Transient expression is widely used as an alternative method for stable transformation of plants, especially cereals, trees and recalcitrants. In this method, multiple copies of transgene are introduced in to the plant cell and then are transcribed and translated. As introduced gene is not integrated in the genome, its expression is not affected by epigenetic factors and the location of insertion. Transient expression is a convenient method for functional analysis of genes including overexpression, silencing, gene expression networks, protein localization, promoter analysis, identification of biosynthesis pathways and so on. Additionally, this method is applied in biotechnological industries like production of recombinant proteins. Transient transformation is performed by different methods, however, many of them have overlap. So, a combination of these methods can be used for transient expression. In this review, various transient transformation techniques, their advantages and disadvantages, latest progress regarding transient expression in model and crop plants and potential and limitations regarding the application of transient expression technique in science and industry will be discussed.

Transient expression of genes using Agrobacterium (Agroinfiltration) is a powerful tool for the analysis of foreign gene expression and function in different gene expression systems in plant before stable plant transformation. However, the production of stable transgenic plants is a lengthy process, in transient assays, it is possible to measure gene expression within a very short time. With simple infiltration of Agrobacterium cells carrying appropriate gene constructs into bean intact leaves, transient expression can be performed within 3 days without using expensive instruments or complicated procedures. In this study, We analysed different regulatory gene expression factors affecting transient expression following agroinfiltration in bean by injection suspensions of the Agrobacterium carrying the plasmid constructs of gene interest. After three days the amount of soluble protein in the leaves was measured and compared. This study proposed to evaluate the best systems expression in transgenic bean plants, using agroinfiltration technique. The results demonstrate that between the three systems expression the highest level of protein is producted by the system that makes the transfer and accumulation of proteins in the endoplasmic reticulumin this study. The efficiency of agroinfiltration was 24-50%.

Barley is one of the most important crops in the world and is also important in agricultural biotechnology as a recombinant protein production platform. In plants such as cereals, whose regeneration and transformation are difficult, in some cases transient gene expression is considered as a substitute for the stable method. Agrobacterium-based transient expression (agroinfiltration) is an inexpensive and quick way to study the function of genes and to evaluate the elements that affect gene expression (untranslated regions, introns, transcription factors and promoters). By using this method, it is possible to evaluate the function of gene constructs without time-consuming process of plant regeneration only within a few days. In order to optimize this method in barley, the effect of different treatments such as tissue type, plant age, leaves position, acetosyringone concentration, type of induction medium, time of assay after induction, Agrobacterium strain, OD of induction medium, type of procedure, duration and frequency of vacuum pump operation and barley genotype on transient expression of the ß-glucuronidase reporter gene in leaves were evaluated in this research. Also, the effect of type of induction medium, Agrobacterium strain and barley genotype on transient expression of the reporter gene in buds were evaluated. Transient gene expression of ß-glucuronidase were observed in all bud samples of Bahman cultivar and EC-82-6 genotype of barley which were inoculated with GV3301 strain harboring the reporter gene construct suspended in the induction medium with acetosyringone concentration of 200 uM by using insulin syringe. According to the results of this study, an effective and low-cost Agrobacterium-based transient expression system in barley is introduced.

Human tissue-type plasminogen activator (tPA) is one of the most important therapeutically proteins involved in the breakdown of blood clots of brain and heart blood vessels following stroke. Therefore, several modern approaches in plant biotechnology such as nuclear and chloroplast transformation have been used to produce the valuable protein. Plant transient expression is a rapid, flexible and reproducible approach to obtain high level of expression of valuable recombinant pharmaceutical proteins. It is shown that posttranscriptional gene silencing (PTGS) process influences the expression level. Therefore, the effect of co-expression of gene silencing suppressor gene P19, derived from tomato bushy stunt virus (TBSV), has been studied on transient expression of tPA in Nicotiana tabacum cv. Xanthi. To serve this purpose, the expression proportion of injected Agrobacterium tumefaciens containing a binary vector pTRAc-tPA-ERH with Agrobacterium containing pCambia-P19 have been studied comparison with the expression level from Agrobacterium containing only the binary vector pTRAc-tPA-ERH. ELISA results from co-agroinjection experiments have shown that the expression level of tPA using p19 was 10μg/g of leaf fresh weight, which this expression level was about two times more than that from leaves injected with Agrobacterium without the vector harboring p19. Therefore, comparing to the stable transformation which needs more time to produce the protein, in transient expression, depends on the number of the injectable leaves per plant and production of 10μg of tPA per gram leaf fresh weight, a higher level of expression can be achieved less than one week. In conclusion, it seems transient expression could be utilized to express tPA usefully and the expression level could ameliorate using virus coded gene silencing suppressor to achieve the expression proportion of 10μg/g of leaf fresh weight.

Transient gene expression is fast، easy and not influenced by positional effects that potentially affect on gene expression levels in stable gene transformation. Transient expression can be applicable using agroinfilteration، biolistic and viral vectors. Agrobacterium mediated transient expression have been shown as an efficient and versatile method for analyzing transgene expression، gene scilencing، host-pathogen interactions، protein-protein interaction، and cis-element/transfactor interaction. A control vector consists of an interon containing reporter gene under control of a common promoter is often required for Cis-acting elemen analysis to standardize the variation. This study was carried out to construct a control vector based on two parental binary vectors، pGPTV and pCAMBIA3301. The constructed vector، pGCGi had an interon containing GUS reporter gene under control of the full sequence of CaMV 35S promoter. The GUS staining results revealed that the GUS intron containg gene cannot produce an active B- glucronidase enzyme in agrobacterial cells. The functional gene expression analysis of the new vector was done using agroinjection and prticle delivery system in tobacco and onion eoidermal cells respectively. The histochemical GUS staining results showed pGCGi could express the reporter gene in plant cells and culd be used as control vector in transient expression experiments.

