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Localized transient expression of GUS in leaf discs following cocultivation with Agrobacterium
Abstract
A chimaeric gene has been constructed that expresses beta-D-glucuronidase (GUS) in transformed plant tissues, but not in bacterial cells. This gene has proved extremely useful for monitoring transformation during the period immediately following gene transfer from Agrobacterium tumefaciens. GUS expression was detectable 2 days after inoculation, peaked at 3-4 days and then declined; if selection was imposed expression increased again after 10-14 days. The extent of transient expression after 4 days correlated well with stable integration as measured by kanamycin resistance, hormone independence, and gall formation. Histochemical staining of inoculated leaf discs confirmed the transient peak of GUS expression 3-4 days after inoculation. The most surprising result was that the blue staining was concentrated in localized zones on the circumference of the disc; within these zones, essentially all the cells appeared to be expressing GUS. We suggest that the frequency of gene transfer from Agrobacterium is extremely high within localized regions of leaf explants, but that the frequency of stable integration is several orders of magnitude lower.
... The Agrobacterium-based genetic transformation undergo two specific mechanisms; stable (T-DNA integration in the genome stably inherited) [84][85][86][87] and transient transformation (absence of T-DNA integration in the genome still capable to perform transcription and translation) [52,[88][89][90][91][92][93][94]. The expression of T-DNA carrying the gene of interest occur in either transient or stable manner was demonstrated by Krenek et al. [30], Janssen and Gardner [95]. Both stable and transient transformation methods can be utilized in genome editing [96], to study the gene and protein function [97,98], molecular processes [99] and are applicable to numerous plant species [100]. ...
... Another method AGROBEST (Agrobacterium-mediated Enhanced Seedling Transformation) has also been developed during the year 2014 by Wu et al. [94] to obtain high transient transformation efficiency in whole seedlings. Presently Agrobacterium-based transient transformation is a safe, high-level, and rapid transient transgene expression method [95,[116][117][118]. Moreover, agroinfiltration is a promising technology for in planta transient expression of high-value recombinant proteins [119,120]. ...
... (i) Exemption of tissue culture [33] (ii) Temporary expression [52] (iii) T-DNA position effect elimination [33] (iv) Simple, quick, economical, and effective [109] (v) The transient transformation frequency was at least 1000-fold greater than that of stable transformation [29,95] (vi) Transient transformation is often versatile, quick, and efficient as compared to stable transformation [99]. ...
Agrobacterium-mediated transformation is one of the most commonly used genetic transformation method that involves transfer of foreign genes into target plants. Agroinfiltration, an Agrobacterium-based transient approach and the breakthrough discovery of CRISPR/Cas9 holds trending stature to perform targeted and efficient genome editing (GE). The predominant feature of agroinfiltration is the abolishment of Transfer-DNA (T-DNA) integration event to ensure fewer biosafety and regulatory issues besides showcasing the capability to perform transcription and translation efficiently, hence providing a large picture through pilot-scale experiment via transient approach. The direct delivery of recombinant agrobacteria through this approach carrying CRISPR/Cas cassette to knockout the expression of the target gene in the intercellular tissue spaces by physical or vacuum infiltration can simplify the targeted site modification. This review aims to provide information on Agrobacterium-mediated transformation and implementation of agroinfiltration with GE to widen the horizon of targeted genome editing before a stable genome editing approach. This will ease the screening of numerous functions of genes in different plant species with wider applicability in future.
... Agrobacterium tumefaciens strain EHA105 carrying the binary vector pKIWI105 (Janssen and Gardner, 1990;Ben Zvi et al., 2012) was used for transient transformation. For stable transformation of Solidago, we used A. tumefaciens strain AGLO pCGN7001 (Ben Zvi et al., 2012). ...
... pKIWI105 and pCGN7001 carried the uidA gene encoding β-glucuronidase (GUS) driven by either an 35S promoter (pKIWI105) or a mannopine synthase promoter (pCGN7001). The GUS-encoding gene is not expressed in agrobacterium cells carrying pKIWI105 due to lack of a bacterial ribosome-binding site, making this plasmid suitable for evaluation of transient transformation (Janssen and Gardner, 1990). Solidago PAP1overexpressing lines were obtained using A. tumefaciens strain AGLO carrying 35S:PAP1 in the binary vector pCGN1559 (Ben Zvi et al., 2012). ...
... To examine the applicability of agrobacteria to transformation of Solidago, leaf explants of the three cultivars were inoculated transiently with Agrobacterium strain AGLO carrying the plasmid pKIWI105-35S:uidA. The uidA gene in this plasmid encodes GUS, which is not expressed by Agrobacterium cells, making it suitable for transient transformation studies (Janssen and Gardner, 1990). Histochemical assay revealed a transformation efficiency (number of GUS-expressing leaf explants out of total number of inoculated explants) of ca. ...
Floral pigmentation is of major importance to the ornamental industry, which is constantly searching for cultivars with novel colors. Goldenrod (Solidago canadensis) has monochromatic yellow carotenoid-containing flowers that cannot be modified using classical breeding approaches due to a limited gene pool. To generate Solidago with novel colors through metabolic engineering, we first developed a procedure for its regeneration and transformation. Applicability of different cytokinins for adventitious regeneration was examined in the commercial cv. Tara, with zeatin yielding higher efficiency than 6-benzylaminopurine or thidiazuron. A comparison of regeneration of commercial cvs. Tara, Golden Glory and Ivory Glory revealed Tara to be the most potent, with an efficiency of 86% (number of shoots per 100 leaf explants). Agrobacterium-based transformation efficiency was highest for cv. Golden Glory (5 independent transgenic shoots per 100 explants) based on kanamycin selection and the GUS reporter gene. In an attempt to promote anthocyanin biosynthesis, we generated transgenic Solidago expressing snapdragon (Antirrhinum majus) Rosea1 and Delila, as well as Arabidopsis thaliana PRODUCTION OF ANTHOCYANIN PIGMENT 1 (PAP1) transcription factors. Transgenic cv. Golden Glory expressing cauliflower mosaic virus 35S-driven PAP1 generated red flowers that accumulated delphinidin and its methylated derivatives, as compared to control yellow flowers in the GUS-expressing plants. The protocol described here allows efficient engineering of Solidago for novel coloration and improved agricultural traits.
... Transient expression in N. benthamiana [46] was performed with the A. tumefaciens strain GV3101. Cultures carrying the corresponding constructs were grown for 20−24 h in YEB liquid medium. ...
... GUS histochemical staining was performed on N. benthamiana disks infiltrated by a protocol previously described in [46] and was quantified as the inverse of the median gray values measured with the Fiji software tool [37]. Photographs were taken using a Leica MZ9.5 magnifying glass with a DCF280 camera. ...
Plants have evolved different mechanisms to increase their tolerance to aluminum (Al) toxicity and low pH in the soil. The Zn finger transcription factor SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1) plays an essential role in the adaptation of plants to Al and low pH stresses. In this work, we isolated the ScSTOP1 gene from rye (Secale cereale L.), which is located on chromosome 3RS. The ectopic expression of ScSTOP1 complements the Arabidopsis stop1 mutation in terms of root growth inhibition due to Al and pH stress, as well as phosphate starvation tolerance, suggesting that rye ScSTOP1 is a functional ortholog of AtSTOP1. A putative STOP1 binding motif was identified in the promoter of a well-known STOP1 target from rye and Arabidopsis and was later corroborated by genomic DAP-seq analyses. Coexpression analyses verified that ScSTOP1 activated the promoter of ScALMT1. We have also identified a putative phosphorylatable serine in STOP1 that is phylogenetically conserved and critical for such activation. Our data indicated that ScSTOP1 also regulated Al and pH tolerance in rye.
... In turn, the integration pattern can affect the stability and expression level of the transgene and this is of concern in agricultural and bio-industrial applications [18,20]. While the transient nature of T-DNA expression following transformation was addressed before [13,21,22], the corresponding number of unintegrated T-DNA molecules could not be directly determined, mainly due to their temporal instability and numerical heterogeneity among individual cells. A plausible approach to resolve this complication could be the direct imaging of the T-DNA molecules throughout the steps of transfection. ...
... Furthermore, when taking into consideration the fact that this imaging system does not differentiate between integrated and non-integrated dsT-DNA foci, it also suggests that part of the (maybe all) observed foci represent non-integrated T-DNA molecules. This is consistent with the works of Janssen et al. and De Buck et al. who determined dominancy of transient T-DNA molecules in the initial stages of protein expression [21,38]. Another puzzling observation made here is that, contrary to our initial expectation to identify mobile dsT-DNA intermediates, the foci that were observed exhibited constrained diffusion dynamics. ...
Plant transformation mediated by Agrobacterium tumefaciens is a well-studied phenomenon in which a bacterial DNA fragment (T-DNA), is transferred to the host plant cell, as a single strand, via type IV secretion system and has the potential to reach the nucleus and to be integrated into its genome. While Agrobacterium-mediated transformation has been widely used for laboratory-research and in breeding, the time-course of its journey from the bacterium to the nucleus, the conversion from single- to double-strand intermediates and several aspects of the integration in the genome remain obscure. In this study, we sought to follow T-DNA infection directly using single-molecule live imaging. To this end, we applied the LacO-LacI imaging system in Nicotiana benthamiana, which enabled us to identify double-stranded T-DNA (dsT-DNA) molecules as fluorescent foci. Using confocal microscopy, we detected progressive accumulation of dsT-DNA foci in the nucleus, starting 23 h after transfection and reaching an average of 5.4 and 8 foci per nucleus at 48 and 72 h post-infection, respectively. A time-course diffusion analysis of the T-DNA foci has demonstrated their spatial confinement.
... Initial works on transient assay was done by leaf disc transformation, where analysis of transformation efficiency can be performed two days after the co-culture [90]. Janssen and Gardner (1990) reported that gene transfer of transiently expressed bglucuronidase (GUS) was 1 000-fold more efficient than stable integration [91] . Since then, transient assay has gained wider adoption as a platform to achieve rapid production of recombinant proteins within a matter of days. ...
... Initial works on transient assay was done by leaf disc transformation, where analysis of transformation efficiency can be performed two days after the co-culture [90]. Janssen and Gardner (1990) reported that gene transfer of transiently expressed bglucuronidase (GUS) was 1 000-fold more efficient than stable integration [91] . Since then, transient assay has gained wider adoption as a platform to achieve rapid production of recombinant proteins within a matter of days. ...
Dengue has been ranked as one of the top emerging diseases in Asia and Latin America. Current epidemiological data may not even reflect the true burden of disease due to under-reported figure Vector control programmes have failed to contain the disease and worst of all, no specific treatment is available at the moment. Thereby, this pushes the demand for a dengue vaccine as a long-term protective approach. Despite there are numerous vaccine candidates ahead, they could be held back by different aspects in promoting vaccine implementation. Particularly for developing nations, logistics and cost are the major hurdles that need to be addressed in order to provide a quick yet affordable medical relief. As an alternative, plant-based vaccine production system is able to offer an attractive prospect given to its advantages of biocontainment warranty, low operation cost, rapid scalability and logistics flexibility. Researches that have embarked on this scope are laid out and reviewed in terms of the feasibility of plant system to serve as a biofactory for dengue vaccine.
... This method has the advantages of simplicity, rapidity, efficiency, and accuracy, which can be used to evaluate the function of the target gene and the expected phenotype of transgenic plants in an early manner. Furthermore, the transient transformation efficiency can sometimes reach 1000 times that of stable transformants [37]. In recent years, many plant leaves, fruits, petals, roots, suspension cells, cell embryos, and calli have also been gradually used in the study of transient expression systems, which have been successfully used in phalaenopsis [38], roses [39,40], grapes [41], strawberries [42], carnations [43], and cassava [44]. ...
