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Review

Plant Transformation and Genome Editing for Precise Synthetic Biology Applications

by
Sharathchandra Kambampati
,
Pankaj K. Verma
and
Madhusudhana R. Janga
*
Institute of Genomics for Crop Abiotic Stress Tolerance, Department of Plant and Soil Science, Texas Tech University, Lubbock, TX 79409, USA
*
Author to whom correspondence should be addressed.
SynBio 2025, 3(3), 9; https://doi.org/10.3390/synbio3030009 (registering DOI)
Submission received: 29 May 2025 / Revised: 14 June 2025 / Accepted: 25 June 2025 / Published: 27 June 2025
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Abstract

Synthetic biology (SynBio) is an emerging interdisciplinary field that applies engineering principles to the design and construction of novel biological systems or the redesign of existing natural systems for new functions. As autotrophs with complex cellular architectures, plants possess inherent capabilities to serve as “living factories” for SynBio applications. Recent advancements in genetic engineering, genome editing, and transformation techniques are improving the precision and programmability of plant systems. Innovations, such as CRISPR systems, prime editing strategies, and in planta and nanoparticle-mediated delivery, are expanding the SynBio toolkit for plants. However, the efficient delivery of genetic constructs remains a barrier due to plant systems’ complexity. To address these limitations, SynBio is increasingly integrating iterative Design–Build–Test–Learn (DBTL) cycles, standardization, modular DNA assembly systems, and plant-optimized toolkits to enable predictable trait engineering. This review explores the technological foundations of plant SynBio, including genome editing and transformation methods, and examines their integration into engineered systems. Applications, such as biofuel production, pharmaceutical biosynthesis, and agricultural innovation, are highlighted, along with their ethical, technical, and regulatory challenges. Ultimately, SynBio could offer a transformative path toward sustainable solutions, provided it continues to align technological advances with public interest and global sustainability goals.

