Agroinfiltration Mediated Scalable Transient Gene Expression in Genome Edited Crop Plants

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.


Introduction
The CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic repeat DNA Sequences-CRISPR associated), a Nobel prize-winning technology for the year 2020 pioneered by Emmanuelle Charpentier and Jennifer Doudna in 2012 proved to be an indispensable tool for editing genomes in the agricultural biotechnology field. Besides genome editing, this tool has been applied for studying gene regulation, epigenetic editing and chromatin engineering also [1]. The discoveries of meganucleases, zinc finger nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs) followed by the breakthrough discovery of CRISPR-Cas9 have revolutionized the genome editing research. It involves targeted base editing of the genome through the use of Streptococcus pyogenes Cas9 endonuclease and short guide RNA molecule (sgRNA) that revolutionized genetics and functional genomics [2,3]. The Cas9 endonuclease and sgRNA lead to the generation of DNA double-stranded breaks in the targeted genome sequences and ensure successful editing of plant genomes [4].
Plant biotechnology which involves the generation of genetic modifications of host cells through the introduction of foreign genes encased in a vector leads to the production of transgenic plants. These genetic modifications can be carried out through numerous methods namely indirect gene transfer methods like Agrobacterium-mediated, floral dip, agroinfiltration, and direct gene transfer methods like polyethylene glycol method (PEG)mediated method, particle bombardment (biolistics), microinjection, and electroporation ( Figure 1). These serve as a key for the development of genetically engineered plants with the trait of interest in a limited time that circumvents the conventional plant breeding approaches. In all the above-mentioned methods, widely acceptable genetic transformation method includes indirect transfer using Agrobacterium [5]. It is one of the most preferred techniques for the generation of genetically modified plants [6] as Agrobacterium-a natural genetic engineer, can deliver the desired gene of interest naturally due to its innate ability to transfer T-DNA. Scientists have explored their vision for using the genome editing process in conjugation with the Agrobacterium-mediated process through the use of Cas9 and sgRNA in the T-DNA to perform specific base editing. Recently, Sandhya et al. [14] reviewed numerous CRISPR/Cas9 based delivery methods such as Agrobacterium-mediated, biolistics, PEG-mediated, and floral dip or pollen-tube pathway method. But all these methods require sophisticated strategies for the selection of desired transformants possessing novel characteristics. Moreover, numerous confirmation protocols are required to ascertain the successful transfer of the gene and proper functioning of the gene of interest in the host plant [15]. The Agrobacterium-based floral dip method undergoes a stable transformation that targets the germinal cells while Agrobacterium-based transient approach i.e., agroinfiltration target somatic cells, hence not transferred to the next generation. The genetic engineering technology mostly suffers from major limitations like low transformation efficiency, high time requirements, and event of insertion of foreign DNA in the host plant leading to stable transformation [16].
Keeping in view the generation of stable transformation through Agrobacterium-based technique that involves tissue culture, we simply focus on Agrobacterium-based transient agroinfiltration approach which does not involve time-consuming tissue culturing step [17]. As the CRISPR/Cas9 DNA editing still has regulatory and biosafety concerns [18,19], therefore, transient expression through agroinfiltration-based genome editing holds promise in the present era that will overcome the legislation concerns due to its temporary expression for a limited time~within 3 days [20].

