water quality. In the present work, alkaline phosphatase activity (APA) and abundance of phosphatase-active bacteria (PAB) in Lake Baikal pelagic zone and in the mouths of its major tributaries was studied. In the pelagic zone, APA and PAB abundance decreased with depth, indicating that the main processes of phosphate generation occurred in the trophic layer of the lake. In the main tributaries, both APA and PAB abundance were considerably higher than in the pelagic zone. These results indicate active biochemical processes of transformation of organophosphorous compounds occur in the estuarine zones of the rivers. The degradation processes result in regeneration of phosphates, which are completely incorporated in the biological turnover, providing for phytoplankton development. [ABSTRACT FROM AUTHOR]

Agrobacterium tumefaciens mediated plant transformation is a versatile tool for plant genetic engineering following its discovery nearly half a century ago. Numerous modifications were made in its application to increase efficiency, especially in the recalcitrant major cereals plants. Recent breakthroughs in transformation efficiency continue its role as a mainstream technique in CRISPR/Cas-based genome editing and gene stacking. These modifications led to higher transformation frequency and lower but more stable transgene copies with the capability to revolutionize modern agriculture. In this review, we provide a brief overview of the history of Agrobacterium-mediated plant transformation and focus on the most recent progress to improve the system in both the Agrobacterium and the host recipient. A promising future for transformation in biotechnology and agriculture is predicted. [ABSTRACT FROM AUTHOR]

Plant genetic transformation has emerged as a key platform for both fundamental and translational research. The reporter genes are widely used to visualize gene expression patterns, promoter analysis, localization, and stable transformation studies. Marker genes would be expected to exhibit characteristics such as robustness, chemical affordability, visualization without any special equipment, and a non-destructive detection approach. Betalains are tyrosine-derived red-yellow pigments found in the caryophyllale plant group that are capable of being used as a visual reporter marker to identify plant transformation. In this study, we transformed Nicotiana tabacum with a RUBY vector construct consisting of three gene clusters, CYP76AD1, DODA, and 5-GT, along with a hygromycin selective marker for visible reporter study. After three weeks of transformation, the betalain pigments were easily detected in the explant leaf tissue. The RUBY-transformed shoots displayed a reddish color that could easily be distinguished from the non-transgenic green shoots during tissue culture. Besides the reddish color, RUBY transformed shoots displayed phenotypically similar to nontransformed shoots and did not show any negative effect during multiple shoots differentiation, proliferation, regeneration, and in the initial stage of plant development. However, the RUBY-expressed plants exhibited slightly reduced plant height and delayed flowering time compared to the wild-type plant. Transgenic plants were further confirmed by PCR amplification of a 341 bp inter DNA fragment of the gene construct. The betalain pigments were observed during all stages of plant development (stem, root, and leaves), beginning with the in vitro shoot buds of genetically transformed explants. Additionally, microstructural analysis of the leaf clearly indicates that betalain pigments accumulated more in the vascular bundle, lamina, trichome, and especially stomata guard cell. A comparative cross-section of the stem and root clearly revealed the presence of betalain pigment within the cortex, endodermis, epidermis, xylem, and phloem tissues of the RUBY-transformed plant. Our findings provide conclusive evidence for the potential application of the RUBY for the visual screening of genetic transformations.Key message: The transgenic tobacco (Nicotiana tabacum L.) plants were produced with a non-invasive visual reporter (RUBY) gene. The accumulation of betalin pigment was observed in various plant parts during the different developmental stages. [ABSTRACT FROM AUTHOR]

With the changing environmental conditions due to natural and anthropogenic activities, plants are inevitably subjected to various biotic and abiotic stresses. These create immense pressure for their survival during unfavourable conditions. Drought is a significant abiotic stress that hinders plant development and productivity. Metallothioneins (MTs) are cytoplasmic proteins that actively regulate metal homeostasis, detoxification, developmental processes, and different plant abiotic stresses. Mustard is an economically important oilseed crop confronting drought stress at different life cycle stages. This study emphasizes the role of the chickpea Metallothionein1 (MT1) gene for drought stress tolerance in mustard (Brassica juncea L.). The qRT-PCR analysis showed a higher expression level of chickpea MT1 gene up to 10 to 14 folds in T1 generation transgenic lines of mustard under drought stress as compared to wild-type control plants. Further, the various antioxidant enzymes ascorbate peroxidase (APX), catalase (CAT), glutathione S-transferase (GST), superoxide dismutase (SOD), monodehydroascorbate reductase (MDHAR) showed enhanced activities in developed over-expressing lines. The low levels of stress markers, TBARS and H2O2, were reported during the study, enabling plants to sustain under harsh conditions. Moreover, the physiological parameters water use efficiency (WUE), stomatal conductance (gs), photosynthesis rate (A), PSII (Fv/Fm), and transpiration (E) were also found improved in transgenic lines, which assisted in enduring varying stressed conditions. The study concludes that the over-expression of the chickpea MT1 gene increases the drought tolerance capacity in mustard by regulating biochemical and physiological traits. Therefore, with the aid of genetic engineering, this could be further explored to develop crop varieties with improved stress tolerance under drought conditions.Key message: The chickpea metallothionein1 (MT1) gene overexpression in mustard assists in counteracting drought stress through regulation of antioxidant enzyme activities, different physiological traits, and reduction in oxidative load, therefore improving stress tolerance. [ABSTRACT FROM AUTHOR]