The tree peony, a traditional flower in China, has a short and concentrated flowering period, restricting the development of the tree peony industry. To explore the molecular mechanism of tree peony flowering-stage regulation, PoEP1, which regulated the flowering period, was identified and cloned based on the transcriptome and degradome data of the early-flowering mutant Paeonia ostii ‘Fengdan’ (MU) and Paeonia ostii ‘Fengdan’ (FD). Through bioinformatics analysis, expression pattern analysis, and transgene function verification, the role of PoEP1 in the regulation of tree peony flowering was explored. The open-reading frame of PoEP1 is 1161 bp, encoding 386 amino acids, containing two conserved domains. PoEP1 was homologous to the EP1 of other species. Subcellular localization results showed that the protein was localized in the cell wall and that PoEP1 expression was highest in the initial decay stage of the tree peony. The overexpression of PoEP1 in transgenic plants advanced and shortened the flowering time, indicating that PoEP1 overexpression promotes flowering and senescence and shorten the flowering time of plants. The results of this study provide a theoretical basis for exploring the role of PoEP1 in the regulation of tree peony flowering.
... β-glucuronidase (GUS, Sigma-Aldrich) staining was performed according to [27]. Callus induced from explants co-cultured with A. tumefaciens containing pCAMBIA1301-GUS-PgF3 5 H were used for the histochemical analysis of GUS expression. ...
Gypsophila paniculata is one of the most popular cut flowers in the world whose major cultivars are blooming white. As is well known, blue flowers could be generated via the overexpression of the gene encoding flavonoid 3′5′-hydroxylase (F3′5′H) in species that naturally lack it. In this study, we established the regeneration and a genetic transformation system for the commercial cultivar ‘YX4’ of G. paniculata and introduced the F3′5′H of Platycodon grandiflorus (PgF3′5′H) successfully into ‘YX4’ using the established protocol. A total of 281 hygromycin (Hyg)-resistant plantlets were obtained, and 38 of them were polymerase chain reaction (PCR) positive, indicating a 13.5% transformation efficiency. Shoot apex without meristem was more suitable for explant due to its high regeneration capacity, and the supplement of thidiazuron (TDZ) provided the most efficient promotion of adventitious bud induction, whereas the supplement of 6-Benzyladenine (6-BA) and 1-naphthaleneacetic acid (NAA) did not affect much. Additionally, the combination of 1 day (d) pre-culture, 5 d co-culture, 10 min infection, 30 mg·L−1 additional acetosyringone (AS) supplement, and 10 mg·L−1 Hyg selection formed the optimized system for ‘YX4’ transformation. This reliable and efficient agrobacterium-mediated transformation of the valuable commercial cultivar ‘YX4’ will contribute not only to the creation and improvement of G. paniculata cultivars, but also to the function research of genes associated with important ornamental traits.
... Transient gene expression for recombinant protein production can be mediated by infiltration with Agrobacterium tumefaciens (agroinfiltration) harboring a binary vector containing the gene construct as part of the T-DNA to be transferred to the plant cells for transient expression [34] (Figure 1). Transient expression using agroinfiltration often yields much higher levels of recombinant proteins that the stably integrated genes in transgenic plants [35,36]. Transgenes to be expressed can also be engineered into the genomes of plant viruses such as Tobacco mosaic virus (TMV) and introduced into infected plants [37] ( Figure 1). ...
The production of therapeutic and industrial recombinant proteins in plants has advantages over established bacterial and mammalian systems in terms of cost, scalability, growth conditions, and product safety. In order to compete with these conventional expression systems, however, plant expression platforms must have additional economic advantages by demonstrating a high protein production yield with consistent quality. Over the past decades, important progress has been made in developing strategies to increase the yield of recombinant proteins in plants by enhancing their expression and reducing their degradation. Unlike bacterial and animal systems, plant expression systems can utilize not only cell cultures but also whole plants for the production of recombinant proteins. The development of viral vectors and chloroplast transformation has opened new strategies to drastically increase the yield of recombinant proteins from plants. The identification of promoters for strong, constitutive, and inducible promoters or the tissue-specific expression of transgenes allows for the production of recombinant proteins at high levels and for special purposes. Advances in the understanding of RNAi have led to effective strategies for reducing gene silencing and increasing recombinant protein production. An increased understanding of protein translation, quality control, trafficking, and degradation has also helped with the development of approaches to enhance the synthesis and stability of recombinant proteins in plants. In this review, we discuss the progress in understanding the processes that control the synthesis and degradation of gene transcripts and proteins, which underlie a variety of developed strategies aimed at maximizing recombinant protein production in plants.
... While the stable genetic transformation endows transformed plants with hereditable new characteristics, transient plant transformation mediated by A. tumefaciens is widely applied for high-throughput gene function studies due to its simplicity, rapidity and high efficiency [2][3][4]. In comparison to stable transformation, introduced DNA segments are often expressed at much higher levels, albeit transiently, when introduced into plant cells via transformation [5]. Most of the widely used transient transformation assays involve introduction of Agrobacterium into plants using vacuum-aided infiltration technique or a disposable needless syringe [6,7]. ...
Background
The Agrobacterium -mediated transient transformation, which proved effective in diverse plant species, has been widely applied for high-throughput gene function studies due to its simplicity, rapidity, and high efficiency. Despite the efforts have made on Artemisia annua transient expression, achieving high-throughput gene functional characterization basing on a fast and easy-manipulated transient transformation system in A. annua remains challenging.
Results
The first pair of true leaves of A. annua is an ideal candidate for Agrobacterium injection. EHA105 was the optimal strain that can be used for the development of the transient expression system. The supplementation of Triton X-100 at a concentration of 0.005% greatly improved the transient expression frequency. According to the histochemical β-Glucuronidase (GUS) staining assay, high transient expression level of the reporter gene ( GUS ) maintained at least a week. Dual-luciferase (Dual-LUC) transient assays showed that the activity of cauliflower mosaic virus 35S ( CaMV35S ) promoter and its derivates varied between A. annua and tobacco. In A. annua , the CaMV35 S promoter had comparable activity with double CaMV35S promoter, while in tobacco, CaMV35 S exhibited approximately 50% activity of double CaMV35S promoter. Otherwise, despite the CaMV35S promoter and double CaMV35S promoter from GoldenBraid Kit 2.0 displayed high activity strength in tobacco, they demonstrated a very low activity in transiently expressed A. annua . The activity of UBQ10 promoter and endogenous UBQb promoter was investigated as well. Additionally, using our transient expression system, the transactivation of AaGSW1 and AaORA on AaCYP71AV1 promoter was confirmed. Dual-LUC assays demonstrated that AaHD8 activated the expression of two glandular secreting trichomes-specific lipid transfer protein genes AaLTP1 and AaLTP2 , indicating that AaLTP1 and AaLTP2 might serve as downstream components of AaHD8-involved glandular trichome initiation and cuticle formation, as well as artemisinin secretion in A. annua .
Conclusions
A simple, rapid, good-reproducibility, high-efficiency and low-cost transient transformation system in A. annua was developed. Our method offered a new way for gene functional characterization studies such as gene subcellular localization, promoter activity and transcription activation assays in A. annua , avoiding the aberrant phenotypes resulting from gene expression in a heterologous system.
... Apart from the stable transformation of crop species, Agrobacterium has been widely used for transient transformation in Nicotiana and Arabidopsis for subcellular localization, PPI, and plant-pathogen interaction, promoter-gus-reporter studies, etc., with constantly improved protocol (Janssen and Gardner 1990;Sheludko et al. 2007;Tsuda et al. 2012;Norkunas et al. 2018;Zhang et al. 2020a, b). Additionally, we also used this approach for studying the individual subcellular localization of RPBF and RISBZ to verify our results. ...
Proteins regulate cellular and biological processes in all living organisms. More than 80% of the proteins interact with one another to perform their respective functions; therefore, studying the protein–protein-interaction has gained attention in functional characterization studies. Bimolecular fluorescence complement (BiFC) assay is widely adopted to determine the physical interaction of two proteins in vivo. Here, we developed a simple, yet effective BiFC assay for protein–protein-interaction using transient Agrobacterium-mediated-transformation of onion epidermal cells by taking case study of Rice-P-box-Binding-Factor (RPBF) and rice-seed-specific-bZIP (RISBZ) in vivo interaction. Our result revealed that both the proteins, i.e., RISBZ and RPBF, interacted in the nucleus and cytosol. These two transcription factors are known for their coordinate/synergistic regulation of seed-protein content via concurrent binding to the promoter region of the seed storage protein (SSP) encoding genes. We further validated our results with BiFC assay in Nicotiana by agroinfiltration method, which exhibited similar results as Agrobacterium-mediated-transformation of onion epidermal cells. We also examined the subcellular localization of RISBZ and RPBF to assess the efficacy of the protocol. The subcellular localization and BiFC assay presented here is quite easy-to-follow, reliable, and reproducible, which can be completed within 2–3 days without using costly instruments and technologies that demand a high skill set.
... The substrate, X-gluc (5-bromo-4-chloro-3-indolyl-β-glucuronide), was prepared in 20-mM sodium phosphate buffer (pH 7.2) containing 0.1% Triton X-100, 10-mM EDTA, 50-mM potassium ferricyanide and 50-mM potassium ferrocyanide. Infiltrated leaves were cut into small discs and incubated with the substrate overnight at 37°C following the published protocol (Janssen and Gardner 1990). Subsequently, the tissues were cleared with serial transfers in 70% alcohol to remove excess blue colour, for photography. ...
Squalene epoxidase (SQE) is a crucial regulatory enzyme for the biosynthesis of several important classes of compounds including sterols and triterpenoids. The present paper identified and characterised five SQE genes (GgSQE1 to GgSQE5) from Glycyrrhiza glabra through transcriptome data mining and homology-based cloning, for the first time. The phylogenetic analysis implied their functional divergence. The ORF corresponding to one of the five SQEs, namely, GgSQE1, was cloned and studied for its function in a heterologous system, following transient and stable expressions. The transient expression followed by GgSQE1 encoding protein purification suggested approximately 58.0-kDa protein following the predicted molecular mass of the deduced protein. The gene expression profiling based on qRT-PCR indicated its highest expression (6.4-folds) in the 10-month-old roots. Furthermore, ABA (12.4-folds) and GA3 (2.47) treatments upregulated the expression of GgSQE1 in the shoots after 10 and 12 hours, respectively, which was also reflected in glycyrrhizin accumulation. The inductive effects of ABA and GA3 over GgSQE1 expression were also confirmed through functional analysis of GgSQE1 promoters using GUS fusion construct. Stable constitutive expression of GgSQE1 in Nicotiana tabacum modulated the sterol contents. The study could pave the way for understanding the metabolic flux regulation concerning biosynthesis of related sterols and triterpenoids.
... By comparing several different reagents in this manner the most efficient one can be selected and used to generate the gene edited plant. Currently, the most common transient delivery systems involve protoplasts (Lin et al., 2018) or leaf infiltrations (Janssen and Gardner, 1990;Ali et al., 2018). While both are effective, each has its own associated drawbacks. ...
The production of transgenic or gene edited plants requires considerable time and effort. It is of value to know at the onset of a project whether the transgenes or gene editing reagents are functioning as predicted. To test molecular reagents transiently, we implemented an improved, Agrobacterium tumefaciens -based co-culture method called Fast-TrACC (Fast Treated Agrobacterium Co-Culture). Fast-TrACC delivers reagents to seedlings, allowing high throughput, and uses a luciferase reporter to monitor and calibrate the efficiency of reagent delivery. We demonstrate the use of Fast-TrACC in multiple solanaceous species and apply the method to test promoter activity and the effectiveness of gene editing reagents.
... It has been shown through transient expression kinetics that T-DNA strand is converted to ds T-DNA (Janssen and Gardner 1989) and, then in case of T-DNA repeat formation, recombines before integration in genome (De Buck et al. 1999). Inter-chromatid and inter-homologue recombination mechanism has been explained in A. thaliana by Molinier et al. (2004). ...
Malnutrition is a severe public health challenge in several underdeveloped countries worldwide. Thus, sustainable plant productivity and its easy availability in the coming years shall be a major constraint for food and nutritional security for the teeming millions. Moreover, various abiotic and biotic stresses in plants contribute to yield penalty. The conventional breeding techniques for improvement/enhancement of growth, yield, and quality traits are tedious, time-consuming, and impermanent. On the contrary, microbe-assisted genetic manipulation of crop plants has revolutionized the crop improvement through incorporation of value-added traits of agronomic and nutritional importance. It is now possible to transfer genes(s) of interest, irrespective of its origin to crop plants through direct or indirect (vector or vectorless) approaches. Indeed, Agrobacterium has become the most effective vector for gene transfer in the arena of transgenic technology. The success of a transgene of nutritional importance depends upon its high expression level and stability in plant system. To give effect to this hypothesis, various strategies have been deployed including elimination of destabilizing elements of the transgene; removal of putative polyadenylation sequences, cryptic splicing sites, and codon biasness; and incorporation of elements for high-level expression (strong promoter(s), 5′ untranslated leader sequence, translation initiation context). In this chapter, we have discussed the importance of Agrobacterium and various genetic engineering approaches for enhancing the expression of foreign genes including the current scenario and advancements in the biofortification of crops. This chapter also summarizes state of the art of nutrition enhancement in crops, major challenges, and future prospects.