Genome Editing Methods
The CRISPR/Cas9 system has been successfully applied in various plant species for site-specific base modifications through various genetic transformation methods with varying editing efficiency. It has been observed that the editing efficiency of 9.6%, 18.4%, and 31.9% can be obtained in the edited lines depending on the Agrobacterium-mediated transformation, ribonucleoprotein complexes, and transient expression in protoplasts respectively [21]. Another advancement of the CRISPR/Cas9 genome editing, the Transiently Expressed CRISPR/Cas DNA (TECCDNA) is a simple and efficient genome-editing approach in which mutant plants are regenerated after transient expression of CRISPR/Cas9 DNA [22]. The CRISPR/Cas9 DNA (plasmid constructs) or RNA (in vitro synthesized transcripts) can be delivered through biolistic technique. The circumvention of lengthy and labour-intensive herbicide selection steps on a medium supplemented with antibiotic was escaped concluding that the foreign DNA does not get integrated into the genome. Generally, the plant genome editing tools require additional cycles of plant regeneration under antibiotic selection medium but TECCDNA approach excluded the addition of herbicide or antibiotics addition to the medium meant for the selection of transformed plants. After biolistic delivery of TECCDNA, embryos were transferred to callus induction, regeneration, and rooting medium that generated a large number of T 0 seedlings about 1 week later provided no selective agents were used during the tissue culture process. After TECCDNA, CRISPR-Cas ribonucleoprotein (RNP) served as a simple, convincing, and promising tool for precision plant breeding [23] (Figure 2). This technique has been used for targeted genomic modifications without causing any genomic disturbances or off-target effects [24]. Contrastingly, agroinfiltration involves the direct introduction of a foreign gene in the intercellular [7] and extracellular [25] leaf spaces thereby circumventing the requirement of expensive biolistic equipment for both TECCDNA and CRISPR-Cas RNP approaches. The use of lengthy procedures and sophisticated/ specialized equipment can be avoided through the implementation of the agroinfiltration approach. Specific reviews [29][30][31][32][33] are available on transient gene expression in different host organisms. Sheludko [29] reviewed the biological characteristics of the transient expression process and application for recombinant protein production. Tyurin et al. [33] also reviewed the main and critical steps involved in various methods for transient gene expression in plants. Another recent review by Zlobin et al. [4] elaborated the utilization of Agrobacteriumbased floral dip transformation for genome editing. However, the floral dip transformation method falls short for targeting a single gene due to several specific problems such as nonreproducibility of the process, generation of false-positive results, and low transformation efficiency. Therefore, the agroinfiltration transient approach has several applications in genome editing which have numerous advantages over stable transformation. The primary

Types of Agrobacterium-Based Genetic Transformation
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]. Heenatigala et al. [99] have developed efficient protocols for stable and transient gene transformation for Wolffia globosa using Agrobacterium that has application in a wide range of scientific and commercial processes. Stable genetic transformation process is more appropriate to study long-term analysis of gene function or for long-term production of specific compounds but has the requirement of several months. However, if the target is to introduce a gene of interest in a short period to produce desired protein products, the preferred method is transient transformation [99]. Hence, transient transformation is a preferred method to study short-term gene expression analysis [101].

Stable Genetic Transformation
The transfer of T-DNA through Agrobacterium leading to the inheritance in the next generation referred as stable transformation. Generally, this method confers the gene transfer through Agrobacterium via two means; co-cultivation method (including tissue culture technology) and floral dip method (devoid of tissue culture). The co-cultivation method using Agrobacterium suspension containing desired gene of interest involves culturing the explant. Although it leads to the formation of stable transformants yet it suffers from several disadvantages such as non-uniform transformants can be obtained due to genetic and epigenetic changes that occur during repeated culturing. The induction of callus during tissue culture is a cumbersome approach leading to the generation of undesirable somaclonal variations thus regeneration protocols have not been developed for all plant species.
The widely adapted Agrobacterium-mediated stable gene transfer method is the floral dip method. The floral dip method avoids the subsequent tissue culturing steps that may lead to generation of somaclonal variants such that desired transformants can be obtained within a short time period as compared to the co-cultivation method [52].

