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Effect of ACC deaminase and GABA transaminase activity on the transfer of T-DNA. (A) Map of a plasmid for the expression of ACC deaminase (acdS) and GABA transaminase (gabT) in A. tumefaciens. lacP, lac gene promoter from E. coli; acdS, ACC deaminase gene from Psedomonas sp (Sheehy et al., 1991, Accession No. M73488); gabT, GABA transaminase gene from E. coli (Accession No. CP040667); pBBR1 Rep, replication protein for the broad-host-range plasmid pBBR1 from Bordetella bronchiseptica; pBBR1 oriV, replication origin of the broad-host-range plasmid pBBR1 from B. bronchiseptica; pBBR1 Rep, protein for replication required by pBBR1 oriV, GmR, Gentamicin resistance gene. (B) Growth curve of A. tumefaciens. Open and solid circles represent A. tumefaciens C and V1, respectively. Open and solid squares represent V3 and V4, respectively. (C) ACC deaminase activity in A. tumefaciens. (D) Detection of GABA transaminase activity in A. tumefaciens. C, GV2260 (pBBRMCS1-5); V1, A. tumefaciens GV2260 (pBBRacdS); V3, A. tumefaciens GV2260 (pBBRgabT); V4, A. tumefaciens GV2260 (pBBRacdSgabT). Bars represent the standard deviation (n = 3). Different characters indicate values that were statistically different in the one-way ANOVA and Tukey-Kramer method, multiple comparison method (P < 0.01).
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Agrobacterium tumefaciens has been utilized for both transient and stable transformations of plants. These transformation methods have been used in fields such as breeding GM crops, protein production in plant cells, and the functional analysis of genes. However, some plants have significantly lower transient gene transfer and stable transformation...
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Overgrowth of Agrobacterium tumefaciens has frequently been found in Agrobacterium-mediated plant transformation. This overgrowth can reduce transformation efficiency and even lead to explant death. Therefore, this research investigates an alternative way to mitigate or eliminate Agrobacterium after transformation using a bacteriophage. To develop...
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... To conduct this study, we grew an Agrobacterium starter culture (3 mL) from a single colony overnight. We then started Agrobacterium cultures with varying volumes of the starter culture in order to catch the Agrobacterium in exponential phase (OD 600 = $1.0 (Nonaka et al., 2019;Semeniuk et al., 2014)) or stationary phase (OD 600 = $3.0). We confirmed that Agrobacterium in the exponential phase was more efficient at transforming C. roseus seedlings (Figure 2a). ...
Agrobacterium‐mediated transient expression methods are widely used to study gene function in both model and non‐model plants. Using a dual‐luciferase assay, we quantified the effect of Agrobacterium‐infiltration parameters on the transient transformation efficiency of Catharanthus roseus seedlings. We showed that transformation efficiency is highly sensitive to seedling developmental state and a pre‐ and post‐infiltration dark incubation and is less sensitive to the Agrobacterium growth stage. For example, 5 versus 6 days of germination in the dark increased seedling transformation efficiency by seven‐ to eight‐fold while a dark incubation pre‐ and post‐infiltration increased transformation efficiency by five‐ to 13‐fold. Agrobacterium in exponential compared with stationary phase increased transformation efficiency by two‐fold. Finally, we quantified the variation in our Agrobacterium‐infiltration method in replicate infiltrations and experiments. Within a given experiment, significant differences of up to 2.6‐fold in raw firefly luciferase (FLUC) and raw Renilla luciferase (RLUC) luminescence occurred in replicate infiltrations. These differences were significantly reduced when FLUC was normalized to RLUC values, highlighting the utility of including a reference reporter to minimize false positives. Including a second experimental replicate further reduced the potential for false positives. This optimization and quantitative validation of Agrobacterium infiltration in C. roseus seedlings will facilitate the study of this important medicinal plant and will expand the application of Agrobacterium‐mediated transformation methods in other plant species.
... Hence, they used ethylene biosynthesis inhibitors (aminoethoxyvinylglycine) and ethylene perception inhibitors (AgNO3 and silver thiosulphate) to improve the T-DNA transferring procedure [53]. Another alternative technique is the application of ACC deaminase that can break down the ethylene precursor and α keto-butyrate [54]. Songstad et al. [55] tested the function AgNO3 in maize tissue culture for the first time and found out that AgNO3 has the ability to increase the plant regeneration frequency by reducing the ethylene effect. ...