... It was stated that the genes could be transiently expressed up to 1,000-fold higher than in stable transformants. 7 Moreover, it is possible to measure gene expression within a very short time, independent of the regeneration of a transformed cell in transient assays. 8 Agrobacterium tumefaciens infiltration (agroinfiltration) is used as an efficient method for transient gene expression in intact plant leaves. ...
Low transformation efficiency and long generation time for production of transgenic Gerbera jemosonii plants leads to vulnerable gene function studies. Thus, transient expression of genes would be an efficient alternative. In this investigation, a transient expression system for gerbera petals based on the Agrobacterium infiltration protocol was developed using the reporter genes β-glucuronidase (gus) and green florescence protein (gfp). Results revealed the incapability of using the gfp gene as a reporter gene for transient expression study in gerbera flowers due to the detection of green fluorescent color in the non-infiltrated gerbera flower petals. However, the gus reporter gene was successfully utilized for optimizing and obtaining the suitable agroinfiltration system in gerbera flowers. The expression of GUS was detectable after three days of agroinfiltration in gerbera cultivars “Express” and “White Grizzly” with dark pink and white flower colors, respectively. The vacuum agroinfiltration protocol has been applied on the cultivar “Express” for evaluating the transient expression of the two genes involved in the anthocyanin pathway (iris-dfr and petunia-f3'5'h), which is responsible for the color in flowers. In comparison to the control, transient expression results showed change in the anthocyanin pigment in all infiltrated flowers with color genes. Additionally, blue color was detected in the stigma and pollen grains in the infiltrated flowers. Moreover, blue colors with variant intensities were observed in produced calli during the routine work of stable transformation with f3'5'h gene.
... Transient expression also has the potential to be used for the production of large amounts of protein as a mainstream production platform, but ultimately has limited scaling up potential compared with transgenic plants (Vaquero et al., 2002). The rapidity of the system nevertheless provides the potential for a rapid response, for example, in response to a pandemic, since the need for full transformation is eliminated (Marsian & Lomonossoff, 2016). is much more efficient than that of integrated genes, reported to be at least 1,000 fold higher (Janssen & Gardner, 1990) VLPs do not contain infectious genomic material, so they are considered safe, yet they are similar enough to virus particles to successfully elicit an immune response (Marsian & Lomonossoff, 2016). ...
Societal Impact Statement
Therapeutic protein production in plants is an area of great potential for increasing and improving the production of proteins for the treatment or prevention of disease in humans and other animals. There are a number of key benefits of this technique for scientists and society, as well as regulatory challenges that need to be overcome by policymakers. Increased public understanding of the costs and benefits of therapeutic protein production in plants will be instrumental in increasing the acceptance, and thus the medical and veterinary impact, of this approach.
Summary
Therapeutic recombinant proteins are a powerful tool for combating many diseases which have previously been hard to treat. The most utilized expression systems are Chinese Hamster Ovary cells and Escherichia coli, but all available expression systems have strengths and weaknesses regarding development time, cost, protein size, yield, growth conditions, posttranslational modifications and regulatory approval. The plant industry is well established and growing and harvesting crops is easy and affordable using current infrastructure. Growth conditions are generally simple: sunlight, water, and the addition of cheap, available fertilizers. There are multiple options for plant expression systems, including species, genetic constructs and protein targeting, each best suited to a particular type of therapeutic protein production. Transient expression systems provide a mechanism to rapidly transfect plants and produce therapeutic protein in a matter of weeks, rather than the months it can take for many competing expression systems, while proteins targeted to cereal seeds can be harvested, stored and potentially purified much more easily than in competing systems. Current challenges for plant expression systems include a lack of regulatory approval, environmental containment concerns and nonhuman glycosylation, which may limit the scope of the type of therapeutic proteins that can be manufactured in plants. The specific strengths of plant expression systems could facilitate the production of certain therapeutic proteins quickly and cheaply in the near future.
... They mainly include Agrobacterium-mediated transient transformation and direct gene transfer by biolistic bombardment, electroporation or protoplast transfection. Janssen and Gardner (1990) showed that a short period of cocultivation with A. tumefaciens led to the active transcription of many copies of the transgene in the mesophyll cells. In grapevine, this technology was successfully used to monitor elicitor-induced calcium changes by overexpressing apoaequorin in V. vinifera cv. ...
Context of the review: The manipulation of the genetic basis controlling grapevine adaptation and phenotypic plasticity can be performed either by classical genetics or biotechnologies. In the last 15 years, considerable knowledge has accumulated about the grapevine genome as well as the mechanisms involved in the interaction of the vine with the environment, pests and diseases. Despite the difficulties associated with genetic mapping in this species (allele diversity, chimerism, long generation intervals...), several major controlling important vegetative or reproductive traits have been identified. Considering the huge genotypic and phenotypic diversities existing in Vitis, breeding offers a substantial range of options to improve the performances of cultivars. However, even if marker-assisted selection was largely developed to shorten breeding programs, the selection of improved cultivars, whether for agronomic traits or disease tolerances, is still long and uncertain. Moreover, breeding by crossing does not preserve cultivar genetic background, when the wine industry and market are still based on varietal wines.Significance of the review: In grapevine, pioneering biotechnologies were set up in the 1960s to propagate and/or clean the material from micro-organisms. In the 1990s, the basis of genetic engineering was primary established through biolistic or Agrobacterium with several derived technologies refined in the last 10 years. The latest advance is represented by a group of technologies based on genome editing which allows a much more precise modification of the genome. These technologies, so-called NBTs (new breeding technologies), which theoretically do not deconstruct the phenotype of existing cultivars, could be potentially better accepted by the wine industry and consumers than previous GMO (genetically modified organism) approaches. This paper reviews the current state-of-the-art of the biotechnologies available for grapevine genome manipulation and future prospects for genetic improvement.
... Breeding for disease resistance can be achieved through utilizing effectors of plant pathogens (Lauge et al. 1998;Vleeshouwers and Oliver 2014). This can be achieved by transient expression of candidate effector genes in plant leaves by Agrobacteriummediated expression and by a virus vector such as Potato virus X (Chapman et al. 1992;Janssen and Gardner 1990). Subsequently, plants can be screened for the occurrence of cell death-associated defence reaction, known as the hypersensitive response, which indicates effector recognition by a matching plant immune receptor . ...
... For rapid screening of hpRNAi constructs, transient expression by agroinfiltration is the method of choice, as it is fast and suitable for wide range of crops and is independent of in vitro regeneration protocols [18,29,56]. Transient expression was reported to be far more efficient than [27]. The transient expression also offers several advantages like, assay speed, cost effectiveness and it is less laborious to screen the transgene construct without position effect bias [31]. ...
Plant viruses are the most devastating pathogens causing substantial economic losses in many crops. Current viral disease management relies on prophylactics, roguing and insect vector control, since in most crops resistant gene pools for resistance breeding are unavailable. RNA interference, a sequence dependent gene silencing mechanism holds great potential in imparting virus resistance. In this study, the efficacy of a RNAi gene construct developed against four viruses commonly infesting tomato and chilli viz., capsicum chlorosis virus, groundnut bud necrosis virus, cucumber mosaic virus and chilli veinal mottle virus was evaluated. A 3546 bp dsRNA-forming construct comprising sense-intron-antisense fragments in binary vector pBI121 (hpRNAi-MVR) was mobilized into Agrobacterium tumefaciens. Cowpea (Vigna unguiculata) was used as an indicator plant for GBNV agroinfiltration to evaluate the efficacy of hpRNAi-MVR construct in conferring GBNV resistance. The type of agroinfiltration, bacterial concentration and incubation-temperatures were optimized. Vacuum infiltration of three pulses of 20–30 s at 66.66 kPa were effective than syringe infiltration. Of the five Agrobacterial concentrations, OD600 0.5 was more efficient. Incubation temperature of 31 ± 1 °C was favorable for development of disease symptoms than 20 ± 1 °C and 26 ± 1 °C. ELISA revealed a 35% decline in viral load in hpRNAi-MVR infiltrated plants compared to vector control plants. Quantitative real time PCR results have shown a viral gene silencing to the extent of 930–990 folds in hpRNAi-MVR infiltrated plants compared to vector control. This approach is simple, rapid and efficient to screen the efficacy of RNAi constructs developed for the RNAi mediated plant virus management.
... With modern advances in transgenesis and vector design, the use of plant biomass for the cost-effective manufacture of bioproducts continues to improve. Today, transient transformation using Agrobacterium tumefaciens is by far the preferred method of protein production as it provides safe, high-level and very rapid transgene expression in comparison to transgenic plants [1][2][3]. Commonly, the expression cassette containing the gene of interest is carried by recombinant agrobacteria and delivered into the extracellular leaf spaces by physical or vacuum infiltration, a process known as agroinfiltration. In many cases, researchers have relied on a single expression host for this purpose, namely Nicotiana benthamiana, because of its amenability to transformation and innate ability to support high levels of heterologous gene expression [4]. ...
Background
Agroinfiltration is a simple and effective method of delivering transgenes into plant cells for the rapid production of recombinant proteins and has become the preferred transient expression platform to manufacture biologics in plants. Despite its popularity, few studies have sought to improve the efficiency of agroinfiltration to further increase protein yields. This study aimed to increase agroinfiltration-based transient gene expression in Nicotiana benthamiana by improving all levels of transgenesis.
Results
Using the benchmark pEAQ-HT deconstructed virus vector system and the GUS reporter enzyme, physical, chemical, and molecular features were independently assessed for their ability to enhance Agrobacterium-mediated transformation and improve protein production capacities. Optimal Agrobacterium strain, cell culture density and co-cultivation time for maximal transient GUS (β-glucuronidase) expression were established. The effects of chemical additives in the liquid infiltration media were investigated and acetosyringone (500 μM), the antioxidant lipoic acid (5 μM), and a surfactant Pluronic F-68 (0.002%) were all shown to significantly increase transgene expression. Gene products known to suppress post-transcriptional gene silencing, activate cell cycle progression and confer stress tolerance were also assessed by co-expression. A simple 37 °C heat shock to plants, 1–2 days post infiltration, was shown to dramatically increase GUS reporter levels. By combining the most effective features, a dual vector delivery system was developed that provided approximately 3.5-fold higher levels of absolute GUS protein compared to the pEAQ-HT platform.
Conclusions
In this paper, different strategies were assessed and optimised with the aim of increasing plant-made protein capacities in Nicotiana benthamiana using agroinfiltration. Chemical additives, heat shock and the co-expression of genes known to suppress stress and gene silencing or stimulate cell cycle progression were all proven to increase agroinfiltration-based transient gene expression. By combining the most effective of these elements a novel expression platform was developed capable of producing plant-made protein at a significantly higher level than a benchmark hyper-expression system.
Electronic supplementary material
The online version of this article (10.1186/s13007-018-0343-2) contains supplementary material, which is available to authorized users.
... However, the adoption of stable transformation is often held back by the long developmental time involving plant regeneration procedures, along with issues like gene silencing and potential outcrossing to food crops (Rybicki, 2009). In the 1980s, Janssen and Gardner (1989) already discovered that the frequency of gene transfer achieved by transientlyexpressed β-glucuronidase (GUS) was 1,000-fold higher than stable integration. At that time, transient expression was not popular and was merely used for testing efficiency of constructs before undertaking stable transformation. ...