This study aims to establish an Agrobacterium-mediated transformation system for use with the 'MiniMax'soybean cultivar. MiniMax is a mutant soybean whose growth cycle is around 90 days, half that of most other soybean varieties, making it an optimal model cultivar to test genes of interest before investing in modification of elite lines. We describe an efficient protocol for Agrobacterium-mediated transformation using MiniMax seeds. It uses a modified 'half seed' regeneration protocol for transgenic soybean production, utilizing the rapid generation MiniMax variety to obtain T1 seeds in approximately 145 days. Addition of phloroglucinol (PG) to the regeneration protocol was key to obtaining high-efficiency rooting of the regenerated shoots. Transfer to soil was accomplished using an organic soil amendment containing nutrients and mycorrhiza for plants to thrive in the greenhouse. This combination of genotype and stimulants provides a transformation protocol to genetically engineer MiniMax seeds with a transgenic lab-to-greenhouse production efficiency of 4.0%. This is the first report of MiniMax soybean whole plant transformation and heritable T1 transmission. This protocol provides an ideal resource for enhancing the genetic transformation of any soybean cultivar. [ABSTRACT FROM AUTHOR]

The type VI secretion system (T6SS) assembles into a contractile nanomachine to inject effectors across bacterial membranes for secretion. The Agrobacterium tumefaciens species complex is a group of soil inhabitants and phytopathogens that deploys T6SS as an antibacterial weapon against bacterial competitors at both inter-species and intra-species levels. The A. tumefaciens strain 1D1609 genome encodes one main T6SS gene cluster and four vrgG genes (i.e., vgrGa-d), each encoding a spike protein as an effector carrier. A previous study reported that vgrGa-associated gene 2, named v2a, encodes a His-Me finger nuclease toxin (also named HNH/ENDO VII nuclease), contributing to DNase-mediated antibacterial activity. However, the functions and roles of other putative effectors remain unknown. In this study, we identified vgrGc-associated gene 2 (v2c) that encodes another His-Me finger nuclease but with a distinct Serine Histidine Histidine (SHH) motif that differs from the AHH motif of V2a. We demonstrated that the ectopic expression of V2c caused growth inhibition, plasmid DNA degradation, and cell elongation in Escherichia coli using DNAse activity assay and fluorescence microscopy. The cognate immunity protein, V3c, neutralizes the DNase activity and rescues the phenotypes of growth inhibition and cell elongation. Ectopic expression of V2c DNase-inactive variants retains the cell elongation phenotype, while V2a induces cell elongation in a DNase-mediated manner. We also showed that the amino acids of conserved SHH and HNH motifs are responsible for the V2c DNase activity in vivo and in vitro. Notably, V2c also mediated the DNA degradation and cell elongation of the target cell in the context of interbacterial competition. Importantly, V2a and V2c exhibit different capacities against different bacterial species and function synergistically to exert stronger antibacterial activity against the soft rot phytopathogen, Dickeya dadantii. [ABSTRACT FROM AUTHOR]

Okra is an important vegetable crop of the Malvaceae family and is infected by varying numbers of viruses of the genus Begomovirus. Regardless of the importance of the crop, very little consideration has been given to its genetic improvement. RNA interference (RNAi), a potent biotechnological tool, is known to control Begomovirus in many crops. For the implementation of successful RNAi, there is a need for an efficient genetic transformation system in okra. In the present study, we developed a procedure for Agrobacterium-mediated tissue culture–dependent regeneration of okra plants for the application of RNAi. Eleven transgenic okra RNAi plants were regenerated by utilising hypocotyls as explants. Transformed plants were screened with hygromycin at the regeneration stage and the presence of transgenes (AC1, AC2 & AC4 codes for replication-associated protein, transcriptional activator protein and suppressor of PTGS) in putative transformed plants was confirmed by polymerase chain reaction (PCR). Wild and transgenic lines were challenged with a dimeric Begomovirus clone or viruliferous whiteflies and the level of resistance was estimated with quantitative real-time reverse transcriptase PCR (qRT-PCR) by utilising viral gene–specific primers. The resistant transgenic lines accumulated very low titres of viral gene products according to the qRT-PCR assays compared to the control plants. This is the first report of tissue culture–mediated RNAi-derived resistance in okra against Begomovirus infection. [ABSTRACT FROM AUTHOR]