An efficient plant transformation technique is considered as an essential requirement in developing genetically modified (GM) maize plants that can encounter a wide range of biotic and abiotic stresses caused by climate changes. Compared to different plant transformation methods, Agrobacterium-mediated transformation is perceived to be cheap, simple, and has stable gene integration with minimum rearrangements. Accordingly, this method serves as the best option for developing countries in the continents of Asia and Africa. In light of these, Agrobacterium-mediated gene delivery into maize has been constantly improved, making it as a foremost technology not only in genetic engineering but also in genome editing over the past decades. This can be further enhanced by practicing advanced and novel techniques during the transformation procedure. Agrobacterium-mediated transformation of maize plants and the subsequent recovery are affected by several factors including maize genotypes, explant types, bacterial strains, vector systems, and co-cultivation medium. Ensuring optimal conditions would increase efficiency and effectivity of the transformation procedure. Moreover, as a developing nation, biosafety regulations in Sri Lanka are evolving to keep pace with advancements in biotechnology and international standards. However, Sri Lanka face multiple challenges in implementing the guidelines and policies. Addressing these challenges are crucial not only for protecting the environment and public health, but also for promoting innovation, trade, transparency, and trust. Though some of the farmers are cultivating traditional maize varieties in Sri Lanka, majority of the farmers cultivate imported hybrid varieties. Hence, by performing efficient gene transformation technique, traditional maize varieties can be improved to have high yield, tolerant to climatic changes and resistant to pest and disease.
... These results match those of Oltmanns et al. (2010), whom also discovered GV3101 to be the most effective of all tested strains. EHA105, GV3101, and GV2260 are considered similar in their genetic background (Nonaka et al. 2019). Nevertheless, significant differences in transformation frequencies were observed in this study. ...
Submergence stress in plants subjected to flooding is a significant issue in agriculture. Decades of previous research have elucidated many molecular and physiological responses of plants to this stress. Since Arabidopsis thaliana has been established as a model organism in this area of research, in this study, we started by replicating published results of seven sensitive and tolerant ecotypes of Arabidopsis to dark-submergence stress to assess its generalizability and consistency, adapted a leaf-damage index to quantify the progression of stress over time, and measured the recalcitrance of all those ecotypes to Agrobacterium transformation. By using photographic comparisons, median lethal time (LT50), and leaf-damage index, we ascertained that the order of tolerance reported by previous studies is robust enough to allow independent replications (Cvi-0 < Ita-0 < Bay-0 < Col-0 < Kin-0 < Lp2-6 < C24), supporting their consistency. We continued by testing the transformation recalcitrance of all seven ecotypes to three different Agrobacterium strains and identified C24 and Cvi-0 as the most suitable ecotypes useful in a transformation platform despite their low transformation frequencies (<0.1%). The results from this study can be useful in developing transgenic methodologies that enhance plant responses to stress and transitioning these methodologies from the laboratory to the field.
... Improvisation of transformation techniques: Many reports have been mentioned about inefficient DNA delivery by Agrobacterium, which is being tried to overcome by recent developments of super-binary vectors and highly virulent strains (141,142). Also, analysis of host-pathogen interaction should be followed for characterization of peanut genotypes and Agrobacterium strains which may be compatible partners for transformation and are expected to improve transformation efficacy (143). Chemical compounds like acetosyringone (AS) and thiols are supplemented individually in a co-cultivation medium, have been reported to significantly increase the number of transformed plantlets in rice (144) and peanuts (145). ...
... Chemical compounds like acetosyringone (AS) and thiols are supplemented individually in a co-cultivation medium, have been reported to significantly increase the number of transformed plantlets in rice (144) and peanuts (145). Acetosyringone stimulates vir gene in the Ti plasmid of Agrobacterium which enhances the scope of infection to explant (142). Thiol compounds have been reported to inhibit peroxidase and polyphenol oxidase in explants during cocultivation which facilitates T-DNA delivery to explants (146). ...