Dengue fever has emerged as one of the fastest growing health problems in recent years. Transmission of the mosquito-borne disease is widespread throughout the tropics, causing intriguing effects to the poorer populations with limited access to healthcare. At present, most vector control programmes have failed to contain the disease and no specific treatment is available yet. The existence of dengue virus as four distinct serotypes poses a significant threat as secondary infection is often manifested in a more severe form leading to hospitalisation and even death. Hence, this pushes the demand for a dengue vaccine as a long-term protective approach. Particularly for developing nations, cost is a major factor that needs to be meticulously addressed in order to provide a quick yet affordable medical relief. With that, this study aimed at producing a safe and cost-effective plant-based dengue subunit vaccine to protect against the febrile illness. A consensus sequence of the dengue envelope glycoprotein domain III (namely cEDIII) was selected as the antigenic determinant. cEDIII was expressed in two forms, i.e. as recombinant proteins with fusion to green fluorescent protein (sGFP) or cholera toxin B subunit (CTB), and as an epitope display on hepatitis B core antigen (HBcAg) virus-like particles (VLPs). All the constructs were cloned into pEAQ-HT vector and transient expression was achieved via agroinfiltration of Nicotiana benthamiana plants. Following the successful detection of heterologous proteins in N. benthamiana, purification procedures were carried out to harvest the recombinant proteins and chimeric HBcAg VLPs-displaying cEDIII, correspondingly. The recombinant fusion of cEDIII to CTB was shown to preserve the ability to fold into its active pentamer and bind with native gangliosides. Meanwhile, assembly of the chimeric HBcAg VLPs-displaying cEDIII was verified via transmission electron microscopy. These purified recombinant proteins and chimeric VLPs were then used for immunogenicity testing in BALB/c mice. Following vaccination with the recombinant protein, the results showed that successful production of anti-cEDIII specific response with neutralising potency against four dengue serotypes was obtained. T cell analyses suggested that the cEDIII induced a predominant T helper (Th) 1 response while the fusion with CTB could skew the response towards a mixed Th1/Th2. Immunisation with the chimeric VLPs-displaying cEDIII also achieved induction of cEDIII-specific responses, however, further evaluations are needed to warrant the successful use of these VLPs-based vaccines. Overall, the findings in this study have provided solid evidence that the development of a plant-based dengue vaccine is feasible. This is of crucial importance to battle against the upsurge of disease burden, and the production of a local vaccine could complement with Malaysian government’s efforts in combating dengue disease.
... There is also evidence for the existence of extrachromosomal ds T-DNA molecules after Agrobacterium infection. The first piece of evidence is the rapid and broad transient expression of T-DNA genes in infected leaves (Janssen and Gardner 1990), no matter if the transferred ss T-DNA is the coding or noncoding strand (Narasimhulu et al. 1996). In addition, experiments involving homologous recombination between extrachromosomal T-DNA constructs delivered as noncomplementary strands suggested that at least one of the ss T-DNA constructs must have been converted to ds T-DNA prior to recombination (Offringa et al. 1990). ...
The mechanism of T-DNA integration into plant genomes during Agrobacterium-mediated genetic transformation is still not understood. As genetic transformation of plants via Agrobacterium has become a routine practice among plant biologists, understanding T-DNA integration remains important for several reasons. First, T-DNA is the final step in one of the unique cases of inter-kingdom horizontal gene transfer in nature. Second, understanding T-DNA integration is important for biotechnological applications. For example, better knowledge of this process may help develop methods to transform species that are currently not susceptible to Agrobacterium-mediated transformation. In addition, regulatory agencies usually require “clean” and “precise” transgenic insertion events, whereas transgenic insertions are commonly complex unpredictable structures. Furthermore, whereas T-DNA integration under natural conditions occurs randomly, technology to direct T-DNA to specific sites in the genome is highly desired. A better understanding of T-DNA integration may help develop methods to achieve more desirable results. Finally, gene targeting methods that require a foreign DNA template for precise DNA modifications in plants often utilize Agrobacterium to deliver the DNA template. Better understanding of the fate of T-DNA in the plant nucleus may help utilize T-DNA for more efficient gene targeting. For introducing gene targeting reagents, efficient delivery of T-DNA without ectopic integration would be useful. The following review summarizes current knowledge related to T-DNA integration. Five major open questions related to T-DNA integration are being presented. Finally, different models for T-DNA integration are being discussed, and a revised model is proposed.
... resulting in good efficiency, while higher concentrations led to seedling death and low concentrations resulted in few or no plants showing transient expression; the optimal co-cultivation period was 6 h, with shorter or longer incubation time negatively affecting efficiency and/or leading to seedling browning or death (Additional file 10). Previous studies have shown that stable expression requires T-DNA integration into the host genome, and that the use of selection results in an increase in transgene expression levels 10-14 days post infection of plant tissues [34]. Indirect evidence indicates that transient expression predominantly occurs from T-DNA copies that are not integrated into the host genome [35,36]. ...
Background
Genetically modified cotton accounts for 64% of the world’s cotton growing area (22.3 million hectares). The genome sequencing of the diploid cotton progenitors Gossypium raimondii and Gossypium arboreum as well as the cultivated Gossypium hirsutum has provided a wealth of genetic information that could be exploited for crop improvement. Unfortunately, gene functional characterization in cotton is lagging behind other economically important crops due to the low efficiency, lengthiness and technical complexity of the available stable transformation methods. We present here a simple, fast and efficient method for the transient transformation of G. hirsutum that can be used for gene characterization studies. ResultsWe developed a transient transformation system for gene characterization in upland cotton. Using β-glucuronidase as a reporter for Agrobacterium-mediated transformation assays, we evaluated multiple transformation parameters such as Agrobacterium strain, bacterial density, length of co-cultivation, chemicals and surfactants, which can affect transformation efficiency. After the initial characterization, the Agrobacterium EHA105 strain was selected and a number of binary constructs used to perform gene characterization studies. 7-days-old cotton seedlings were co-cultivated with Agrobacterium and transient gene expression was observed 5 days after infection of the plants. Transcript levels of two different transgenes under the control of the cauliflower mosaic virus (CaMV) 35S promoter were quantified by real-time reverse transcription PCR (qRT-PCR) showing a 3–10 times increase over the levels observed in non-infected controls. The expression patterns driven by the promoters of two G. hirsutum genes as well as the subcellular localization of their corresponding proteins were studied using the new transient expression system and our observations were consistent with previously published results using Arabidopsis as a heterologous system. Conclusions
The Agrobacterium-mediated transient transformation method is a fast and easy transient expression system enabling high transient expression and transformation efficiency in upland cotton seedlings. Our method can be used for gene functional studies such as promoter characterization and protein subcellular localization in cotton, obviating the need to perform such studies in a heterologous system such as Arabidopsis.
... The Agrobacterium strains can be infiltrated directly in leaves of various plants. In this case, multiple T-DNA copies are transferred to the nuclei and can persist for a period of time, providing a transient expression of the transgene restricted to the infiltrated zone(Janssen and Gardner 1990). Transient expression by agroinfiltration can be achieved quickly compared to creating stable transgenic plants; therefore, it allows to screen the functions of many effector candidate genes, including those which are toxic for the plant (e.g. ...
Les effecteurs jouent un rôle fondamental lors des interactions antagonistes plantes-pathogènes en supprimant les défenses de la plante, permettant ainsi aux parasites de se développer. De tels effecteurs ont été caractérisés chez les insectes herbivores mais leur rôle dans la spécialisation à la plante reste méconnu. Les pucerons se nourrissent de la sève du phloème et injectent dans la plante des effecteurs salivaires. L'étude des patrons d’évolution des effecteurs, ainsi que la caractérisation de leurs fonctions sont nécessaires à la compréhension des mécanismes de spécialisation chez les pucerons. Au cours de ces travaux, nous avons cherché à identifier les effecteurs salivaires impliqués dans l'adaptation du puceron du pois, Acyrthosiphon pisum, à ses hôtes.Des approches évolutives, basées sur un nouveau catalogue de 740 effecteurs candidats surexprimés dans les glandes salivaires de A. pisum, ont révélé que certains d'entre eux évoluent rapidement et que l'expansion de familles multigéniques apparaît comme une source importante de diversité des effecteurs. En parallèle, ces travaux ont permis d'optimiser l'expression transitoire médiée par Agrobacterium dans le pois. Ce nouvel outil d'analyse fonctionnelle permet maintenant l'étude des effecteurs candidats afin d'identifier les effecteurs du puceron du pois impliqués dans l'adaptation à la plante hôte.
... Transient expression provides a rapid method for assaying the function of some types of genes; transgene can often be assayed within a few days of infiltration (Janssen and Gardner, 1989). The results herein showed that the co-infiltration of plasmid carrying viral silencing suppressor Protein (P19) with plasmid carried the corresponding transgene sequences and GFP gene clearly resulted in a stronger expression of GFP gene in infiltrated leaves. ...
... Avant même son intégration dans le génome de la plante, l'ADN-T peut être transcrit, on parle alors d'expression transitoire (Janssen and Gardner 1989 (Klapwijk et al. 1978). La bactérie détourne ainsi la machinerie biosynthétique de la plante à son profit. ...
La rue officinale (Ruta graveolens L) est une plante connue comme étant particulièrement riche en métabolites secondaires et produisant notamment des molécules d'intérêt pharmaceutique comme les furocoumarines. Nous avons tenté par une approche de génie métabolique d'augmenter la teneur en furocoumarines produites dans les plantes. La mise en place de telles approches nous a également permis de mieux comprendre les mécanismes de régulation de la voie de biosynthèse des phénylpropanoïdes. Pour atteindre ces objectifs nous avons transformé la rue avec différents gènes placés sous le contrôle d'un promoteur constitutif fort, le promoteur 35S du CaMV. Pour chaque série de transformants nous avons étudié la teneur en furocoumarines et analysé les variations de composés phénylpropanoïdes (rutine, umbelliférone, ferulate, scopolétine). Parallèlement à cette analyse métabolique, une corrélation a été réalisée avec le niveau d'expression des transgènes et de certains endogènes par l'utilisation d'approche de PCR quantitative. Les séries de plantes transgéniques surexprimant les gènes codants pour la Coumaroyl ester 3'-Hydroxylase de rue (CYP98A22) et d'A. thaliana (CYP98A3) présentent toutes les deux une augmentation significative d'un facteur 3 de la teneur en furocoumarines. Par contre si les premières sont caractérisées par une diminution de la production en rutine et en umbelliférone, les secondes présentent une augmentation importante de la teneur en Scopolétine et en umbelliférone. Ces résultats suggèrent la coexistence de deux C3'H chez R. graveolens ayant des fonctions différentes, l'une d'entre elles étant impliquée directement ou non dans la synthèse de scopolétine. Si la transformation génétique de rues avec des gènes de la famille CYP98A induit des modifications du métabolisme secondaire, la surexpression d'un gène spécifique à la voie de biosynthèse des furocoumarines (gène cyp71AJ1, codant pour la psoralène synthase d'A. majus) permet d'augmenter uniquement la teneur en furocoumarines (X4). L'ensemble de ces travaux a permis de montrer l'intérêt d'une approche de génie métabolique pour générer des plantes présentant un intérêt potentiel pour la production de molécules d'intérêts pharmaceutiques
... Once effectors have been identified for a given pathogen, these effectors can be used to screen germplasm for novel recognition specificities, and thus new resistance sources (Vleeshouwers and Oliver 2014). This can be done by transient expression of effector genes in germplasm by Agrobacteriummediated expression or potato virus X (PVX) (Chapman et al. 1992;Janssen and Gardner 1990). In this manner, many effectors can be tested in parallel, circumventing the limitations of disease assays, while simultaneously providing the possibility of screening germplasm that is a non-host to the pathogen or the possibility of screening effectors of pathogens with a complex lifecycle. ...
... Taken together, these results demonstrate that root transformation is rapid and associated with a root zone located 0.5-2 cm behind the root tip, suggesting that transformation is transient and renewed over time in the same responsive root zone. This is maybe reflecting T-DNA transfer to root cells without genome integration as proposed by Janssen and Gardner (1989) and reviewed in Krenek et al. (2015) for other transient transformation methods. ...