1. Introduction

Plants, as autotrophs, possess unique capabilities to produce food, fuel, fiber, pharmaceuticals, and other commercially valuable specialized metabolites. Their complex developmental programs, compartmentalized cellular architecture, and photosynthetic machinery make them ideal hosts for the biosynthesis of a wide range of compounds [1]. These features position plants as promising “living factories” for synthetic biology (SynBio) applications. As illustrated in the schematic diagram (Figure 1), plant synthetic biology involves a workflow starting from creating synthetic gene constructs to generating enhanced transgenic plants and novel bioproducts. Engineered DNA constructs are designed with specific regulatory elements (promoters, enhancers, and terminators) and optimized coding sequences, which are then assembled into plasmid vectors carrying transgenes. These plasmids are introduced into plant cells via transformation methods [2]. Once the transgenes stably integrate into the plant genome and are expressed, effectively turning the host cell into a biofactory that produces the corresponding proteins or metabolites. Through this process, transgenic plants can be developed that exhibit enhanced traits (e.g., improved stress tolerance or yield), or they can be engineered as production platforms to synthesize valuable outputs, such as renewable biofuels and high-value metabolites [1,3]. This illustrates how synthetic gene design and delivery can be translated into tangible outcomes in crop improvement and industrial biotechnology.
Synthetic biology is an interdisciplinary field that applies engineering principles to the design and construction of new biological entities and systems or to the redesign of existing natural systems for novel functions. However, the inherent complexity of plant systems has posed significant challenges for implementing SynBio approaches in plants. The recent advancements in genetic engineering; improved transformation techniques; the development of standardized genetic parts; and the integration of genome editing tools, such as CRISPR-Cas systems [2], offer new opportunities to overcome these challenges and unlock the full potential of SynBio in plant systems.
CRISPR-Cas systems are providing a growing toolkit of molecular components, including synthetic promoters, transcriptional activators, and modular DNA assembly systems, now enabling the rational programming of plant traits with precision and control [4]. Recent advancements in CRISPR systems, such as Miniature CRISPR systems like ISYmu1, allow for faster and simpler editing [5], while new approaches like the TwinPE prime editing strategy [6] are improving the efficiency of specific edits in rice [7]. Additionally, systems like IPGEC are enabling in planta genome editing without tissue cultures [8], and nanotechnology is being explored for the enhanced delivery of CRISPR components [9]. While earlier genome editing was considered very difficult and unpredictable, the advent of CRISPR-Cas technology has transformed it into a highly precise and programmable tool, allowing researchers to rewrite plant genomes, build novel functions, and redesign biology [10]. However, the efficient delivery of these constructs into plant systems remains challenging due to their rigid cell walls, complex tissue structures, and lengthy regeneration life cycles. Despite these barriers, various transformation techniques, such as Agrobacterium-mediated transformation and both physical and biological delivery methods, are advancing the integration of genome-edited circuits into plant hosts [3]. To fully harness the potential of these rapidly evolving genome editing technologies, it is essential to integrate them into robust synthetic biology frameworks that emphasize engineering principles and leverage efficient plant transformation platforms. The integration of engineering principles, like modularity, standardization, and predictability, along with the use of iterative Design–Build–Test–Learn (DBTL) cycles, can enable the precise refinement of genetic constructs for plant development and metabolism [11,12]. These engineering-driven workflows require model systems that support rapid and reliable genetic manipulation. Thus, the availability of plant species with standardized transformation protocols, such as fast-growing, easily transformable plants and photosynthetic organisms like cyanobacteria, provides the essential platforms for advancing synthetic biology. Tobacco, in particular, is widely used due to its high transformation efficiency and compatibility with in vitro culture systems [13].
As the technical capabilities of plant synthetic biology continues to expand, they are increasingly intersecting with societal values, raising important questions about the broader implications of plant synthetic biology. A mix of excitement and concern has arisen in the public sphere, as enthusiasts see in it the potential to create climate-resilient crops, edible vaccines, and sustainable biofactories, while critics worry about ecological disruption, the unintended consequences, and corporate control of genetic resources. Public attitudes have varied widely across regions, often influenced by cultural perspectives on nature, historical experiences with GMOs, and the transparency of scientific communication [12]. These societal reflections underscore the urgency of advancing plant synthetic biology not only as a technical endeavor but also as a transformative discipline that must align scientific progress with public interest and global sustainability goals.
Today, SynBio stands at the forefront of modern biology, merging engineering principles with biology to enable the rational design of novel functions and systems. It has seen rapid progress in microbial systems, owing to their simplicity and ease of manipulation [14]. Its expansion into the plant kingdom could open transformative opportunities beyond its microbial capabilities. In the face of a growing global population and escalating climate challenges, SynBio could offer a pathway to resilient, efficient, and sustainable agriculture. Yet, this progress must be guided by ethical foresight, transparency, and public trust. At its core, plant synthetic biology is a human pursuit that is driven by a desire to understand and improve life. By reimagining the genetic potential of plants, researchers aim to enhance food security, reduce reliance on fossil fuels, and restore ecological balance. For this vision to succeed, the field must prioritize rigorous modeling, standardization, plant-optimized toolkits, and integration with machine learning and omics data. Equally important are open-access platforms and an inclusive dialogue to ensure engineered plants align with societal values.
Considering these opportunities and responsibilities, this review delves into the technological foundations and application potential of plant synthetic biology. In this review, we explore the key transformation methods and genome editing advancements, and their integration into synthetic biology. We highlight their applications in agriculture, biofuel production, and pharmaceutical biosynthesis, while addressing the challenges and future directions in the field. The combination of synthetic biology and genome editing will pave the way for innovative and sustainable solutions in plant biotechnology.