Peanut (Arachis hypogaea L.) or the common ‘peanut’ is a worldwide popular, affordable food containing high protein, calories, vitamins, and minerals. Several biotic and abiotic stresses are responsible for reaching the expected production of peanuts worldwide. Especially, the fungi are the major constraints that not only hamper the production but also that is deadly health hazardous for both human consumption and poultry-livestock. Approaches from various dimensions like cultural management, diseases free cultivar development, hybridization, tissue culture, and genetic transformations have been tried to overcome such challenges. This review epitomizes the total scenario from the plant physiological basis of fungal diseases to the peanut development approaches, which aimed to develop a concrete understanding of sustainable management of peanut production. Comparisons of Genetic Engineering methods such as Agrobacterium-mediated and direct gene gun (particle bombardment- mediated) with traditional hybridization have been compiled here, furthermore, candidate genes transformed to achieve fungus-resistance in peanuts have been listed up to provide an overview. Along with, the limitations of transformation attempts and the techniques for improvisation of transformation techniques have been discussed in sustainable peanut production. This study provides, comprehensive information on fungal-resistant peanut development so that, further research in this arena could be guided in an integrated way, which may serve for the thrust of sustainable improvisation in peanut cultivation.
... Its phytopathogenic properties depend on the presence of a specific plasmid known as the Ti (tumor-inducing) plasmid [5]. Crown gall disease causes extensive damage in many agricultural species, such as those from the families Rosaceae (rose, apple, cherry, and pear), Vitaceae (grape), and the genus Juglans (walnut) [6]. ...
Soil-borne pathogenic microorganisms are known to cause extensive crop losses. Agrobacterium tumefaciens, a member of the Proteobacteria, causes the neoplastic crown gall disease in plants. Plant protection is mainly based on toxic chemicals that are harmful to the environment. The use of cold atmospheric-pressure plasma is an attractive method for microbial eradication. Its antimicrobial mechanism includes the formation of large quantities of reactive oxygen species (ROS). The advantages of eradicating bacteria using cold plasma are not needed for chemicals, short treatment, and environmental temperatures. This study examined the impact of plasma corona discharge exposure on A. tumefaciens viability, membrane permeability, relative cell size, and ROS formation. The results showed that 90 s of plasma exposure led to a reduction by four orders of magnitude when the initial concentration was 1 × 107 CFU/mL and in a dry environment. When the initial concentration was 1 × 106 CFU/mL, 45 s of exposure resulted in total bacterial eradication. In a liquid environment, in an initial concentration of 2.02 × 106 CFU/mL, there was no complete bacterial eradication even at the most prolonged examined exposure (90 s). The influence of plasma treatment on the membrane permeability of A. tumefaciens, and their possible recovery, were analyzed using flow cytometer analysis using propidium iodide (PI). When the plasma-treated bacteria were suspended in Luria–Bertani (LB) (rich medium), the PI-positive count of the plasma-treated bacteria after two hours was 12 ± 3.9%. At the 24th hour, this percentage was only 1.74 ± 0.6%, as the control (0.7 ± 0.1%). These results may indicate the repair of the plasma-treated bacteria that were suspended in LB. At the 24th hour, the relative cell size of the treated bacteria shifted to the right, to ~3 × 104 forward side scatter (FSC), about 0.5-fold higher than the untreated cells. Measurement of the ROS showed that the intracellular fluorescence of the 90-s plasma-treated cells led to significant fluorescence formation of 32 relative fluorescence units (RFU)/cell (9 × 104 fold, compared to the nontreated cells). This study showed that cold plasma is a useful method for A. tumefaciens eradication. The eradication mechanism involves ROS generation, membrane permeability, and changes in cell size.
... Enhancing the virulence of R1000 and R1500 by adding the pTVK291 cosmid that carries part of the vir region of pTiBO342 (conferring the supervirulent phenotype to the A. tumefaciens strain A348) permitted the generation of hybrid poplar hairy roots and the subsequent regeneration of shoots from those hairy roots [70]. A similar modification was carried out for A. tumefaciens, i.e., the stable transformation rate of tomato (Solanum lycopersicum) with the resulting strain was increased 3.6-fold compared with that for the original GV2260 strain [86]. ...