Medicago truncatula is widely used as a model legume for symbiotic and pathogenic microbial interaction studies. Although a number of Agrobacterium-mediated transformation methods have been developed for M. truncatula, a rapid root transformation system was not yet available for this model plant. Here, we describe an easy method for rapid transgene expression in root hairs of M. truncatula, using young seedlings co-cultivated with the disarmed hypervirulent A. tumefaciens strain AGL1. This method leads to efficient expression of various GUS and fluorescent reporters in M. truncatula root hairs. We showed that transgene expression is detected as soon as 2 days following co-culture, in root hairs of a particular responsive zone lying 0.5–2 cm behind the root tip. This method can be used with a variety of M. truncatula genotypes, and is particularly useful for rapid investigation of the sub-cellular localization of fluorescent fusion proteins. Moreover, combining distinct Agrobacterium strains during the initial co-culture step efficiently generates co-transformed root hairs, suitable for co-localization of different fluorescent fusion proteins in the same cell.
... After 48 hrs, gus expressing cells showed blue colour as a result of chromogenic cleavage by X-Gluc, representing successful integration of gus gene and its expression in leaf sections as depicted in Figure 3. The gus assay was performed 8 days after the bombardment, this was done to minimize the transient expression and increase the chances to see the stable transformation, as transient expression is maximum till 96 hrs of bombardment and after that the chances to appear for the stable expression are more (Janssen and Gardner, 1990). Gus gene showed sufficient expression 8 days after bombardment which means that there are enough chances for the stable transformation. ...
Wild crop relatives are important source of genetic diversity. Plant tissue culture provides proficienct means of conservation of endangered wild plants sustaining biodiversity, improving plant traits by virtue of induced somaclonal variation and trangensis. The objective of study was to establish direct in vitro regeneration and biolistic transformation protocol for wild tobacco N. plumbaginifolia using uidA gene. For direct in vitro regeneration, six combinations of IAA and kinetin were used. Then, biolistic transformation was carried out using BioRad PDS 1 000 (He) gun and 1 100-psi rupture discs. Transformation was confirmed through histochemical gus assay. A proficient and reproducible in vitro regeneration system was standardized for wild tobacco named as Nicotiana plumbaginifolia . In present study 1 mg/L kinetin in combination with 0.25 mg/L IAA proved best for direct in vitro shoot regeneration from mature leaf-based explants. Success in direct in vitro regeneration circumvented the callogensis step, accelerating the process of in vitro regeneration and increasing the chances of obtaining true to type plants. Individual shoots were placed on MS medium with/without IBA for rooting. Individual shoots were successfully rooted on MS medium without IBA. Then, explants were transformed with uidA gene using gene gun. Transient assay of bombarded calli showed gus activity on histochemical assay even after a week. Analysis of gus expression has been found as a principal method of plant transformation optimizations.
... These molecules are capable of being transcribed and further processed to express the transgene in a transient manner (Kapila et al., 1997). More interestingly, expression given by these nonintegrated transgene molecules (i.e., the transient expression) is often ~1000 fold higher than the stable expression of the integrated transgene (Janssen and Gardner, 1989). Apart from this quantitative advantage, the transient expression system provides several other advantages, including, less requirement of time to analyse the effect of the transgene and freedom from the biased position effect (Kapila et al., 1997). ...
... Functional genomics research often involves transgenic approaches that overexpress or silence genes, and among those approaches, both stable and transient transformation methods are valid for studying the functions and regulatory mechanisms of genes (Parinov and Sundresan 2000;Wroblewski et al. 2005). Compared to stable transformation, transient transformation is a more attractive alternative that allows transgenes to be assayed more rapidly and easily (Janssen and Gardner 1989;Kapila et al. 1997), so transient transformation has become the main strategy in functional genomics research and has been increasingly employed for research in various species (Ben-Amar et al. 2013;Bhaskar et al. 2009;Wu et al. 2014;Yang et al. 2008). ...
Background
Because most transient transformation techniques are inadequate for functional genomics research in roots, we aimed to develop a simple and efficient Agrobacterium-mediated transient transformation system that utilized root absorption for research in flowering Chinese cabbage.
Results
Both semi-quantitative and fluorescent quantitative RT-PCR confirmed that the target gene BcAMT1;3 was more highly expressed in plants that were infected with the transformed Agrobacterium strain (EHA105-p35S-BcAMT1;3) than in control plants that were infected with the control strain (EHA105-p35S). Furthermore, GUS staining analysis conformed the availability of this transient transformation system. In addition, we found that the highest transformation efficiency was achieved using an Agrobacterium cell density of OD600 = 0.3 for 3–6 h, without hyperosmotic pretreatment, and under these conditions, the peak transformation efficiency was observed at 2 and 4 d after infection.
Conclusions
The transformation method developed by the present study is simple and convenient, since no special equipment is required, and since the method causes no damage, the plants can be used for subsequent experiments.
... In the plant biotechnology community, the term transformation is used to describe the insertion of engineered gene sequences into a plant cell, leading to a change in the genetic makeup of the target cell and its derivatives (i.e., reverse genetics). The foreign molecule can function for a short time in the nucleus as an extrachromosomal entity (transient transformation) [57], or the integration into the genetic material of the target cell can be necessary for long-term functionality and expression (stable transformation) [58,59]. Therefore, transient expression has been used to evaluate factors that influence the stability or consistency of gene expression [60]. ...
The aim of this chapter is to provide a description of the latest scientific advances in the field of gene functional analysis in grapevine. It provides general information about the studies conducted during the past decade to understand the natural variation of this plant and how this information has been exploited for the understanding of traits of interest. Likewise, it is exposed how the use of biotechnology tools have helped to characterize the mechanisms of gene expression and its regulation, as well as the subcellular localization of proteins and their interactions with other molecules. Finally, an approximation to the new technologies of gene editing and their potential application in the functional study of grapevine has been carried out.
Transient in planta transformation is a fast and cost-effective alternative for plant genetic transformation. Most protocols for in planta transformation rely on the use of Agrobacterium-mediated transformation. However, the protocols currently in use are standardized for small-sized plants due to the physical and economic constraints of submitting large-sized plants to a vacuum treatment. This work presents an effective protocol for localized vacuum-based agroinfiltration customized for large-sized plants. To assess the efficacy of the proposed method, we tested its use in cacao plants, a tropical plant species recalcitrant to genetic transformation. Our protocol allowed applying up to 0.07 MPa vacuum, with repetitions, to a localized aerial part of cacao leaves, making it possible to force the infiltration of Agrobacterium into the intercellular spaces of attached leaves. As a result, we achieved the Agrobacterium-mediated transient in planta transformation of attached cacao leaves expressing for the RUBY reporter system. This is also the first Agrobacterium-mediated in planta transient transformation of cacao. This protocol would allow the application of the vacuum-based agroinfiltration method to other plant species with similar size constraints and open the door for the in planta characterization of genes in recalcitrant woody, large-size species.
Attenuated strains of the naturally occurring plant pathogen Agrobacterium tumefaciens can transfer virtually any DNA sequence of interest to model plants and crops. This has made Agrobacterium‐mediated transformation (AMT) one of the most commonly used tools in agricultural biotechnology. Understanding AMT, and its functional consequences, is of fundamental importance given that it sits at the intersection of many fundamental fields of study, including plant‐microbe interactions, DNA repair/genome stability, and epigenetic regulation of gene expression. Despite extensive research and use of AMT over the last 40 years, the extent of genomic disruption associated with integrating exogenous DNA into plant genomes using this method remains underappreciated. However, new technologies like long‐read sequencing make this disruption more apparent, complementing previous findings from multiple research groups that have tackled this question in the past. In this review, we cover progress on the molecular mechanisms involved in Agrobacterium‐mediated DNA integration into plant genomes. We also discuss localized mutations at the site of insertion and describe the structure of these DNA insertions, which can range from single copy insertions to large concatemers, consisting of complex DNA originating from different sources. Finally, we discuss the prevalence of large‐scale genomic rearrangements associated with the integration of DNA during AMT with examples. Understanding the intended and unintended effects of AMT on genome stability is critical to all plant researchers who use this methodology to generate new genetic variants.
Protoplasts preparation and purification have been frequently used in plant genetics and breeding studies, whereas application of protoplasts in woody plants is still in its infancy. Although transient gene expression using purified protoplasts is well-documented and widely used in model plants and agriculture crops, no instance of either stable transformation or transient gene expression in the woody plant Camellia Oleifera has as of yet been reported. Here, we developed a protoplast preparation and purification method using C. oleifera petals by optimizing osmotic condition with D-mannitol and polysaccharide-degrading enzyme concentrations for petal cell wall digestion, to reach a high efficiency of protoplast productivity and viability. The achieved protoplasts yield was approximately 1.42 × 107 cells per gram of petal material and the viability of protoplasts was up to 89%. In addition, we explored influencing factors of protoplast transformation, including concentrations of PEG4000 and plasmid DNA. The transformation efficiency of 81% could be reached under the optimized condition. This protoplast isolation and transient expression system were deployed to further identify the functional regulation of C. oleifera related genes and the subcellular distribution of their encoded products. In summary, the protoplast isolation and transient expression system we established using oil-tea tree petals is an efficient, versatile and time-saving system, being suitable for gene function characterization and molecular mechanism analysis.
Key message
OsGSTU5 interacts and glutathionylates the VirE2 protein of Agrobacterium and its (OsGSTU5) overexpression and downregulation showed a low and high AMT efficiency in rice, respectively.
Abstract
During Agrobacterium-mediated transformation (AMT), T-DNA along with several virulence proteins such as VirD2, VirE2, VirE3, VirD5, and VirF enter the plant cytoplasm. VirE2 serves as a single-stranded DNA binding (SSB) protein that assists the cytoplasmic trafficking of T-DNA inside the host cell. Though the regulatory roles of VirE2 have been established, the cellular reaction of their host, especially in monocots, has not been characterized in detail. This study identified a cellular interactor of VirE2 from the cDNA library of rice. The identified plant protein encoded by the gene cloned from rice was designated OsGSTU5, it interacted specifically with VirE2 in the host cytoplasm. OsGSTU5 was upregulated during Agrobacterium infection and involved in the post-translational glutathionylation of VirE2 (gVirE2). Interestingly, the in silico analysis showed that the ‘gVirE2 + ssDNA’ complex was structurally less stable than the ‘VirE2 + ssDNA’ complex. The gel shift assay also confirmed the attenuated SSB property of gVirE2 over VirE2. Moreover, knock-down and overexpression of OsGSTU5 in rice showed increased and decreased T-DNA expression, respectively after Agrobacterium infection. The present finding establishes the role of OsGSTU5 as an important target for modulation of AMT efficiency in rice.
Plants are severely affected by many biotic stresses, which cause a reduction in crop quality and quantity. One of the strategies to manage biotic stresses is the generation of transgenic plant lines that can be used as biosensors. These biosensor plants can trigger an early warning upon any pathogen infection. Two promoters with β-glucuronidase reporter gene fusions were constructed. The first contained the flagellin sensing 2 gene promoter, whereas the second contained synthetic promoter containing four repeats of cis-acting elements from the pathogen-related protein 1 gene and two transcription enhancers from the 35S promoter. Transformed leaves were treated with a phytohormone salicylic acid to mimic the occurrence of biotic stress. Validation of reporter gene expression induced from both constructs in transformed potato leaves displayed an increase upon salicylic acid treatment. The results reflect that both constructs could serve in the production of potato biotic stress biosensors.
Particle bombardment or biolistic transformation is an efficient, versatile method. This method does not need any vector for the gene transfer and is not dependent on the cell type, species, and genotype. The success of any transformation technique depends on the starting experimental materials or the explants. Here, we describe the factors that have influenced the choice of explants in biolistic transformation. Many general factors in the selection of explants in the development of transgenic plants are presented here. Therefore, this chapter provides extensive guidelines regarding the choice of explants for researchers working on various plant genetic transformation techniques.
While the use of stable transformation technology has led to great insight into gene function, its application in high-throughput studies remains arduous. Although techniques such as agro-infiltration have been widely used in species such as Nicotiana benthamiana for the rapid detection of gene expression and protein interaction analysis, this technique does not work efficiently in the other plant species, including Arabidopsis thaliana. As an efficient high-throughput transient expression system is currently lacking in the model plant species Arabidopsis thaliana, we describe a method that is characterized by high efficiency, reproducibility and suitability for a variety of functional proteins in a number of plant species, including Arabidopsis thaliana, Brassica oleracea, Capsella rubella, Thellungiella salsuginea, Thellungiella halophila, Solanum tuberosum, Capsicum annuum and Nicotiana benthamiana. Efficiency of this method was independently verified in three independent research facilities pointing to the robustness of this technique. Furthermore, in addition to demonstrating the utility of this technique in a range of species, we also present a case study wherein we assess protein-protein interactions in the sucrose biosynthesis pathway in Arabidopsis.