Transient (Temporary) Genetic Transformation
The temporary integration of the exogenous DNA in the plant progeny is the basis of transient transformation of plant tissues [102]. Agrobacterium-mediated genetic transformation involves transfection of millions of plant cells by T-DNA in a short period. It allows only a short-term (transient) expression of the gene constructs in a plant cell. The transient approach holds the fact that disarmed Agrobacterium strains lead to modification of nonreproductive or somatic tissue which is not inherited and hence transient in nature [103]. It has been reported that heritable mutations adhering to Mendel's law only have the potential to transfer to the next generations [104] but this is not the case in agroinfiltration because transgenes are transiently expressed in somatic cells of plant tissues and not germ cells hence not heritable. Transient gene expression has been demonstrated to produce large amounts of recombinant proteins in a very short time of few days [105,106]. The somatic cells in the zone of agroinfiltration have the potential to express the transgenes under the control of the constitutive CaMV-35S promoter within 3-5 days after agroinfiltration. A transient expression is an approach for the verification of transformation construct activity and the validation of the formation of small amounts of recombinant protein. Potrykus [107] reviewed the most widely used Agrobacterium infiltration method utilized for transient transformation in Nicotiana benthamiana. The generation of simple insertion events for the T-DNA marked by the border (left and right) sequences in a binary vector is the hallmark of generating transgenics with a reduced frequency of transgene silencing [108]. Zheng et al. [109] have developed the conventional Agrobacterium-mediated transient gene expression system in which the seedlings and plantlets were co-cultivated with Murashige and Skoog medium containing Agrobacterium for several days exhibiting transient expression of the β-glucuronidase (GUS) gene. Further, it is a shorter and efficient way that can help reduce the time required for the development of rice transgenics [110].
Fischer et al. [111] have also reported that transient gene expression is a convenient method as compared to time-consuming stable transformation methods for the production of recombinant proteins. It has been reported that 100% transient transformation efficiency can be achieved in Nicotiana benthamiana and lettuce [112]. The biggest advantage of plant transient transformation is the gene expression for a short time, in which the target gene can express within 12 h and high expression could be attained within 2~4 days in the plant cells [113]. The non-integrated T-DNA copies remain transiently present in the nucleus that can transcribe and translate, leading to T-DNA transient gene expression. The transient genetic transformation can unveil the complex functional genomics in conifer species [114] and medicinal plants [115].
Li et al. [92] have developed a novel transient assay Fast Agro-mediated Seedling transformation (FAST) in the year 2009 which was based on co-cultivation of Arabidopsis seedlings with Agrobacterium tumefaciens in the presence of a Silwet L-77 surfactant. They have successfully expressed the constructs driven by different promoters in cotyledons but not roots of Arabidopsis seedlings in diverse genetic backgrounds through this approach. 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]. Also, A. tumefaciens based transient gene expression tends to affect plant signal transduction that could be used for studying various regulatory processes and defense mechanisms in the plant system [121].

Stable vs. Transient Genetic Transformation
The transient genetic transformation exhibits upto 21.8% high transformation efficiency as compared to 0.14% stable genetic transformation efficiency [99]. Stable transformation being labour intensive process that takes even 15 months [122] or even more for the production of transformed plants. On the other hand, transient transformation is a rapid method with a high expression level. The transient expression holds promise before implementing the tedious and time-consuming stable expression methods [30,94,123]. The transient transformation has several advantages over stable transformation as mentioned below: (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].