The production of pharmaceutical compounds having role in treating diseases like AIDS is necessary. In this regard, griffithsin (GRFT) (an algae lectin) can inhibit HIV entry to the cell at picomolar concentration. To production of recombinant GRFT protein, alfalfa, soybean and lettuce were used as hosts for transient expression. Effect of KDEL, signal peptide of carrot extensin and SP subunit of ZERA signal peptide that target protein to endoplasmic reticulum, apoplastic space and space around the cell wall, respectively was studied. 72 hours after agroinfiltration, qRT- PCR analysis showed high potential of lettuce to express of GRFT at transcript level while alfalfa had lowest transcription level. ELISA result using polyclonal anti-mouse anti-GRFT antibody confirmed that targeting of GRFT to apoplast space of soybean led to highest amount of GRFT. Lettuce despite of high level of gene expression, showed low level of GRFT accumulation which may be due to posttranscriptional and post translational modifications. Our results suggest using of soybean leaf tissue for fast and reliable production of pharmaceutical using transient expression.

Transient expression is an efficient and fast system to express recombinant proteins which has been used in different eukaryotic hosts such as mammalian and plant cells. Several applications of this system have so far been used which expression of proteins of interest is one of them. Recently, plants have attracted attention for being used as hosts for the production of recombinant pharmaceutical proteins such as antibodies and vaccines due to their lower price of production, compared to the mammalian systems. Many studies have conducted on the rapid production of vaccine candidate proteins either as a monomer or virus-like particles during epidemics of fast-spreading diseases such as influenza. Virus-like particles have been expressed in prokaryotic and eukaryotic systems are demonstrated to be one of the best antigen presenting systems which can carry antigens in a safe repetitive format on a particle to induce immunity.. Here, we present recent advances in applying transient expression in plants that can be used to produce naked or enveloped virus-like particles as vaccine candidate as well some clinical trial studies.

Transient expression through viral vectors has emerged as a preferred method for the production of plant recombinant protein. The short-term production of recombinant protein in this method, compared to time-consuming production of stable transgenic plants along with being cheaper and easier strategy pave the way for commercial production of recombinant proteins. In this research, Anti-Vascular endothelial growth factor (AntiVEGF) protein was produced in Chenopodium album plant using Zucchini yellow mosaic virus (ZYMV) virus-based transient expression method. Target plants were initially infected using a viral vector containing the GFP reporter gene (pZYMVGFP) to ensure the transgene expression. After the appearance of viral symptoms on plant leaves and also confirmation of plant infection by RT-PCR and dot blot methods, target gene cloning was performed in the pZYMVGFP virus vector. In the next step, the target plants were inoculated using a recombinant viral vector (pZYMVAntiVEGF) and the target gene expression was examined by RT-PCR at the transcription level and by Dot blot, Western blot and ELISA tests at translation level. Various molecular analysis confirm the recombinant AntiVEGF expression in this plant. Therefore, ZYMV viral-based transient expression system would be a suitable platform for recombinant proteins production in Chenopodium album plants.

Bacillus anthracis as a spore-forming bacterium is the causative agent for anthrax. Three proteins from B. anthracis including protective antigen (PA), lethal factor (LF) and edema factor (EF) are causative factors of this bacteria. To produce plant-based vaccine, fusion protein of PA 4 domain and LF 1 domain via furin cleavage site joined to subunit B Vibrio cholera toxin (CTB) as an inducer of immune system. In this research we used 3’and modified 5’ untranslated regions of cowpea mosaic virus (CPMV) as translation enhancer and silencing inhibitor gene 2b of cucumber mosaic virus (CMV) to construct binary vector and transfer gene. Transient expression of tobacco and lettuce performed via agrobacterium and transient expression was confirmed at the level of transcriptome and proteome through RT-PCR and ELISA assay, respectively.

Rabies virus is a neurotropic virus that causes fatal, but, a preventable disease in mammals. Administration of rabies immunoglobulin (RIG) is essential for the post-exposure of the prophylaxis to prevent the disease. However, replacement of polyclonal RIGs with alternative monoclonal antibodies (MAbs) that are capable of neutralizing rabies virus has been recommended.
Here, we have investigated the transient expression of the full-size human MAb against rabies virus glycoprotein; the MAb SO57 in the tobacco plants using vacuum agro-infi ltration. Previously, stably transformed plants expressing the MAb have been reported.
In this study three vectors carrying the codon-optimized genes for the heavy or light chain and p19 silencing-suppressor were constructed. These vectors were co-infi ltrated into Nicotiana tabacum leaves and the transgenes were expressed.
Dot blot, Western blotting, ELISA, and in vitro neutralization assays of the plant extracts showed that the human MAb could assemble in tobacco leaves and was able to neutralize rabies virus.
This study is the fi rst report of transient expression of human MAb SO57 gene in tobacco plant within a few days after vacuum agro-infiltration.