2. The Principles and Progress of Synthetic Biology in Plant Systems

Synthetic biology (SynBio) is an interdisciplinary field that focuses on the engineering of living systems and organisms. It applies engineering principles to the design and construction of novel biological parts, devices, and systems, or to reengineer existing biological components for new functions [15]. At its core, synthetic biology seeks to make biology easier to engineer, viewing living systems as potentially programmable machines built from standardized, interchangeable parts [11]. In synthetic biology, the focus shifts from merely asking questions like “How does it work?” to “How can we build it?” and “How can we make it do something useful?” [12]. The genesis of synthetic biology is often traced back to landmark studies around the turn of the millennium, particularly the construction of simple genetic circuits like the toggle switch in Escherichia coli in 2003 [16]. These early successes, which often emerged from engineering departments in universities, established the key principles that would later influence plant science. While much of the initial synthetic biology research focused on microbial systems, the potential to apply these principles to plants was soon recognized, and research started roughly a decade later, making it a relatively young field [12]. While the field was initially grounded in microbial research, its expansion into plant systems has introduced new complexities that require systematic and iterative engineering approaches. A central goal of synthetic biology is the Design–Build–Test–Learn (DBTL) cycle (Figure 2). This iterative process involves designing a biological system based on the desired function, building the genetic constructs, testing their performance in a biological host, and learning from the results to inform subsequent design iterations [17].
The cycle begins with the design phase, where biological parts are selected and assembled into synthetic circuits. In the build phase, these are physically assembled using molecular cloning and introduced into a host. The test phase involves evaluating the system’s behavior and its responses, followed by the learn phase, in which opportunities arise to identify mistakes and make corrections in future designs [19,20]. The goal of SynBio is to accelerate this cycle, moving towards more predictable and robust engineering outcomes. This concept underpins experimental design and analysis in plant synthetic biology, driving the field towards increasingly complex and functional systems [21]. A key aspect of synthetic biology is its focus on standardization and modularity, much like traditional engineering [22]. The aim is to build a library of well-defined biological parts like promoters, coding regions, and terminators that can be combined to create genetic circuits [23]. Though standardization began in microbes with tools like BioBricks, plant synthetic biology now uses its own methods, such as Type IIS-based assembly, to enable the easier sharing and reuse of genetic parts [24]. This push for modularity and standardization is seen as essential for building complex biological systems in a predictable manner.
The concept of a biological “chassis”, defined as the host organism into which synthetic pathways or circuits are introduced, is fundamental to synthetic biology [13,25]. In plant systems, Arabidopsis thaliana and Nicotiana benthamiana are widely employed due to their compact genomes, rapid life cycles, and availability of robust genetic tools [1]. N. benthamiana is favored for transient expression studies owing to its amenability to agroinfiltration and high protein expression capacity [26,27]. Beyond these models, species such as rice, maize, and potato, as well as unconventional hosts like Physcomitrella patens and microalgae, are being evaluated for specialized synthetic biology applications [1,28]. A distinguishing feature of synthetic biology, as compared to traditional genetic engineering, lies in its scale and complexity, which emphasize the coordinated expression of multi-gene networks rather than single-gene modifications [11,12]. Influenced by engineering principles, particularly the analogy of biological systems as programmable circuit boards, synthetic biology enables the modular, rational design of novel functions within a plant chassis [29,30]. While the analogy of biological systems as electronic circuits has guided synthetic biology, experts in plant systems have highlighted its limitations. Though conceptually useful, it oversimplifies the inherent complexity and context-dependence of biological networks. In plants, factors such as pleiotropy, epistasis, and chromatin architecture often cause genetic parts to behave unpredictably, underscoring the need for plant-specific tools and frameworks [12]. Despite these challenges, plant synthetic biology offers a transformative potential to address global issues, such as food security, climate change, and sustainable resource use. The vision of a “Green Revolution 2.0” involves engineering crops with higher yields, reduced environmental inputs, and the capacity to produce biofuels and biochemicals. Achieving this goal requires integration with systems biology to support iterative design and data-driven optimization [11,31]. However, there are still several barriers, such as complex genomes, slow development cycles, and limited trait predictability. In addition to these biological challenges, technical and societal hurdles persist. Although DNA synthesis has become more affordable, there is still a need for reliable plant-specific regulators, such as synthetic promoters and riboswitches. Additionally, public acceptance and regulatory frameworks continue to influence how these technologies are deployed in agriculture [31].
In the current scenario, plant synthetic biology is transitioning from foundational research on model systems toward tackling global challenges in agriculture, sustainability, and biotechnology. Driven by rapid technological progress and a growing toolkit, the field is expanding its ambition to engineer plant systems for societal and environmental benefit [11]. This evolution will require not only scientific innovation but also thoughtful engagement with ethical, regulatory, and societal dimensions [12]. By shifting from descriptive biology to a predictive and modular engineering framework, synthetic biology will enable the rational design of novel traits, metabolic pathways, and regulatory circuits in plants. Central to this effort is genome editing, which allows for precise, site-specific modifications to endogenous genes and facilitates the targeted integration of synthetic constructs [32]. These capabilities will lay the foundation for building increasingly sophisticated and reliable plant systems with improved performance, resilience, and new functionalities.

3. Rewriting the Code of Life Using Genome Editing in Synthetic Biology

To make genome editing possible, several technologies were developed (Table 1), such as zinc-finger nucleases (ZFNs) (Figure 3a), transcription activator-like effector nucleases (TALENs) (Figure 3b), and the recently developed clustered regularly interspaced short palindromic repeats (CRISPR) system. ZFNs, developed in the 1990s, and TALENs, introduced in the early 2010s, were the first tools used for targeted genome editing in plants. These technologies enabled gene modifications in crops like tobacco and rice [33,34]. ZFNs rely on modular zinc-finger domains, each recognizing about three base pairs, while TALENs use tandem repeats that recognize individual bases, both fused to the FokI nuclease. However, designing a new protein for each target site makes these methods labor intensive and time consuming [35]. Additionally, TALEN constructs are quite large (around 3 kb per monomer), which complicates their delivery into plant cells and limits their practical applications.