Agroinfiltration
The transient expression methods include transfection of protoplasts using PEGmediated or electroporation [124], biolistics [125], and agroinfiltration [126], but agroinfiltration is a simple and effective method for transfer of the gene of interest in the host cell [118]. It involves direct delivery of recombinant agrobacteria carrying the gene of interest in the extracellular leaf spaces by physical or vacuum infiltration [25]. Agroinfiltration led to a transfer of single-stranded T-DNA from Agrobacterium to the plant cells which is trafficked through chaperones to the nucleus. During integration, a small percentage of T-DNA is integrated into the host chromosomes that lead to the development of stable transformation [36]. On the other hand, part of T-DNAs that do not integrate into chromosomes results in the high production of short-lived recombinant protein production [127]. The host factors (karyopherin α and VIP1), bacterial factors (VirD2, VirE2, and VirE3), and plant factor KU80 play a vital role in T-DNA integration in Agrobacterium-based genetic transformation methods [6]. Moreover, this method applies to leaf tissue as well as other plant organs, like fruits and berries and recalcitrant plant species [33,128]. Agroinfiltration has high transformation efficiency, high scalability, and ease of multiple transient expression assays with multiple transgenic vectors and multiple R genes carrying the gene of interest on a single leaf [129,130]. The transient expression of two resistance genes Cf-9 and Cf-4 has been observed in tomato plants through agroinfiltration approach in tobacco. It has also been observed that co-expression of R gene with the corresponding Avr gene trigger host-defence responses in a hypersensitive response in Nicotiana tabacum. It resulted in the formation of the necrotic sector through the expression of both genes in the overlapping region that led to the transient production of R and Avr proteins in the infiltrated leaf area through agroinfiltration [131]. Kapila et al. [7] have first worked on the intact petunia leaves for the expression of heterologous proteins through the infiltration of A. tumefaciens suspensions of harbouring a binary vector into leaf interstitial spaces. The gene transfer through agroinfiltration enabled the transfer of T-DNA in all cell layers of the leaf including intercellular spaces in the upper epidermis, lower epidermis, and palisade parenchyma, spongy parenchyma, and interstitial cell layers. Li et al. [92] have developed the transient agro-infiltration method for rapid production of recombinant protein, an attribute fundamental for functional genomics studies. The process of Agroinfiltration is temporary as it remains in the in vivo system for a limited period [132], therefore, there occurs no T-DNA integration into the plant genome [133,134]. The implementation of transient genetic transformation in the research studies will serve as a pilot experiment before following stable transformation which will provide information about the knock-out effect of the gene in question. It could be used in replacement of the in vitro transcription and cleavage assay of sgRNA and Cas9 protein of protoplast transfection method. It has been reported that Agrobacterium-based transient agroinfiltration can achieve success within 3 days without the need for any cumbersome procedures and equipment. Moreover, Agrobacterium-based transient approach agroinfiltration is the one that saves time, labour will lead to the temporary transformation which will make genome editing "safer technology". A synonym term agroinoculation implies the use of a virus-based vector while agroinfiltration is based on the implementation of binary vector marks the difference between both approaches [135]. In comparison with the plant breeding based back crosses approach that has the potential to remove the CRISPR-Cas9 vector insertion and retain only the beneficial gene-edited alleles but agroinfiltration is an efficient approach that can be performed as a preliminary screening technique for somatic or germinal cells and later the stable transformation technique can be followed.
The transient protein production in the cytoplasm due to the absence of insertion of genes into the crop genome will have an exemption from transgenic regulation and hence greater public acceptance [131]. Agroinfiltration results in the direct creation of markerfree tobacco plants [136]. It is the method that results in the rapid functional analysis of transgenes directly [137]. The molecular and functional analysis can also be achieved through agroinfiltration [138]. The genetic background and the tissue culture conditions are the important measures for a successful agroinfiltration experiment [139].

Methods of Agroinfiltration
Agroinfiltration can be carried out through numerous methods like syringe infiltration, vacuum infiltration, hydrogen peroxide-based agroinfiltration, special agroinfiltration method, leaf disc vacuum infiltration, spray-based agroinfiltration, and detached leaf-based infiltration approach which will be briefly summarised here. Generally, agroinfiltration is carried out using two methods; syringe and vacuum infiltration applicable for a variety of plant species (Tables 1-3). Among these methods, vacuum infiltration requires specialized equipment but exhibits improved transformation potential or yield [98]. An advanced modified version of the vacuum infiltration technology namely leaf disc vacuum infiltration has also been developed that involves pulling off the syringe plunger for the creation of a small vacuum in the syringe [140]. Likewise, Xu et al. [141] have developed a special agroinfiltration method to attain gene expression within 5 days. This method involved the injection of the gene of interest into the interfaces between the adaxial epidermis and mesophyll using a plastic syringe with a needle. This leads to the formation of an agroinfiltration bubble (approximately 1 cm 2 area) with a high ratio of agrobacteria carrying gene of interest to be infiltrated in the epidermal cells.
Shin and Park [142] have developed another agroinfiltration method namely hydrogen peroxide-based agroinfiltration. The use of this abrasive chemical induces wounds in the treated tissue which results in the improvement in the transformation efficiency in Chinese cabbage. A novel spray-based agroinfiltration is the technology that is applicable without the need of a vacuum chamber [143]. The detached leaf-based infiltration approach is also developed to overcome the necrosis, photo-bleaching, browning, and senescence cell death problem in intact leaves [144].