Plant viral movement proteins (MPs) serve a critical role in facilitating intercellular transport, pivotal for propagating infections from afflicted cells to their neighboring counterparts. Leveraging MPs within transient expression systems holds the potential to augment the expanse of protein expression zones. In this investigation, we present evidence showcasing the synergistic effects of fusing a transient expression system vector with the movement protein of tomato mosaic virus (ToMV MP), thus broadening the scope of protein expression within plant stem tissues. Through agroinfiltration, we achieved transient expression of green fluorescent protein (GFP) in tomato stem tissues, which was subsequently confirmed via immunoblotting with an anti-GFP antibody. Quantitative analysis unveiled that the co-expression of ToMV MP alongside GFP led to an over twofold increase in GFP production within stem tissues distal from the primary infection site. This research underscores the efficacy of combining ToMV MP with transient expression systems as a potent strategy for achieving transient expression of target genes within tomato plants. [ABSTRACT FROM AUTHOR]

Tea has historically been one of the most popular beverages, and it is currently an economically significant crop cultivated in over 50 countries. The Northwestern Caucasus is one of the northernmost regions for industrial tea cultivation worldwide. The domestication of the tea plant in this region took approximately 150 years, during which plantations spreading from the Ozurgeti region in northern Georgia to the southern city of Maykop in Russia. Consequently, tea plantations in the Northern Caucasus can serve as a source of unique genotypes with exceptional cold tolerance. Tea plants are known to be recalcitrant to Agrobacterium-mediated transfection. Research into optimal transfection and regeneration methodologies, as well as the identification of tea varieties with enhanced transformation efficiency, is an advanced strategy for improving tea plant culture. The aim of this study was to search for the optimal Agrobacterium tumefaciens-mediated transfection protocol for the Kolkhida tea variety. As a result of optimizing the transfection medium with potassium phosphate buffer at the stages of pre-inoculation, inoculation and co-cultivation, the restoration of normal morphology and improvement in the attachment of Agrobacterium cells to the surface of tea explants were observed by scanning electron microscopy. And an effective method of high-efficiency Agrobacteria tumefaciens-mediated transfection of the best local tea cultivar, Kolkhida, was demonstrated for the first time. [ABSTRACT FROM AUTHOR]

Industrial hemp Cannabis sativa L. is an economically important crop mostly grown for its fiber, oil, and seeds. Due to its increasing applications in the pharmaceutical industry and a lack of knowledge of gene functions in cannabinoid biosynthesis pathways, developing an efficient transformation platform for the genetic engineering of industrial hemp has become necessary to enable functional genomic and industrial application studies. A critical step in the development of Agrobacterium tumefaciens-mediated transformation in the hemp genus is the establishment of optimal conditions for T-DNA gene delivery into different explants from which whole plantlets can be regenerated. As a first step in the development of a successful Agrobacterium tumefaciens-mediated transformation method for hemp gene editing, the factors influencing the successful T-DNA integration and expression (as measured by transient β-glucuronidase (GUS) and Green Florescent Protein (GFP) expression) were investigated. In this study, the parameters for an agroinfiltration system in hemp, which applies to the stable transformation method, were optimized. In the present study, we tested different explants, such as 1- to 3-week-old leaves, cotyledons, hypocotyls, root segments, nodal parts, and 2- to 3-week-old leaf-derived calli. We observed that the 3-week-old leaves were the best explant for transient gene expression. Fully expanded 2- to 3-week-old leaf explants, in combination with 30 min of immersion time, 60 µM silver nitrate, 0.5 µM calcium chloride, 150 µM natural phenolic compound acetosyringone, and a bacterial density of OD600nm = 0.4 resulted in the highest GUS and GFP expression. The improved method of genetic transformation established in the present study will be useful for the introduction of foreign genes of interest, using the latest technologies such as genome editing, and studying gene functions that regulate secondary metabolites in hemp. [ABSTRACT FROM AUTHOR]

The octoploid-cultivated strawberry variety Benihope (Fragaria × ananassa Duch cv. Benihope) is an important commercial plant. It is highly susceptible to different diseases, which ultimately leads to a reduction in yield. Gene-editing methods, such as CRISPR/Cas9, demonstrate potential for improving disease resistance in the strawberry cv. Benihope. Establishing a plant regeneration system suitable for CRISPR/Cas9 gene editing is crucial for obtaining transgenic plants on a large scale. This research established a callus induction and plant regeneration system for Agrobacterium-mediated CRISPR/Cas9 gene editing in strawberry cv. Benihope by evaluating multiple types of explants and various plant growth regulators throughout the entire tissue culture process. The results showed that the efficiency of callus induction is strongly influenced by the type of explant and is highly sensitive to the combination of plant growth regulators. Among the different plant growth regulators employed, thidiazuron (TDZ), in combination with 2,4-dichlorophenoxyacetic acid (2,4-D), effectively induced callus formation and plant regeneration from explants derived from nutrient tissues such as runner tips and crowns. In addition, the regeneration experiment demonstrated that the addition of polyvinylpyrrolidone (PVPP) to the shoot regeneration medium could inhibit tissue browning. The gene-edited plants in which some or all of the Fvb7-1, Fvb7-2, Fvb7-3, and Fvb7-4 genes in the MLO (Mildew resistance Locus O) gene family were knocked out by CRISPR/Cas9 system were obtained by applying the plant regeneration system developed in this study. [ABSTRACT FROM AUTHOR]