CRISPR-Cas Systems

The discovery and adaptation of the CRISPR and CRISPR-associated (Cas) protein systems from bacterial adaptive immunity revolutionized genome editing, due to their simplicity, versatility, and ease of programmability [40]. CRISPR/Cas9 technology has been effectively applied to several crop species, including cotton, to enable precise gene editing for trait improvement (Figure 3c). CRISPR-based approaches have been validated using GFP-reporter systems, confirming the editing efficiency and mutation types in transformed cotton tissues [41]. Similarly, the targeted knockout of genes like GoPGF using CRISPR/Cas9 has led to a glandless phenotype, showing the potential of genome editing for modifying complex traits [42]. CRISPR-based approaches have also been used for targeted mutations for maize improvement [43]. In contrast, CRISPR-Cas systems have greatly simplified genome editing. Instead of engineering a new protein for each target, CRISPR uses a common Cas9 protein guided by a customizable single guide RNA (sgRNA) that includes a 20-nucleotide target sequence, making the design faster, cheaper, and more scalable [44]. This has allowed CRISPR to be widely adopted in crop improvement programs for many species due to its precision, efficiency, and ability to edit multiple genes at once [35].
The most widely used CRISPR system employs the Cas9 endonuclease, which is guided to a specific DNA target site by an sgRNA molecule. The sgRNA consists of a sequence complementary to the target DNA (spacer) and a scaffold sequence that binds to Cas9 [40]. Cas9 introduces a double-strand break (DSB) at the target site, typically located immediately upstream of a protospacer adjacent motif (PAM) sequence. The primary advantages of CRISPR-Cas9 are its remarkable ease of design, i.e., simply changing the sgRNA sequence, and its ability to target virtually any DNA sequence, followed by the appropriate PAM. Crucially, a single Cas9 protein can be programmed to target multiple sites simultaneously by simply expressing multiple sgRNAs, enabling multiplex genome editing. This feature is invaluable for targeting multiple genes in a pathway, gene families, or homologous genes in polyploid species. CRISPR-Cas9 systems have been successfully established and widely used in numerous plant species, including Arabidopsis thaliana, rice, maize, wheat, and Nicotiana benthamiana [45,46]. Beyond simple gene knockout via NHEJ, modifications of Cas9 have expanded its capabilities. Catalytically inactive Cas9 (dCas9) can be fused to transcriptional activators or repressors to modulate gene expression without cutting DNA. Cas9 nickases (cut only one DNA strand) can be used in pairs to increase specificity by requiring two binding events for a DSB [47].
Despite its widespread success, CRISPR-Cas9 has limitations, such as the requirement for a specific PAM sequence (e.g., NGG for SpCas9), which can restrict the target sites [48]. Off-target cutting at sites with a partial similarity to the target sequence remains a concern, although strategies like using paired nickases or truncated sgRNAs can mitigate this. Moreover, the efficiency of editing can also be influenced by the chromatin structure of the target site [49,50]. Beyond SpCas9, other Cas nucleases from different bacterial species have been adapted for genome editing, offering different PAM specificities and properties (e.g., Cas12a, Cas12b). Systems targeting RNA (e.g., Cas13) are also being developed. Furthermore, the fusion of dCas9 with enzymes that chemically modify bases (base editors) (Figure 3d) or reverse transcriptases (prime editors) (Figure 3e) allow for precise single-nucleotide changes or small insertions/deletions without creating DSBs. These technologies offer unprecedented precision for introducing specific point mutations [47].
Despite the remarkable progress, several challenges persist in plant genome editing. Achieving high editing efficiency, particularly for HDR-mediated precise modifications, remains difficult in many species. Off-target editing, although reduced with improved SSNs, can still occur and require careful validation. Delivering editing reagents efficiently and uniformly across different plant tissues and developmental stages is crucial for maximizing the chances of obtaining edited germline cells [47]. Polyploid genomes, common in many crops, like wheat, cotton, and peanut, present additional complexity as multiple homologous genes (homoeoalleles) may need to be edited simultaneously to achieve a desired phenotype [51]. Regulatory hurdles and public perception surrounding genetically modified organisms also impact the development and deployment of gene-edited crops [12,31].