Factors Affecting Agroinfiltration
The success of agroinfiltration depends on numerous factors that need to be optimized to increase the transformation efficiency [97,140]. These factors influence the transfer of T-DNA from Agrobacterium to plant cells [145] and include plant genotype, type of explant, Agrobacterium strain, cell density in the inoculation medium, inoculation conditions, and co-culture. A comprehensive detail about these factors as discussed in previous reports has been presented in Table 1. Apart from these factors, numerous physical and molecular factors need to be considered during Agrobacterium-mediated transformation. The physical factors include ambient and leaf temperature, light source, pH, osmotic conditions, explant type, bacterial strain, density, and co-cultivation time that may affect the Agrobacteriumbased transformation [146][147][148]. The infiltration time for 30 min has been observed to improve the transformation in Medicago leaves while less than 25 min was considered to be best for Trifolium leaves [149]. The surfactants such as Silwet L-77 or dark treatment after infiltration can improve the transformation efficiency during transient transformation [25]. The effects of the infiltration method, Agrobacterium strain, and age of the donor plant are the critical factors in the transient transformation studies [150]. Secondly, molecular factor involving foreign gene transfer followed by transgene expression may be altered by the inherent host plant mechanism of post-transcriptional gene silencing [151]. It has been observed that gene silencing results in the loss of protein expression so the co-expression of gene silencing inhibitor (e.g., p19 protein from tomato bushy stunt virus, NS1 protein from human influenza A virus and P1/HC-Pro protein of Tobacco etch potyvirus) can lead to 50 fold enhancement in the expression of the candidate protein through agroinfiltration process [152,153].  Cytoplasmic RNA silencing may occur during agroinfiltration transient expression [176]. Recently, Norkunas et al. [118] have identified the effects of chemical additives and heat shock pretreatment to confer improved transformation through agroinfiltration in Nicotiana tabacum. The construct comprised of the geminiviral replication system and a double terminator including heat shock protein terminator combined with an extensin terminator can also increase transient protein expression during the agroinfiltration process [177]. Hyperosmotic pretreatment with sucrose (25% w/v) containing 1/2 MS solution (pH 5.8) for 3 h before agroinfiltration greatly improved transient expression efficiency [109]. Sheludko [29] reviewed the optimization of transient expression protocol for high-scale protein production. The leaf position of apical leaves showed high transient GFP expression [140]. The transient transformation efficiency of 99-100% in recalcitrant banana plants was achieved by using hydroponic solution followed by syringe-based infiltration [178].

Advantages of Agroinfiltration Method over Other Transient Genetic Transformation Methods
Various genetic transformation methods for transient genetic transformation in plants include PEG-mediated, biolistics, viral vectors, and agroinfiltration. Agroinfiltration exhibits numerous advantages over the above-mentioned transient methods which are briefly summarized here. Agroinfiltration process does not involve isolation of protoplast which is a pre-requisite for the PEG-mediated genetic transformation. Further, agroinfiltration is a simple and cost-effective method that simply involves the infiltration of transgenes into the intercellular layers of plant cells [7,118]. Moreover, agroinfiltration is less time-consuming compared to the protoplasts-based genetic transformation [179].
The high-velocity biolistics method also exhibits a transient expression of an introduced gene(s) in the plant cells. This method was first demonstrated in the laboratory of J.C. Sanford at Cornell University in the late 1980s [8]. But this rapid and versatile method suffers from a major limitation of the requirement of special equipment PDS-1000/He [32]. Moreover, the transformation efficiency of biolistics is relatively low as compared to agroinfiltration [180]. The efficiency of promoters can be tested efficiently in agroinfiltration as compared to the biolistics genetic transformation method [181].
The transient expression was also achieved using viral vectors but this method suffered from biosafety and construct-size limitation issues for the insertion of transgenes [152,182]. Contrastingly, agroinfiltration involved the infiltration of the whole leaf with one or multiple target DNA constructs or introducing multiple constructs into the different regions of the same leaf [135]. Thus, agroinfiltration is a better suited transient genetic transformation method compared to biolistics, viral vectors, and PEG-mediated methods in plants.