CRISPR/Cas-mediated genome editing is a powerful tool of plant functional genomics. Hairy root transformation is a rapid and convenient approach for obtaining transgenic roots. When combined, these techniques represent a fast and effective means of studying gene function. In this review, we outline the current state of the art reached by the combination of these approaches over seven years. Additionally, we discuss the origins of different Agrobacterium rhizogenes strains that are widely used for hairy root transformation; the components of CRISPR/Cas vectors, such as the promoters that drive Cas or gRNA expression, the types of Cas nuclease, and selectable and screenable markers; and the application of CRISPR/Cas genome editing in hairy roots. The modification of the already known vector pKSE401 with the addition of the rice translational enhancer OsMac3 and the gene encoding the fluorescent protein DsRed1 is also described.
... Agrobacterium-plant interactions are also affected by gamma-aminobutyric acid (GABA) which occurs by degradation of quorum sensing signals leading to a reduction in horizontal gene transfer of Ti plasmid. A combination of the ACC deaminase and GABA transaminase activity yields a super-Agrobacterium ver. 4 that is known to exhibit increased transformation efficiency (Nonaka et al. 2019). ...
The introduction of genetically modified (GM) cotton in 1996 in the US and its worldwide spread later rejuvenated cotton production in many parts of the world. The evolution is continued since then and currently, the 3 rd and fourth generation of same GM cotton is grown in many parts of the world. The GM cotton introduced in 1996 was simple Bt cotton that expressed a single Cry1Ac gene, the later generation carried multiple Cry genes along with the genes controlling herbicide tolerance. Current day GM cotton does not only give stable resistance against lepidopteran insects but also facilitates the farmers to spray broad-spectrum herbicides without harming the crop. The evolution of GM cotton is continued both on the basic and applied side and interventions have been introduced during the last decade. Earlier the cotton transformation was limited to Cocker strains which are getting possible in many other varieties, too. It is successful with both gene gun, and Agrobacterium and inplanta transformation has made it a routine activity. Apart from overexpression studies for various purposes including biotic, abiotic, and quality traits, RNAi and genome editing are explored vigorously. Through this review, we have tried to explore and discuss various interventions for improving transformation protocols, the applications of cotton transformation, and future strategies being developed to get maximum benefits from this technology during the last decade.
... Therefore, deciphering the epigenetic factors contributing to the pre-harvest sprouting resistance regulation in rice will lay a foundation for a deep understanding of the full machinery in real environmental cues. Moreover, an intensive validation of the existing candidate genes should be processed via CRISPR-Cas9 [129], RNAi [130], and super-Agrobacterium tumefaciens-mediated transformation [131] as a notable example. Altogether, posttranscriptional regulation encompassing, splicing RNA, and epigenetics offer novel avenues for unravelling the mechanism of resistance of pre-harvest sprouting in rice. ...
Pre-harvest sprouting is a critical phenomenon involving the germination of seeds in the mother plant before harvest under relative humid conditions and reduced dormancy. As it results in reduced grain yield and quality, it is a common problem for the farmers who have cultivated the rice and wheat across the globe. Crop yields need to be steadily increased to improve the people's ability to adapt to risks as the world's population grows and natural disasters become more frequent. To improve the quality of grain and to avoid pre-harvest sprouting, a clear understanding of the crops should be known with the use of molecular omics approaches. Meanwhile, pre-harvest sprouting is a complicated phenomenon, especially in rice, and physiological, hormonal, and genetic changes should be monitored, which can be modified by high-throughput metabolic engineering techniques. The integration of these data allows the creation of tailored breeding lines suitable for various demands and regions, and it is crucial for increasing the crop yields and economic benefits. In this review, we have provided an overview of seed dormancy and its regulation, the major causes of pre-harvest sprouting, and also unraveled the novel avenues to battle pre-harvest sprouting in cereals with special reference to rice using genomics and transcriptomic approaches.
... Agrobacterium strains have been improved to achieve increased plant transformation frequency through the development of binary vectors (Zambryski et al., 1982;Hoekema et al., 1983;Bevan, 1984;Komari et al., 2006), the development of ternary helper plasmids and superbinary vectors (Ishida et al., 1996;Anand et al., 2018;Zhang et al., 2019), the upregulation of virulence (vir) gene expression (Ishida et al., 1996;Van Der Fits et al., 2000;Ye et al., 2011;Vaghchhipawala et al., 2018), and the removal of negative factors of T-DNA transfer (Nonaka et al., 2019). Genome editing has also helped improve Agrobacterium strains themselves. ...