4. Targeted Delivery of Genome Editing Components

In plant genome editing and synthetic biology, the efficient delivery of tools like site-specific nucleases (SSNs) and donor DNA is very important (Figure 4, Table 2). The method used for transformation directly affects how well the edits work and whether they are passed on to the next generation [52]. Common methods like Agrobacterium-mediated transformation and particle bombardment are still widely used, but they often depend on the plant species and require tissue culture [51,53,54]. Transient methods, such as agroinfiltration, electroporation, and PEG-mediated protoplast transformation, allow for faster results without genome integration, though obtaining heritable changes can be tough [55,56]. The direct delivery of Cas9-sgRNA as ribonucleoprotein (RNP) complexes is a transgene-free option with fewer off-target effects, but delivering these through tough plant cell walls is still a challenge [47]. Newer approaches, like nanoparticle-based delivery, grafting, and viral vectors like Tobacco rattle virus (TRV), are also being explored to avoid tissue culture and improve efficiency [2,57]. However, achieving a transformation that works across different plant genotypes and ensures proper regeneration remains a major hurdle. In the following section, we will explore the various transformation methods that enable the delivery of genetic material into plant cells.

4.1. Harnessing the Natural Ability of Agrobacterium for Engineering Plant Genomes

The most widely used method for plant genetic transformation is indirect, relying on the natural ability of Agrobacterium species, particularly Agrobacterium tumefaciens, to transfer a segment of its tumor-inducing (Ti) plasmid’s DNA, known as T-DNA, into a plant genome (Figure 4a). Agrobacterium rhizogenes also possesses a similar ability, transferring Ri plasmid T-DNA to induce hairy roots [76]. Researchers have exploited this natural gene transfer mechanism by removing the tumor-inducing genes from T-DNA and inserting the desired foreign genes between the T-DNA borders [31]. Disarmed Ti plasmids, often split into a binary vector system with the T-DNA on one plasmid and the virulence (Vir) genes on another, have become the standard tool for Agrobacterium-mediated transformation. The process begins when phenolic compounds or acidic sugars released from wounded plant tissues activate the Agrobacterium VirA protein, which in turn activates the expression of other Vir genes, leading to the processing of the T-DNA region and its transfer into the plant’s cells. Inside the plant’s nucleus, the T-DNA integrates semi-randomly into the host genome, a process that involves plant DNA repair pathways, particularly non-homologous end joining (NHEJ) [58,77].
Agrobacterium-mediated transformation offers several significant advantages. It typically results in relatively clean integration events with low copy numbers of the transgene, which is desirable for stable expression and the avoidance of gene silencing [53]. It is also highly efficient in certain model species, like Arabidopsis thaliana and Nicotiana tabacum, and in some crops [78]. The floral dip method, for instance, simplifies Arabidopsis transformation by directly immersing flowering plants in an Agrobacterium suspension, bypassing tissue culture entirely and allowing for the recovery of the transformed seeds [71]. This non-tissue culture approach significantly reduces the labor, time, and need for specialized facilities [71].
However, Agrobacterium-mediated transformation is not without its drawbacks. A major limitation is its genotype and species dependence; many important crop species and specific cultivars remain recalcitrant to efficient transformation by this method. The integration of T-DNA is also largely random, which can disrupt endogenous genes or lead to undesirable expression patterns due to insertion location effects [2]. While the floral dip method is revolutionary for Arabidopsis, it is not universally applicable to other flowering plants. For many species, the tissue culture of infected explants (e.g., leaf discs, calli, and immature embryos) followed by regeneration remains necessary, introducing the complexities and limitations associated with in vitro regeneration.

4.2. Particle Bombardment as a Physical Approach to Plant Genetic Transformation

Particle bombardment, also known as biolistic transformation or the gene gun method, is a physical method used to introduce foreign DNA into plant cells. Developed in the late 1980s, it has since become a vital tool in plant biotechnology, particularly for species that are recalcitrant to Agrobacterium-mediated transformation (Figure 4b). Transgenic soybean plants were obtained in 1988 via particle bombardment [79]. Microparticles penetrate the cell walls and membranes, delivering DNA that can integrate into a plant’s genome. Because this method does not rely on a biological vector, it is largely genotype independent. Biolistics enabled the first transformation of several crops previously shown to be recalcitrant to Agrobacterium, including soybean, maize, and rice [80]. Since then, the technique has been applied to many species, from cereals to dicots like potatoes [81]. Notably, biolistic delivery remains the primary method for chloroplast transformation in plants. Other advantages include the ability to introduce multiple genes in one event and even the DNA-free delivery of genome-editing reagents (e.g., CRISPR/Cas9 RNPs) [82].
Despite its versatility, particle bombardment has several limitations. The physical impact of particles can cause tissue damage, necessitating careful optimization to balance DNA delivery and cell survival. Transgene integration is typically random and often multi-copy, which can lead to unpredictable expression or transgene silencing. The transformation efficiency is generally lower than that of Agrobacterium, and the required equipment and supplies (helium and gold particles) are costly [2]. The current research aims to improve its precision and efficiency. The strategies include combining biolistic wounding with Agrobacterium infection to aid the transformation of recalcitrant species, and refining the delivery parameters or using novel nanomaterials to minimize injury. Ongoing innovations in device design and tissue culture are expected to extend biolistics to more difficult crops and achieve more controlled transgene integration [82].