CRISPR-Cas9 Based Genome Editing via Agroinfiltration
The CRISPR/Cas9 system can be effectively coupled with the agroinfiltration transient expression system thereby employing the delivery or use of Cas9 and sgRNA for targeted site modifications in plants (Figure 2). The conjugation of CRISPR/Cas9 (primary choice for plant genome editing) and Agrobacterium-mediated transformation (most preferred method for gene delivery) has the power to deliver the CRISPR/Cas9 DNA constructs efficiently to plants without integration into the plant cells [183]. Generally, agroinfiltration forms a part of the initial screening used as a preliminary experiment to identify whether a particular plant tissue type is amenable to transformation or not which is indirectly validated through the sgRNA expression (genome editing) or genetic transformation (gfp gene or other gene studies). The studies linked to the use of agroinfiltration-based CRISPR-Cas9 genome editing in plants have been given in Table 2. The non-transmissible agroinfiltration approach serves as the first line of experiments to test the extent of transient genome editing before initiating the elaborate genome editing experiments. Contrastingly, a recent comprehensive review is available that emphasizes the use of Agrobacterium-based floral dip stable transformation for genome editing [4]. The focus on the Agrobacteriumbased transient gene transformation agroinfiltration method in genome editing bears better advantages over the Agrobacterium-based stable genetic transformation methods.
The transient agroinfiltration approach is widely employed for the prediction of gRNA efficiency in genome editing constructs in vivo ( Table 2). The combinatorial approach of CRISPR-Cas editing and agroinfiltration showed the result in the transformed tissue within 3 days after infiltration that serve as a reliable assay for testing sgRNAs under native conditions [184].   Among prevailing genome editing tools viz. CRISPR-Cas widely acceptable tool other than ZFNs and TALENs for crop improvement [131,202,203]. Most of the researchers targeted the PDS gene required for carotenoid biosynthesis. Li et al. [185] have utilized agroinfiltration for targeted genome modifications in the AtPDS3 and NbPDS genes of Arabidopsis thaliana and Nicotiana benthamiana. The PDS disruption enhanced the chlorophyll oxidation that led to the development of a visible photobleached phenotype [104,185]. The agroinfiltration was used for CRISPR/Cas9-mediated genome editing targeting PDS gene in various plant species [179,185,[187][188][189][190][191][194][195][196][197]201]. Syombua et al. [201] have employed the Agrobacterium-based transient transformation method; agroinfiltration and genome editing for Yam (Dioscoreaalata) plant genetic improvement. The infiltration of pCas9-gRNA-PDS in leaves tends to show bleached patches that can be analyzed through microscopic examination of an infiltrated leaf section to confirm the transient knockout of the PDS gene, and the effectiveness of both agroinfiltration and genome editing approaches.
Along with PDS, researchers have also utilized another visible marker GFP to analyse the success of the agroinfiltration-based genome editing [186]. The use of non-functional GFP containing the target site cleaved by a Cas9/sgRNA complex resulted in the creation of functional GFP genes to report the success of agroinfiltration-based genome editing [186]. The conversion of frame-shifted GFP to visible GFP due to CRISPR-Cas9 genome editing marks the achievement in agroinfiltration-based genome editing that can be analyzed through fluorescence microscopy of the infiltrated section [201]. Later on, the transient expression can be followed by stable genetic transformation that tend to show phenotype such as dwarf phenotype, albino shoot with a bushy phenotype, and variegated albino plantlets [201]. Besides, fluorescent markers, vectors namely pGD vectors carrying GFP and DsRed elements also ease the interpretation of co-localization and protein-protein interactions in dicots in the agroinfiltration in plants [204]. Moreover, pBYR2HS vector in agroinfiltration applies to leaves and fruits [82]. Sharma et al. [184] have developed a simplified transient transformation assay using agroinfiltration in sorghum and confirmed the gene editing in sorghum leaves using GFP as a marker. The agroinfiltration-based genome editing was used to monocot sorghum which is an unlikely host for Agrobacterium due to the occurrence of epidermal cuticular wax, high silica content, and low volume of intercellular space. However, it was observed to be a successful technique for achieving genome editing in the sorghum. The sgRNA efficiency in orange and purple cabbage has been tested using agroinfiltration [197,200]. Likewise, agroinfiltration transient approach has been tested followed by implementation through stable transformation for gene characterization studies in strawberry [199] (Knockout of Tomato MADS box gene6 generated mutant lines that deciphered its role in anther development), yam [201] (Knockout of PDS gene resulted in the development of albino plants) and cowpea [144] (Knockout of meiosis genes viz. SPO11-1, REC8, and OSD1 showed defects in male and female meiocytes displaying its role for the induction of mitosis from meiosis). Most of the researchers reported the leaf (Table 1) as a host for agroinfiltration-based genome editing while Baltes et al. [192] reported the agroinfiltration-based genome editing using leaf tips with 0.03-70.01% mutation rate conferring geminiviruses resistance.
Further, the repercussions of the genome edits under in vivo conditions are always subtle and not easily detectable. It has been evident that the predicted sgRNAs often have different editing efficiencies in homozygous and heterozygous conditions in each cell. Also, the variation in the knock-outs or sequences in any of the genes after genome editing events will be more useful to study the impact of genome editing or its efficiency rather than obtaining variations in the phenotypic characters. Moreover, the variation in the sequence can be easily identified through Sanger's sequencing technique under in vitro conditions. Moreover, in silico prediction tools have been utilized to analyze the editing efficiency based on the choice and sequence feature of the sgRNA [184].