... Other features may also be manipulated to improve Agrobacterium-mediated transformation and the fate of transformed events. For example, the subversion of host plant factors (Pitzschke, 2013;Sardesai et al., 2013;Hwang et al., 2015), the reduction of vector backbone and transposon integration (Ülker et al., 2008;Kim and An, 2012;Jupe et al., 2019), the increase of T-DNA transfer capacity (Nonaka et al., 2019), the environmental containment of Agrobacterium when used for field applications (Torti et al., 2021), the increase on transient transformation frequency and the use of effective tools for non-invasive monitoring of gene expression and plant transformation (He et al., 2020;Xu et al., 2021). ...
... These technologies have allowed transient expression at an industrial scale by spraying Agrobacterium carrying the viral vector (Torti et al., 2021). Bypassing difficulties, such as low transient gene transfer rates, regeneration difficulties, and host cell integrity issues (Zaidi and Mansoor, 2017;Nonaka et al., 2019), the virus-based vectors could be optimized to allow the development of transient expression strategies for CRISPR-Cas gene editing without the stable integration of foreign DNA. ...
Over the past decades, advances in plant biotechnology have allowed the development of genetically modified maize varieties that have significantly impacted agricultural management and improved the grain yield worldwide. To date, genetically modified varieties represent 30% of the world’s maize cultivated area and incorporate traits such as herbicide, insect and disease resistance, abiotic stress tolerance, high yield, and improved nutritional quality. Maize transformation, which is a prerequisite for genetically modified maize development, is no longer a major bottleneck. Protocols using morphogenic regulators have evolved significantly towards increasing transformation frequency and genotype independence. Emerging technologies using either stable or transient expression and tissue culture-independent methods, such as direct genome editing using RNA-guided endonuclease system as an in vivo desired-target mutator, simultaneous double haploid production and editing/haploid-inducer-mediated genome editing, and pollen transformation, are expected to lead significant progress in maize biotechnology. This review summarises the significant advances in maize transformation protocols, technologies, and applications and discusses the current status, including a pipeline for trait development and regulatory issues related to current and future genetically modified and genetically edited maize varieties.
... Selection of the optimal Agrobacterium strain for the transformation of P. ginseng Transformation efficiency varies between strains of Agrobacterium, and even for the same strain transforming different plant species (Nonaka et al. 2019). For example, the efficiency of sorghum transformation with Agrobacterium strains LBA4404 and AGL1 veraciously differs by more than three times ). ...
Transient gene expression is far superior to stable gene expression in terms of labor time efficiency and is well suited for the study of plant promoters, protein localization, protein–protein interactions, or binary vectors for genome editing. This study investigated the factors affecting the efficiency of transient gene expression in Panax ginseng by analyzing green fluorescent protein (GFP) fluorescence intensity under a confocal laser microscope. The influences of variations in Agrobacterium strain and suspension density, acetosyringone concentration, number of vacuum applications, infiltration buffer composition, and shoot developmental stage on transformation efficiency of P. ginseng leaf parenchyma cells were investigated. We found that the A. tumefaciens EHA105 strain is best suited for P. ginseng transformation, and the highest expression level was observed at the suspension density OD600 = 1.0. The addition of acetosyringone greatly increases transformation efficiency, but only if it does not exceed a concentration of 100 μM. A special mode of physical treatment, which includes five consecutive applications of a relatively deep vacuum, further increased the transformation efficiency. In addition, the use of MES buffer at pH 5.6 and mature leaves for transformation allowed us to achieve the highest level of transit expression, while more than 80% of leaf parenchyma cells were transformed. The optimized transient transformation method allowed us, for the first time, to evaluate the effect of rol gene expression on the accumulation of ginsenosides in P. ginseng leaves. Our study is the first work aimed at the optimization of transient ginseng transformation, and the developed protocol will be of particular importance for ginseng physiology research and modulation of valuable secondary metabolite biosynthesis.