4.3. Other Transformation Methods

Several physical and chemical methods have been developed for plant genetic transformation beyond Agrobacterium and particle bombardment-based transformation. Electroporation uses short electrical pulses to open temporary pores in cell membranes, allowing for DNA uptake, typically into protoplasts (Figure 4c). While it enables the simultaneous transformation of many cells, it requires protoplast isolation and has lower efficiency than Agrobacterium [83]. Microinjections, where DNA is manually injected into cells or nuclei using fine glass needles, allow for precise delivery but are technically demanding, low in efficiency, and limited by plant cell walls (Figure 4d) [84]. Silicon carbide whisker-mediated transformation involves vortexing plant cells with whiskers and DNA; the whiskers puncture the cell walls, allowing for DNA entry (Figure 4e). This simple, low-cost method is mainly used in monocots, but suffers from low efficiency and potential cell toxicity [62,85].
Other innovative methods include the pollen tube pathway, where DNA is applied to flowers during fertilization, allowing for potential transformation without tissue culture (Figure 4f). While simple, it has very low success rates and is species-dependent [86]. Nanoparticle-mediated delivery is an emerging approach, using materials like magnetic nanoparticles or carbon nanotubes to carry genetic material across cell walls (Figure 4g). It offers versatility and minimal tissue damage, but the underlying mechanisms remain poorly understood, and the regeneration of transformed plants remains a major hurdle [70]. Additionally, Agrobacterium rhizogenes-mediated hairy root transformation (ArM-HRT), enables efficient gene transfer by integrating Ri T-DNA into plant genomes, inducing fast-growing, hormone-independent roots (Figure 4i) [87]. Originally described by Chilton, Tepfer, Petit, David, Casse-Delbart, and Tempé [66] in 1982, this method has since become a valuable tool for plant functional genomics and biotechnology. Recent work has shown transformation efficiencies reaching 70–90% in solanaceous crops and up to 75% in citrus, significantly improving the timelines for generating transgenic lines [88]. These roots retain a vascular structure and metabolic functionality, allowing for realistic studies of root biology and pathogen interactions. Moreover, ArM-HRT supports rapid in planta antimicrobial screening against pathogens like Candidatus liberibacter spp., addressing major crop disease challenges [87].

4.4. Cutting-Edge Tools, Such as in Planta and Viral Methods, for Plant Synthetic Biology

To overcome tissue culture bottlenecks, newer in planta methods have been developed. In planta delivery methods avoid the need for tissue culture, which is often a major challenge in many crops. By adding certain growth genes along with CRISPR tools, scientists have been able to edit genes directly in growing parts of plants, in species such as citrus [8]. De Novo Meristem Induction uses gene editing tools and developmental regulators to trigger edited shoot formation directly in plants, and has shown success in crops like tomatoes and grapes [89]. RAPID (Regenerative Activity-Dependent In Planta Injection Delivery), another method, involves the direct injection of Agrobacterium into plant meristems and works well in regenerable species like sweet potatoes [90]. Viral vectors offer a fast, tissue culture-free route for transient gene expression or genome editing (Figure 4h). While viruses can not carry large genes like Cas9, they can efficiently deliver small guide RNAs (sgRNAs), enabling heritable editing when combined with plants expressing Cas proteins [28]. These approaches are promising, but still face challenges like genotype dependence, regeneration issues, and low transformation efficiency.
Plant transformation techniques are continuously evolving to overcome limitations and enhance efficiency. One major approach is the ectopic expression of morphogenic transcription factors (MTFs), like Baby boom (Bbm), Wuschel2 (Wus2), and GRFs, which can enhance regeneration efficiency and expand the range of transformable genotypes [91]. Notably, overexpressing Bbm and Wus2 has enabled the transformation of previously recalcitrant monocot genotypes [92]. To address random DNA integration, targeted insertion strategies are being explored by combining transformation methods (e.g., particle bombardment, Agrobacterium) with sequence-specific nucleases, such as CRISPR/Cas and ZFNs, allowing for precise transgene integration and minimizing disruption to essential genes [2]. The recent advances in plant transformation methods, such as nanoparticle-mediated gene delivery methods, which reduce tissue damage, and improved regeneration using metformin, promise more efficient, genotype-independent transformation. Additionally, emerging in planta methods for plant transformation bypass tissue culture altogether, potentially accelerating the transformation of diverse plant species.