Studies of gene function in apple fruits (Malus domestica) have been hindered by the long juvenile period of apple fruit trees. Transient gene expression is a relatively simple method for gene functional analyses, and has been broadly applied to several plant species; however, few studies have reported a method for systematic transient expression in apple fruits. Here, we developed a highly efficient and robust Agrobacterium-mediated transient expression system for apple fruit, which allows rapid analysis of diverse gene functions. Using a β-glucuronidase gene (GUS) as a reporter for Agrobacterium-mediated transformation, we found that the most suitable infiltration pressure for gene function assays in ‘Red Fuji’, ‘Granny Smith’ and ‘Royal Gala’ fruits was −90 kPa, but −70 kPa in ‘Golden Delicious’. The infiltration efficiency of ‘Golden Delicious’ was higher than that of the other apple cultivars. Transient silencing of MdMYB10, an anthocyanin biosynthesis regulator, altered anthocyanin content in ‘Red Fuji’, demonstrating that vacuum infiltration with Agrobacterium can be used for gene functional analysis. Also a significant positive correlation was found between microcrack density and infiltration efficiency. We conclude that this method represents a general tool for efficient and targeted transient regulation of gene expression in apple fruits.
In the mid 1980s, the first transgenic plants were produced via Agrobacterium-mediated transformation of Nicotiana tabacum (tobacco) protoplasts (Horsch et al., 1984; DeBlock et al., 1984). Since then, over 30 different plant species have been genetically engineered by a variety of techniques (Gasser and Fraley, 1989). The three major breakthroughs in the transformation of dicotyledonous plants were: (1) the development of shuttle vectors for harnessing the natural gene transfer capability of Agrobacterium (Fraley et al., 1986), (2) the methods to use these vectors for the direct transformation of regenerable explants obtained from plant organs (Horsch et al., 1985), and (3) the development of selectable markers. Genetic modification of many field crops, as well as horticultural and tree species, has been achieved with Agrobacterium vectors.
A method for transient gene expression was developed for western white pine (WWP, Pinus monticola Dougl. ex D.Don) using reporter gene uidA encoding β-glucuronidase (GUS). GUS was transiently expressed in cross sections of primary and secondary needles, cotyledons, and current and second year stems of WWP via vacuum-infiltration with Agrobacterium tumefaciens. Histochemical assays of cross sections of secondary needles showed stronger blue color indicating GUS expression at day 1 and 2 than on other days post agroinfiltration (dpa). GUS activity expressed inside WWP cells was confirmed using light microscopy. In fluorometric assays, GUS expression was high at 1 dpa and lasted until 4 dpa in detached secondary needles, while similarly high expression levels only lasted until 2 dpa in attached secondary needles then dropped significantly. Although the length of GUS-staining zones varied among different WWP organs and between growth and dormant seasons, all tested WWP tissues using the protocol had high levels of transient GUS expression. Thus, heterologous candidate genes or endogenous silencing can be expressed in various WWP tissues or organs using this agroinfiltration approach. The current protocol for efficient transient gene expression will aid functional genomics study of WWP and its pathogens and related conifer species.
Genetic transformation of apple and pear is mainly performed via Agrobacterium tumefaciens. The average efficiency of transformation remains low in most laboratories and the success of the transformation experiments is very variable. Improving the efficiency and the reproducibility of apple and pear transformation is thus highly desirable. Adventitious regeneration ability of the explants is not a limiting factor for apple or pear transformation. The present study focuses on improving the frequency of stably transformed cells in the explants following A. tumefaciens inoculation. We report here the results of 36 independent transformation experiments on ‘Gala’ apple, performed with 10 different binary plasmids, comparing three methods of A. tumefaciens inoculation. Agroinfiltration (Agrobacterium-mediated vacuum infiltration) of the explants in a bacterial suspension containing a surfactant (Silwet L-77) at a low concentration (0.002% v/v) significantly increased the average transformation efficiency (mean rate of 5.8% and maximum rate of 30%) and reduced the number of failed experiments (16%) compared to the method of crushing the explants with non-traumatic forceps prior to immersion into the inoculum. The success of the agroinfiltration method was associated with a very high level of GUS expression one month after inoculation.In addition, agroinfiltration dramatically increased the transformation efficiency of pear, reaching rates of transformation between 50 and 80%, compared to inoculation with a scalpel dipped into the inoculum. Altogether, our results demonstrate that the production of large number of transgenic apple or pear lines in a short period of time is feasible using the agroinfiltration method.
Background and objectives: The study of flower color genes function in gerbera
is hampered due to the low efficiency of transformation methods and the long time
span needed for production of stably transformed transgenic plants. For some
functional analysis, the transient expression of genes could be an efficient
alternative.
Materials and methods: This study was conducted in two stages. In first stage, the
agroinfiltration experiment with 3 flower color constructs (1-pBIH-35S-CcF3´5´H:
with one gene, 2-pBIH-35S-Del2: with 3 genes and 3-pBIH-35S-Del8: with 5
genes) in 12 cultivars of gerbera was investigated. Agroinfiltration of gerbera
petals were performed by Agrobacterium tumefaciens strain EHA 101 harboring
binary vectors pBIH that contained one or more genes of flavonoid 3ʹ 5ʹ-
hydroxylase (F3´5´H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase
(ANS), flavanone 3β-hydroxylase (F3H), chalcone isomerase (CHI) and
hygromycin phosphotransferase (hpt). After the establishment of induction and
agroinfiltration media, agrobacterium suspension carrying gene constructs injected
in the base of petals. Then, based on the results of the first experiment, this
experiment was repeated for second time with 4 pink cultivars (‘Aqua Melone’,
‘Bismarck’, ‘Esmara’ and ‘Rosalin’) and mentioned constructs.
Results: Visual observations of injected petals showed that cultivars with pink
color have shifted flower color from pink to blue and produced delphinidin. The
results of HPLC analysis of 4 anthocyanins (delphinidin, cyaniding, pelargonidin
and peonidin) in four cultivars of second experiment showed that the injected
petals of ‘Bismarck’ cultivar with pBIH-35S-Del8 construct have the highest
delphinidin production.
Conclusion: Therefore, ‘Bismarck’ cultivar of gerbera could be suggested as a
promising candidate for sustainable transformation of genes involved in production
of anthocyanins for change of flower color particularly production of delphinidin.
Keywords: Agroinfiltration, Flavonoid 3ʹ 5ʹ-hydroxylase gene, Delphinidin, gerbera
An Agrobacterium-mediated transient gene expression assay was carried out in alfalfa (Medicago sativa) leaves for expression of a chimeric gene encoding a part of capsid protein of Foot and Mouth Disease virus called VP1. The plant leaves were transformed via agroinfiltration procedure. The presence of the foreign gene and its expression in transformed plants were evaluated by polymerase chain reaction (PCR), real time PCR, protein Dot blot and ELISA. Moreover, gene expression in the transformed leaves was quantified by ELISA method. The results obtained in this investigation indicated high level of gene expression in alfalfa leaves, showing that transient gene expression can be applied as an effective and time-saving procedure for the production of recombinant proteins. The procedures for transformation, detection of recombinant protein and its application for molecular experiments are described in the study.
The chapter deals with the biology, breeding, and transgene research of safflower (Carthamus tinctorius L.), an important source of edible oil rich in unsaturated fatty acids. A brief description of its origin, domestication, introduction to other countries, current distribution, and major uses is followed by details about its habit, habitat, taxonomy, cytogenetics, genetic resources, and economic importance. Traditional breeding of this crop has primarily been carried out to improve the oil yield and quality, develop male sterile lines, and incorporate traits for resistance/tolerance to abiotic and biotic stresses. Because of the assiduous and time-consuming nature of the traditional breeding, efforts have been initiated to improve this crop by biotechnological methods, especially genetic transformation. Most of these studies are preliminary and demonstrative in nature, except two: one leading to the development of genetically transformed safflower capable of synthesizing insulin in its seeds and the other incorporating a gene coding for a specific esterase, capable of hydrolyzing a phototoxic from Alternaria carthami, a pathogen causing devastating leaf blight disease of safflower. Besides emphasizing the importance and implications of these studies, transgenes, transformation methods, and selection procedures utilized in such studies have been described. The chapter is concluded by discussing some of the future goals of transgene research on safflower, probable environmental risks because of the products so developed, and possible biosafety measures.
GeneralThe Vectors for Genetic Engineering of Plant CellsTransformation of Plant CellsDirected Genetic Engineering of Plant CellsFactors Affecting Foreign Gene ExpressionProspects: The Future Potential of Genetic Engineering in Plants
The first attempts to transform higher plants were published twenty-five years ago. Hess (1969) described with Petunia hybrida the partial correction of a white flowering mutant to anthocyanin synthesis type by incubating swelling and germinating seeds using wild type DNA; one year later, the transfer of a gene governing leaf shape was reported (Hess, 1970). Furthermore, in 1969 Ledoux and Huart reported on DNA uptake into Arabidopsis seedlings, and some years later on the DNA-mediated correction of thiamineless mutants of the same species (Ledoux, et al., 1974).
We have adapted the "agroinfection" procedure of Grimsley and co-workers [4,5] to develop a simple, efficient, reproducible infectivity assay for the insect-transmitted, split-genome geminivirus, tomato golden mosaic virus (TGMV). Agrobacterium T-DNA vectors provide efficient delivery of both components of TGMV when used in mixed inoculation of wild-type host plants. A greater increase in infection efficiency can be obtained by Agrobacterium delivery of the TGMV A component to "permissive" transgenic plants. These "permissive" plants contain multiple tandem copies of the B component integrated into the host genome. An inoculum containing as few as 2000 Agrobacterium cells can produce 100% infection under these conditions. Further, our results show that there is a marked effect of the configuration of the TGMV A components within the T-DNA vector on time of symptom development. We have also found that transgenic plants carrying tandem copies of the A component do not complement the B component. Possible mechanisms to explain these results and the potential use of this system to further study the functions of the geminivirus components in infection are discussed.
Ninety-two biochemical, physiological and nutritional characters of 70 Agro-bacterium strains and a few representatives of the genera Rhizobium, Pseudomonas, Erwinia, Achromobacter, Klebsiella and Escherichia were subjected to computer analysis. Sixty-five agrobacteria fell into two distinct clusters. All authentic 3-keto-lactose-positive Agrobacterium tumefaciens and A. radiobacter strains grouped in one rather heterogeneous cluster, whereas the atypical 3-ketolactose-negative tumour-inducing agrobacteria, and the named A. rhizogenes strains, grouped in a very tight cluster. Agrobacterium rubi and two other 3-ketolactose-negative tumour-inducing strains were not placed in either cluster. Two other strains were misnamed. Diagnostic characters are presented for distinguishing clusters and groups. The present division of the genus into species is based on phytopathogenicity and does not reflect the natural relationships amongst the agrobacteria.
To test whether virulence mutants of Agrobacterium tumefaciens are capable of promoting T-DNA transfer into plant cells, a tandem array of Cauliflower Mosaic Virus (CaMV) DNA was cloned between T-region border sequences on a wide host range plasmid and introduced into various virulence mutants. The resulting strains were used to infect Brassica rapa cv. Just Right. This assay, recently referred to as agroinfection, is based on the appearance of viral symptoms following transfer of T-DNA to plant cells, and is shown to be at least 100 times more sensitive in detecting T-DNA transfer than tumour formation. Mutants in the loci vir A, B and G, which were avirulent on turnip, failed to induce virus symptoms. Of the two vir D mutants tested, neither induced turnouts, but one was capable of inducing virus symptoms. Mutants in vir E, C and F, which induced respectively no, small and normal tumours on turnip, all induced virus symptoms.
With a stepwise degradation and terminal labeling procedure the 3'-terminal sequence of E. coli 16S ribosomal RNA is shown to be Pyd-A-C-C-U-C-C-U-U-A(OH). It is suggested that this region of the RNA is able to interact with mRNA and that the 3'-terminal U-U-A(OH) is involved in the termination of protein synthesis through base-pairing with terminator codons. The sequence A-C-C-U-C-C could recognize a conserved sequence found in the ribosome binding sites of various coliphage mRNAs; it may thus be involved in the formation of the mRNA.30S subunit complex.