Modifications of Agroinfiltration-Based Genome Editing
Several reports showcasing the use of modified methods for agroinfiltration-mediated genome editing in plants have appeared over the recent decade. Jia and Wang [197,205] have published the first report on targeted genome modification of citrus through Xanthomonas citri subsp. citri (Xcc)-facilitated agroinfiltration to enhance the transient protein expression in a recalcitrant citrus species-Valencia sweet orange. They have recorded the involvement of PthA4Type III effector molecule and transcriptional activator-like effector leading to cell division (hyperplasia) and enlargement (hypertrophy) to be responsible for improved efficacy and success of Xcc-facilitated agroinfiltration. Jia and Wang [197,205] employed Xcc-facilitated agroinfiltration to deliver Cas9, along with a synthetic sgRNA targeting the phytoene desaturase (CsPDS) gene, into sweet orange and confirmed the mutation at the targeted location through DNA sequencing. The CRISPR-Cas9 technology employed agroinfiltration to confer resistance to tobacco rattle virus, bean yellow dwarf virus, and citrus canker accompanying targeting of numerous endogenous plant genes (Table 1) [144,190,[192][193][194][195][198][199][200]206].
Several researchers have employed leaf for both syringe and vacuum infiltration. However, Juranic et al. [144] have illustrated the use of detached leaf rather than the intact leaf. They have interpreted that agro-infiltration of the intact leaves results in a variety of undesirable symptoms including necrosis, photo-bleaching, browning, and senescence or cell death. Hence, they have performed a detached leaf assay of leaflets with fluorescent expression constructs with the objective of meiosis-knock out for asexual seed induction in cowpea to identify gene-specific mutations.
Along with agroinfiltration, researchers have utilized the co-cultivation method as a transient transformation method [207,208]. Li et al. [207] have used this method and successfully employed the CRISPR/Cas9 technology to knock out the self-incompatibilityrelated gene SRK in Chinese cabbage with 10.83% transformation efficiency.