5. Integrating Genome Engineering and Transformation Within Plant SynBio Frameworks

The integration of genome editing technologies, such as CRISPR-Cas systems, with synthetic biology has significantly advanced the precision and scalability of plant engineering. Genome editing allows for the targeted modification or insertion of synthetic constructs, such as gene circuits and metabolic pathways, into defined “safe harbor” loci, leading to their predictable expression and a reduced risk of disrupting essential genes [47]. Although homology-directed repair (HDR) remains inefficient in many plant species, combining genome editing with optimized transformation techniques is progressively enhancing targeted integration outcomes [2]. A major advantage of CRISPR-Cas is its capacity for multiplex editing, enabling the simultaneous modification of multiple genes during a single transformation event. This is particularly valuable for engineering complex traits, redirecting metabolic flux, and fine-tuning regulatory networks [31,51]. Additionally, genome editing facilitates the modification or replacement of cis-regulatory elements, such as promoters, terminators, and untranslated regions (UTRs), enabling fine control over gene expression, a core requirement for constructing synthetic regulatory circuits [4,93]. Genome editing also plays a crucial role in chassis optimization by removing competing endogenous pathways, enhancing precursor availability, and improving tolerance to synthetic products [11,31]. It is an especially promising area for the genetic manipulation of reproductive processes. By targeting the genes associated with male sterility (MS), haploid induction (CENH3, MTL), and meiosis (SPO11-1, REC8, and TAM), researchers are developing strategies for synthetic apomixis, enabling clonal seed production and the stable transmission of hybrid vigor. Furthermore, genome editing enables de novo domestication of wild species by modifying undesirable traits, such as seed shattering or dormancy, while enhancing agronomic traits like yield and stress resistance. This approach broadens the genetic base available for crop improvement by harnessing the valuable traits present in wild relatives [94]. The combination of genome editing with modular DNA assembly systems, such as Golden Gate and Gibson Assembly, further streamlines the DBTL cycle. These tools enable the rapid construction and testing of multi-gene constructs, including SSNs, regulatory elements, and synthetic pathways [11,47]. Together, these advances are positioning genome editing and transformation as foundational components of plant synthetic biology, facilitating the development of programmable, robust, and scalable biological systems for agriculture and biotechnology.

6. SynBio Applications in Agriculture and Biotechnology

The power of plant synthetic biology, augmented by advances in transformation and genome editing, is being directed to a wide array of applications, spanning diverse sectors, from sustainable energy and materials to medicine, nutrition, and agriculture [31].

6.1. Biofuels and Bioproducts

Synthetic biology approaches are being applied to engineering plant biomass composition to make it easier to deconstruct and convert into fuels and chemicals. Lignin, a complex polymer in plant cell walls, is a major barrier to efficient biomass processing [95]. Synthetic biology strategies aim to modify lignin’s structure or content to improve the deconstruction efficiency. For example, engineering plants to incorporate ferulated monolignols can create “zip” lignin with alkali-sensitive ester bonds, making it easier to break down. Reducing the shikimate supply to the lignin pathway can lead to lignin enriched in more easily processed H-monomers [31]. Moreover, plants, with their ability to synthesize complex molecules through photosynthesis, are seen as potential “green factories” for chemical building blocks and platform chemicals [1], such as polyhydroxybutyrate (PHB), a biodegradable bioplastic [96]. Further, the engineering of photosynthetic organisms, such as cyanobacteria, to produce biofuels like isoprene directly from CO2 and sunlight offer a potentially more efficient route to solar fuel production [97,98].

6.2. Medicine and Therapeutics

Synthetic biology enables the plant-based production of recombinant proteins with therapeutic value, such as monoclonal antibodies (mAbs), vaccines, and proteins with human-like glycans [31]. Synthetic biology also enables the production of complex plant natural products with medicinal properties, such as alkaloids and terpenoids, by reconstructing their biosynthetic pathways in an engineered plant chassis. For instance, artemisinin, an antimalarial compound, was produced by reconstructing its pathway in tobacco [1,31] and vaccine adjuvants like QS-21 by reconstructing their complex biosynthesis in Nicotiana benthamiana [26].

6.3. Nutritional Enhancement (Biofortification)

Addressing micronutrient deficiencies, also known as “hidden hunger,” is a critical application of plant synthetic biology [99]. Biofortification aims to increase the levels of essential vitamins, minerals, and health-promoting phytonutrients in staple crops through genetic engineering [100]. A notable success is “Golden Rice,” engineered to produce beta-carotene (provitamin A) in the endosperm by introducing genes from the carotenoid biosynthesis pathway [100,101].