Mutants of Agrobacterium tumefaciens which affect virulence or the ability to catabolize octopine were isolated after Tn5-induced mutagenesis. Of 8,900 colonies tested, 7 mutants with Tn5 insertions in a specific region of other Ti plasmid unable to catabolize octopine were isolated. Thirty-seven mutants affected in tumorigenesis resulted from insertions in the Ti plasmid and the Agrobacterium chromosome. Of these mutations, 12 were chromosomal and 25 mapped on the plasmid. Twenty-three mapped within a 20-megadalton region, which is distinct from the Ti plasmid sequences found stably integrated into the plant cell genome T-deoxyribonucleic acid). Included in these were mutants that were either a virulent or produced tumors with unusual morphologies. Three mutants contained insertions in the T-deoxyribonucleic acid. These three mutants incited tumors which synthesized octopine but had an altered morphology due to either extensive proliferation of shoots or roots from the tumor callus. Three additional mutants not caused by Tn5 contained mutations in the Ti plasmid.
Forty-nine Tn3 and Tn5 transposition insertion mutations were introduced into the virulence region of the pTiA6NC plasmid of Agrobacterium tumefaciens. Five Tn5 transposition mutations from an earlier study (D. Garfinkel and E. Nester, J. Bacteriol. 144:732-743, 1980) were also mapped more accurately. These mutations defined five separate loci within the virulence region. Two Tn3 insertions into one of these loci, virA, result in a strain which is only weakly virulent; however, a Tn5 insertion into this locus eliminates virulence. One Tn5 insertion into another locus, virC, results in a strain which is weakly virulent. Two additional Tn5 insertions into this locus eliminate virulence. Insertions into the remaining three loci eliminate virulence entirely.
Three strains of Agrobacterium rhizogenes were examined for plasmids. Strains 15834 and A4 contained essentially identical large plasmids, pAr15834c and pArA4c, respectively (approximately 260 x 10(6) daltons). These plasmids can dissociate to two smaller plasmid species. Strain TR105 contained only a single plasmid, which was homologous with the dissociation product of pAr15834c, pAr15834b. Plasmid pAr15834c shared little overall sequence homology with other Ti plasmids. One region of conserved homology between pAr15834c and a region of the octopine type plasmid pTiB6806 which contains oncogenicity functions was detected. Lower levels of homology were detected with sequences which are distributed throughout 65% of pTiB6806. Homology with the so-called common deoxyribonucleic acid in the integrated plasmid deoxyribonucleic acid region was detected only after lowering the stringency of hybridization (Tm, -41 degrees C). Furthermore, the A. rhizogenes plasmid is compatible with other Ti plasmids. Therefore, the results suggest that the virulence plasmids of A. rhizogenes are functionally similar to other Ti plasmids, yet have diverged sufficiently from an ancestral Ti plasmid that they now represent a distinct plasmid type based on homology, compatibility, and virulence.
Plant infections with cassava latent virus (CLV) were mediated by the Ti plasmid of Agrobacterium tumefaciens containing either monomeric or dimeric copies of the virus genome. The CLV DNAs caused typical symptoms when they were inoculated in Agrobacterium strains C58, LBA4404 and a virE mutant A1026, but not other Agrobacterium strains with mutations in other vir loci or an E. coli polA strain. Virus-specific DNA forms characteristic of normal CLV infections were found after such infection. Characterization of progeny CLV DNA from selected plants identified several infectious mutants. These were found to be small insertions and/or deletions in the coat protein gene of DNA 1 and in the intergenic region of DNA 2.
The ability of Nicotiana tabacum (cv. Wisconsin 38) pith tissue to give rise to transformed roots after inoculation with Agrobacterium rhizogenes (agropine type strain 1855) has been examined in relation to its state of differentiation. In young plants, all the stem tissues are able to initiate transformed roots while pith of adult plants does not react. Mature pith, composed mostly of polyploid and non-dividing cells, is able to recover susceptibility when placed in vitro on a medium containing auxin and kinetin. The optimal production of transformed roots is obtained after 6-10 days, when proliferating cells redifferentiated "cambial-like" layers and tracheids. The relationships between the inability of fully differentiated pith to give rise to roots and the underlying cell states are discussed.
Successful transformation of plant cells has been obtained utilizing vectors and DNA delivery methods derived from the plant pathogen, Agrobacterium tumefaciens. This soil bacterium is capable of transferring a DNA segment (T‐DNA), located between specific nucleotide border sequences, from its large tumor inducing (Ti) plasmid into the nuclear DNA of infected plant cells. The exploitation of the Agrobacterium/Ti plasmid system for plant cell transformation has been facilitated by (1) the construction of modified Agrobacterium strains in which the genes responsible for pathogenicity have been deleted; (2) the design of intermediate vectors containing selectable drug markers for introducing foreign genes into the Ti plasmid and subsequently into plant cells; and (3) the development of efficient in vitro methods for transforming plant cells and tissues with engineered Agrobacterium strains. These modifications have led to the development of a simple, efficient, and reproducible transformation system from which morphologically normal transformed plants can be readily regenerated. The foreign genes are stably maintained and expressed in the resulting plants and are inherited by progeny as typical Mendelian traits. The availability of transformation systems has already facilitated numerous studies on gene expression and regulation in plants and should eventually allow for the modification of various crop species in an agronomically significant manner. The needs and possibilities for the development of alternate vectors and transformation procedures will be discussed.
Molecular hybrids were prepared between unlabelled DNA from representative strains of eleven genetic races of Agrobacterium and [14C]DNA from typical strains of each of the three main races. The thermal stability of each hybrid was determined. The nature of the hybrids formed varied with the incubation temperature and the kind of DNA used. Hybridization in 2 × SSC-30% dimethylsulphoxide below 59°C yielded two kinds of hybrids: a labile one of unknown nature, denaturing below 59°C, and a more or less stable hybrid denaturing above that temperature. The latter was the only one formed in hybridizations at or above 59°C. There were three kinds of stable hybrids. Within each of the main Agrobacterium races thermal stability of the molecular hybrid was about the same (within 2°C) as for the homo-duplex. Between two races of 50% DNA relatedness, the duplexes were about 6°C less stable. Between races of 10 to 15% DNA relatedness, the duplexes were weak, and the stability was at least 13°C lower. The stability of the hybrids decreased concomitantly with the degree of DNA relatedness. The decreased hybrid denaturation curve is not due to AT-rich sequences. The less two races of agrobacteria appeared to be evolutionarily related, the more mutations occurred within the common part.
A “plant gene vector cassette” to be used in combination with various Escherichia coli gene-cloning vectors was constructed. This cassette contains a replication and mobilization unit which allows it to be maintained and to be transferred back and forth between E. coli and Agrobacterium tumefaciens hosts provided these hosts contain plasmid RK2 replication and mobilization helper functions. The cassette also harbors a transferable DNA unit with plant selectable marker genes and cloning sites which can be combined with different bacterial replicons, thus facilitating the reisolation of transferred DNA from transformed plants in E. coli. The vector cassette contains two different promoters derived from the T-DNA-encoded genes 5 and nopaline synthase (NOS). By comparing the levels of expression of the marker enzymes linked to each of these promoter sequences, it was found that the gene 5 promoter is active in a tissue-specific fashion whereas this is not the case for the NOS promoter. This observation provides the first documented instance of a gene derived from a procaryotic host the expression of which is apparently regulated by plant growth factors.
Twenty-seven local isolates of Agrobacterium and twenty-three isolates supplied by other laboratories were compared by biochemical tests, serology, protein patterns following gel electrophoresis, and by pathogenicity. All isolates fell into two distinct biotypes and this was supported by their serological reactions and by their protein patterns. Biotype 1 contained tumour-inducing, root-proliferating, and non-pathogenic forms; biotype 2 contained both these pathogenic forms but no non-pathogens. We believe the present division of the genus into species based on pathogenicity is untenable. If the genus is to be retained, we propose that it should consist of one species, A. radiobacter, with pathogenicity indicated by a varietal epithet and the biotype specified.
We show here that Agrobacterium tumefaciens virulence (Vir) gene
expression is activated specifically by the plant molecules
acetosyringone (AS) and α-hydroxyacetosyringone (OH-AS). These
molecules induce the entire vir regulon in Agrobacterium as well as the
formation of T-DNA intermediate molecules. AS and OH-AS occur
specifically in exudates of wounded and metabolically active plant cells
and probably allow Agrobacterium to recognize susceptible cells in
nature.
Transformed petunia, tobacco, and tomato plants have been produced by means of a novel leaf disk transformation-regeneration
method. Surface-sterilized leaf disks were inoculated with an Agrobacterium tumefaciens strain containing a modified tumor-inducing plasmid (in which the phytohormone biosynthetic genes from transferred DNA had
been deleted and replaced with a chimeric gene for kanamycin resistance) and cultured for 2 days. The leaf disks were then
transferred to selective medium containing kanamycin. Shoot regeneration occurred within 2 to 4 weeks, and transformants were
confirmed by their ability to form roots in medium containing kanamycin. This method for producing transformed plants combines
gene transfer, plant regeneration, and effective selection for transformants into a single process and should be applicable
to plant species that can be infected by Agrobacterium and regenerated from leaf explants.
The introduction and expression of foreign genes in plants1–3 will advance our understanding of plant molecular biology and should allow the introduction of new agronomically valuable traits into crop plants. The source of potentially useful genes is large (any living organism may be a donor), but identification of these genes remains difficult. We present here an example of how a gene for herbicide tolerance, an agronomically important trait, can be found in a bacterium and introduced into plants. The herbicide glyphosate inhibits a metabolic step in the biosynthesis of aromatic compounds and the aroA gene encodes the inhibited enzyme in bacteria. Therefore, we introduced a mutant allele of this gene that encodes an enzyme less sensitive to glyphosate into tobacco plants. Expression of this gene enhanced tolerance to glyphosate in transformed plants.
The soil bacterium Agrobacterium tumefaciens is a plant pathogen that causes crown-gall tumours after infection of wounded dicotyledonous plants. Large plasmids (Ti-plasmids) are responsible for the oncogenicity of the bacterium1-3. Crown-gall tumours contain a DNA segment, called the T-DNA, which is homologous with a defined part of the Ti-plasmid present in the tumour-inducing bacterium, and is stably integrated into the plant genome4-7. Apart from the T-DNA another region of the Ti-plasmid-called the vir-region, is essential for tumour induction8-11. We report here the interaction of two compatible plasmids, one containing the vir-region, the other carrying the T-DNA on a wide host-range replicon. An A. tumefaciens strain harbouring both plasmids has a normal tumour-inducing capacity, although neither plasmid is functional alone. With this approach, the T-DNA on one plasmid can, because of its size, be easily genetically manipulated using Escherichia coli as a host. Transfer of this plasmid into an A. tumefaciens strain harbouring the plasmid with the vir-region allows introduction of the manipulated T-DNA into plant cells. In this way, sophisticated binary vector systems for plant genetic engineering can be developed.
A gene encoding acetolactate synthase was cloned from a chlorsulfuron-resistant mutant of Arabidopsis. The DNA sequence of the mutant gene differed from that of the wild type by a single base pair substitution. When introduced into tobacco by Ti plasmid-mediated transformation the gene conferred a high level of herbicide resistance. These results suggest that the cloned gene may confer agronomically useful levels of herbicide resistnace in other crop species, and that it may be useful as a selectable marker for plant transformation experiments.
Mutants of E. coli K 12 unable to grow on a -D-glucuronide (methyl--D-glucuronide) and yet able to grow on hexuronates have been isolated; they are deficient for the enzyme: -D-glucuronidase. By conjugation and transduction experiments, these mutations have been mapped in a locus uid A between man and aro D. No or weak cotransduction is found between aro and man: man is shifted from its precedent location to 30.5 minute very close to uid A (linked by 83 to 97% of cotransduction). The order of loci in this region appears to be: ... phe S...pps...aro D...uid A...man.... Some of these mutations can reverse to wild type with a thermosensitive glucuronidase. These reverse mutations are found linked to this locus uid A; these results strongly suggest that uid A is the site of the structural gene of -glucuronidase.