Limitations
Although a useful technology, there are few pitfalls of Agroinfiltration which needs to be overcome through optimization of the genetic transformation factors while conducting the study as mentioned in Table 1 [239]. The major limitation of the transient expression is that it cannot be used for large-scale commercial production in seed banks due to its transient expression [240]. The complex configuration and organization of various types of plant cells foist varying low efficiency to the agroinfiltration process [33]. Also, high variability in the transformation efficiency depends on the type of Agrobacterium strains, target plant species, and specific tissues. Among the most popular methods of Agrobacterium-based genetic transformation, the syringe infiltration technique suffers from poor scalability that hinders experimental success [32]. Further, the plant defence responses are also a critical factor in genetic transformation studies [52]. Molecularly, the post-transcriptional gene silencing limits transient expression. Therefore, several viral suppressors like p19 protein of tomato bushy stunt virus have been introduced to enhance the transient expression [152].

Bio-Safety and Commercialization Aspects
The critical concern in plant biotechnology is the acceptance of genetically modified or genome-edited plants by consumers. Hence, biosafety and regulatory concerns need to be addressed before plant-based research studies. The generation of very high expression levels in a contained facility in an agroinfiltration-based transient approach minimizes the chances of biosafety-related risk [123]. Moreover, this technique has simple operation steps [241]. Although USA has already approved gene editing through Agrobacteriummediated transformations yet the advent of targeting the somatic cells i.e leaves does not allow the foreign gene to be transferred in the next generation, hence, no chance of transgene to be transferred towards the next generation. The biosafety limitations of the genetically modified organisms (GMOs) generated through genetic engineering technology [242] can be efficiently dealt via agroinfiltration transient approach due to its ability to retain transgene temporarily in the plant cells. The transgene(s) thus cannot be inherited to the next generation [243] as these do not get transferred to the reproductive tissues omitting its passage to germline. Chen et al. [32] have reported that agroinfiltration-based platform is an effective, safe, low-cost, and scalable approach for the rapid production of recombinant proteins. Besides these considerable factors, agroinfiltration employing the use of vectors from recombinant plasmids will be performed at BSL-1 level facilities can be considered as transgenics. Keeping in view the biosafety aspects, agroinfiltration can pave towards the production of marker (transgene)-free transgenic plants which will overcome the biosafety concerns and can be efficiently opted for commercialization in future. The commercial production of recombinant protein(s) suffers from biosafety and risk assessment [244]. However, the agroinfiltration technique can potentially resolve these biosafety issues for improving the commercial sustainability for cost and availability of the recombinant protein(s) in the coming eras. Further, the consumer acceptance for these products can be better ascertained as agroinfiltration is a transgene-free approach. Also, it is identified as an efficient approach to develop the marker-free plants utilizing leaf disc agroinfiltration with a marker-free plasmid vector containing the target gene in tobacco plants hence limited biosafety concerns (https://www.isaaa.org/). It is now being widely used to produce commercially important recombinant proteins [245]. The production of low-yielding plant-produced recombinant proteins could be a hindrance to the commercial success of agroinfiltration [246] but the efficient use of optimized agroinfiltration-based factors can circumvent the limitation to increase the transformation efficiency in crop species.

Conclusions
Agroinfiltration coupled with genome editing holds promise for targeted site modification in the genomic sequence without the integration of the construct into the plant genome. The direct injection of Cas9-sgRNA complex in agroinfiltration process, encased in plasmid could edit the genes in the plant. Although CRISPR/Cas9 technology is the "Technology of choice" for editing genes as compared to TALENs and ZFNs, the combination of CRISPR/Cas9 with agroinfiltration, an in planta and transient approach makes it is a breakthrough technology by enhancing the feasibility, efficacy, and ultimately, the consumer acceptability of this technology. Further, the technique avoids the limitation of low transformation efficiency generated during stable genetic transformation methods. Moreover, agroinfiltration-based genome editing can help broaden the scope of targeted genome modification through simple, high throughput and genotype-independent genome editing approach. Thus, the convergence of genome editing (targeted site modification) and agroinfiltration (simplest approach) can improve the ease, versatility, and efficacy of plant genome transformations to achieve commercially viable and consumer-preferred plant products.