Primary hairy root tissues as well as aseptic hairy root culture lines contain specific compounds that have been biologically characterized as opines. These substances are agropine, mannopine, mannopinic acid, and agropinic acid; they have been synthesized and their electrophoretic behavior has been studied. Hairy root tissues also contain agrocinopines. According to the opine content of hairy root tissues, two types of Agrobacterium rhizogenes strains have been identified. Agropine-type strains (A4, 15834, HRI) elicit roots containing agropine, mannopine, mannopinic acid, and agropinic acid, whereas mannopine-type strains (8196, TR7, TR101) elicit roots containing only mannopine, mannopinic acid and agropinic acid. A. rhizogenes strains catabolize the opines whose synthesis they induce in the hairy root tissues. However, strain HRI only catabolizes agropine. Except for strain HRI, all A. rhizogenes strains studied contain three plasmids, of which the largest appears to be a cointegrate of the two others. Transconjugants of A. rhizogenes plasmids in A. tumefaciens have been obtained by selection on opines. Their properties have been studied and related to their plasmid content. In the mannopine strain C58C1(pRi8196), the virulence functions and the opine-related functions are located on the same plasmid (pRi8196). In agropine strains the catabolic functions are dissociated: agropine degradation is specified by the virulence plasmid, which also specifies opine synthesis in hairy root tissue, however, mannopine, mannopinic acid and agropinic acid degradation are specified by the smaller plasmid. Strain HRI contains only the virulence plasmid, which explains its inability to degrade mannopine, mannopinic acid, and agropinic acid.
Southern hybridisation was performed on ninety-six transgenic petunias that had been selected for resistance to kanamycin. Just over half of the plants contained intact copies of the T-DNA. The most common rearrangements (at least 24 plants out of 96) were simple deleted derivatives that had lost one or both ends of the T-DNA. T-DNAs lacking the left border occurred at a frequency of 20%, and estimates of the frequency of T-DNAs lacking the right border were at least this high. Three plants contained grossly rearranged T-DNAs, of which all expressed the kanamycin resistance gene but only one transmitted the gene to progeny. Two plants lacked T-DNA homology altogether and did not express kanamycin resistance in their leaves or their progeny. Circumstantial evidence suggests that plants containing a chimaeric kanamycin resistance gene driven by the ocs promoter do not root efficiently in the presence of kanamycin. There was no correlation between intactness of the T-DNA and Mendelian inheritance of the kanamycin-resistance phenotype. However, a disproportionate number of plants showing non-Mendelian inheritance had a high copy number of their T-DNA.
Cells of certain strains of Agrobacterium colonize plants by transferring a portion of their DNA (the T-DNA) into a host plant cell, so causing it to proliferate and produce substances (opines) which the bacteria can use as food1. Most dicotyledonous plants can act as hosts, but most monocotyledonous species (including the economically important gramineae) are thought not to be susceptible2. We have used agroinfection (Agrobacterium-mediated virus infection3) as a very sensitive assay to test whether DNA transfer at least occurs during Agrobacterium inoculations of maize, a graminaceous plant. Naked DNA of the geminivirus maize streak virus (MSV) is not infectious to plants by itself and the intact virus can only infect if transmitted by an insect vector4. We report here that whole maize plants develop symptoms of viral infection if inoculated with strains of Agrobacterium carrying tandemly repeated copies of MSV genomes in their T-DNA. Mutant Agrobacterium strains defective in transfer of T-DNA cannot transmit MSV DNA in this test. We conclude that cloned MSV DNA is biologically active and that Agrobacterium can transfer DNA to maize.
We have constructed several vectors for the cloning of foreign DNA into plant genomes. These vectors are based on the transformation system of Agrobacterium tumefaciens and incorporate a number of useful features for plant cell transformation. The prototype vector, pEND4K, contains the left and right T-DNA border sequences and a chimeric gene which confers kanamycin resistance on transformed plant cells. The vector also includes the following features that facilitate the bacterial manipulations: the bacteriophage lambda cos site for in vitro packaging of large inserts, alpha complementation for direct screening of cloned inserts, and the wide host range RK2 replication and transfer origins. The vectors have been demonstrated to efficiently transform plant cells by several criteria.
Many crown gall-inducing Agrobacterium tumefaciens strains have multiple plasmids, only one of which, the tumor-inducing (Ti) plasmid, is essential for oncogenicity. For comparison of Ti plasmids, single-plasmid-containing transconjugant or transformant derivatives were used as sources of pure Ti-plasmid DNA. Fingerprinting was undertaken using the restriction endonuclease SmaI because it produced a relatively simple cleavage pattern. Three groups of Ti plasmids are discernible based upon both their genetic characteristics and their SmaI fingerprints: (1) Octopine-type Ti plasmids, which confer oncogenicity and octopine utilization on the bacterium. Tumors incited produce octopine. This group of plasmids is highly conservative; fingerprints of all members were identical except for two minor variations. (2) Nopaline-type Ti plasmids, which confer oncogenicity and nopaline utilization on the bacterium. Tumors incited may or may not produce nopaline; these plasmids have fingerprints that suggest variable degrees of relationship, including one that appears unrelated to the rest (3) Null-type plasmids, which confer oncogenicity but neither utilization trait on the bacterium. Tumors incited by this class of strains produce neither octopine nor nopaline. Only one member of this group has been examined thus far. Fingerprints of plasmids from several nononcogenic strains examined bore no resemblance to fingerprints of any of the Ti plasmids.
Genie genetique chez les plantes via le plasmide Ti d'Agrobacterium tumefaciens, les vecteurs de genes de plantes derives du plasmide Ti, les techniques de transformation et la structure et l'expression des genes dans leur environnement transgenique
Article de synthese sur les processus de transformation des cellules vegetales par l'ADN-T du plasmide Tc des souches d'Agrobacterium, reconnaissance cellulaire et developpement du phenotype de galle en couronne
The nutrient requirements of suspension cultures from soybean root have been investigated, and a simple medium consisting of mineral salts, sucrose, vitamins and 2,4-dichlorophenoxyacetic acid (2,4-d) has been designed.The cells required thiamine, 2,4-d and ammonium in addition to the usual mineral salts and sucrose.Optimum concentrations of nitrate and ammonium were 25 and 2 mM respectively. The highest yield of cells was achieved at an initial pH of 4.5–5.5. During the growth cycle the pH gradually increased to 6.0–6.2.
The nopaline Ti-plasmid pTiC58 of Agrobacterium tumefaciens C58 was characterized by restriction endonuclease analysis. Fragments generated by HindIII (52), HpaI (17), SmaI (37), KpnI (20), BamHI (>50), EcoRI (>50), and XbaI (9) were arranged into a circular map corresponding to a genome size of 132 × 106 D. The map was established by analysis of large overlapping plasmid segments of pTiC58 derived from a partial HindIII digest and cloned in the vector pBR322. Taken together, the segments present in the 33 hybrid plasmids obtained cover the entire pTiC58 DNA except for 2% located between map coordinates 22 and 24.7 T. The restriction map of the pTiC58 region containing the T-DNA is presented in greater detail.
Factors that affect the probability of genetic transformation of Escherichia coli by plasmids have been evaluated. A set of conditions is described under which about one in every 400 plasmid molecules produces a transformed cell. These conditions include cell growth in medium containing elevated levels of Mg2+, and incubation of the cells at 0 degrees C in a solution of Mn2+, Ca2+, Rb+ or K+, dimethyl sulfoxide, dithiothreitol, and hexamine cobalt (III). Transformation efficiency declines linearly with increasing plasmid size. Relaxed and supercoiled plasmids transform with similar probabilities. Non-transforming DNAs compete consistent with mass. No significant variation is observed between competing DNAs of different source, complexity, length or form. Competition with both transforming and non-transforming plasmids indicates that each cell is capable of taking up many DNA molecules, and that the establishment of a transformation event is neither helped nor hindered significantly by the presence of multiple plasmids.
5′-Noncoding sequences have been tabulated for 211 messenger R'JAs from higher eukaryotic cells. The 5′-proximal AUG triplet
serves as the initiator codon in 95% of the mRNAs examined. The most conspicuous conserved feature is the presence of a purine
(most often A) three nucleotides upstream from the AUG initiator codon; only 6 of the mRNAs in the survey have a pyrimidine
in that position. There is a predominance of C in positions −1, −2, −4 and −5, just upstream from the initiator codon. The
sequence (G) thus emerges as a consensus sequence for eukaryotic initiation sites. The extent to which the ribosome binding site in
a given mRNA matches the −1 to −5 consensus sequence varies: more than half of the mRNAs in the tabulation have 3 or 4 nucleotides
in common with the CCACC consensus, but only ten mRNAs conform perfectly.
A broad host range cloning vehicle that can be mobilized at high frequency into Gram-negative bacteria has been constructed from the naturally occurring antibiotic resistance plasmid RK2. The vehicle is 20 kilobase pairs in size, encodes tetracycline resistance, and contains two single restriction enzyme sites suitable for cloning. Mobilization is effected by a helper plasmid consisting of the RK2 transfer genes linked to a ColE1 replicon. By use of this plasmid vehicle, a gene bank of the DNA from a wild-type strain of Rhizobium meliloti has been constructed and established in Escherichia coli. One of the hybrid plasmids in the bank contains a DNA insert of approximately 26 kilobase pairs which has homology to the nitrogenase structural gene region of Klebsiella pneumoniae.
The transfer DNA (T-DNA) mobilized into plant cells by Agrobacterium tumefaciens seems to integrate rather randomly into the plant genome. We analyzed a target site in the genome of Nicotiana tabacum before and after integration of a T-DNA. Clones presenting right and left T-DNA/plant DNA junctions were used as probes to identify and isolate a unique 1.8-kilobase EcoRI fragment corresponding to the plant DNA target site for a T-DNA insertion event. Comparison of the nucleotide sequences of the plant DNA portions of the T-DNA junction clones with the original plant DNA target revealed that several types of rearrangements resulted from insertion of the T-DNA. The most dramatic alteration was a 158-base-pair direct repeat of target plant sequences at the left and right T-DNA junctions. In addition, there were deletion and insertion events at the ends of the right and left copies of the 158-base-pair repeat. The variety of target-site rearrangements suggests that T-DNA insertion is a multistep process of recombination accompanied by local replicative and repair activities mediated by host-cell enzymes.
We have developed a sensitive leaf disc transformation procedure for studying early and/or transient T-DNA expression during Agrobacterium tumefaciens-mediated transformation of plant cells. Using this system, we have examined the function of T-DNA border sequences on the early expression of T-DNA genes and on the stable integration of those genes in infected cells. Deletion of the right border from the T-DNA appears to permit transfer of T-DNA genes from the tumor-inducing (Ti) plasmid but greatly reduces the frequency of their stable integration. A binary vector has been constructed to permit examination of T-DNA border function in trans to the Ti plasmid. In this situation, a single T-DNA border is necessary for early expression of T-DNA genes and is sufficient for stable integration in any orientation.
Transgenic plants are generated in nature by Agrobacterium tumefaciens, a pathogen that produces disease through the transfer of some of its own DNA into susceptible plants. The genes are carried on a plasmid. Much has been learned about how the plasmid is transferred, how the plasmid-borne genes are organized, regulated, and expressed, and how the bacteria's pathogenic effects are produced. The A. tumefaciens plasmid has been manipulated for use as a general vector for the transfer of specific segments of foreign DNA of interest (from plants and other sources) into plants; the activities of various genes and their regulation by enhancer and silencer sequences have been assessed. Future uses of the vector (or others like it that have different host ranges) by the agriculture industry are expected to aid in moving into vulnerable plants specific genes that will protect them from such killers as nonselective herbicides, insects, and viruses.
Cane gall of brambles caused by Phytomonas rubi N. SP
- E M Hildebrand
- EM Hildebrand
Sackin M: Numeri-cal taxonomic analysis ofAgrobacterium
- Kersters K J Deley
- Sneath
- Pha
Kersters K, DeLey J, Sneath PHA, Sackin M: Numeri-cal taxonomic analysis ofAgrobacterium. J Gen Microbiol 78:227-239 (1973).