6.4. Sustainable Agriculture

Synthetic biology can be successfully applied to engineering traits in order to enhance the sustainability of agricultural practices. This includes the production of engineered crops for increased tolerance to biotic and abiotic stresses, such as drought, salinity, and extreme temperatures [102]. Engineering beneficial plant–microbe interactions is also a promising area. Synthetic biology can design plants that better recruit beneficial microbes or engineer microbes to provide services to a plant, such as nitrogen fixation or protection from pathogens [103].

6.5. Novel Traits and Systems

Plant synthetic biology is also exploring the creation of entirely novel plant capabilities and systems. This includes engineering plants as remote sensors for environmental pollutants, explosives, or radiation. These “Plant Sentinels” feature computationally designed sensors and control circuits [12]. As humans prepare for interplanetary missions, synthetic biology will be key to developing plants that can survive the challenges of space travel and provide resources for life support [12,31]. These diverse applications highlight the transformative potential of SynBio.

7. Limitations

7.1. Technical Constraints

Traditional plant transformation relies on genotype-specific tissue culture methods that are slow, laborious, and inefficient [104]. Many crops are recalcitrant to Agrobacterium or biolistic delivery, yielding low transformation rates and mosaic integration. Even as molecular tools improve, transformation and regeneration remain bottlenecks [105].

7.2. Genome Editing Challenges

Precise HDR edits are extremely inefficient in plants, so targeted knockins are rare. Off-target effects are also a concern; CRISPR enzymes can cleave loci with several mismatches, causing unwanted mutations. Polyploid genomes (e.g., wheat, potato) complicate editing further, since all gene copies must be modified simultaneously, often necessitating multiplexed guide strategies [105].

7.3. Standardization Deficits

For example, the iGEM parts registry lists > 20,000 entries, but under 100 are plant-specific [106]. Most genetic parts (promoters, terminators, sensors, etc.) are borrowed from bacteria or model plants, and the experimental data on their behavior in crops is limited. This shortage of standardized parts and design toolkits hampers rational circuit design and sharing.

7.4. Regulatory and Societal Hurdles

Strict GMO regulations and public skepticism limit applications. Many jurisdictions (e.g., EU, New Zealand) regulate CRISPR-edited plants as conventional GMOs, triggering onerous approval processes [107]. Even where regulations are relaxed, consumer resistance to “GMOs” persists. Historically, poor public opinion of transgenic crops and heavy regulatory costs have slowed deployment [107].

7.5. Scalability and Predictability

The long generation times (months to years) slow the design–build–test cycles. Plants consist of diverse cell types and are highly sensitive to environmental conditions, so synthetic circuits may behave unpredictably across tissues or climates [108]. This variability and long life cycle make the reliable, large-scale engineering of plant traits difficult.

8. Challenges, Innovations, and Future Directions in the Field of SynBio

With the advancement of plant synthetic biology, the development of tools and the understanding of fundamental biological processes is paving the way for more predictable, robust, and impactful engineering of plant systems. However, realizing the full potential of plant synthetic biology will require addressing several key challenges and embracing emerging innovations [11,12,31]. This will involve the development of a comprehensive library of well-characterized genes, including promoters, terminators, UTRs, and protein-coding sequences, as well as their regulators, such as synthetic riboswitches and transcription factors, that will allow for the fine-tuned control of gene expression [31]. Moreover, integrating quantitative biology and mathematical modeling into the design process will be essential for predicting system behavior before building and testing [12,31]. Advancements in delivery methods will also be crucial for expanding the reach of plant synthetic biology to a wider range of species and genotypes, particularly those currently recalcitrant to transformation. Non-transgenic and tissue-culture-free methods, such as the nanoparticle-mediated delivery of RNPs or plasmids, and in planta methods leveraging developmental regulators or meristem injection, are promising avenues for the future [2,90,109].
The development of plant-specific biofoundries, automated facilities for high-throughput cloning, assembly, and transformation, will play a key role in this scaling process. These facilities could handle large libraries of genetic constructs and automate the DBTL cycle, significantly increasing the throughput [110]. Exploring and optimizing new plant chassis beyond traditional model organisms will also be critical future directions [13]. This includes developing genetic tools and transformation systems for non-model species that have unique advantages, such as resilience to specific environments or the ability to produce certain compounds [12]. Leveraging plant cell cultures as “bio-factories” for the controlled production of valuable compounds is another promising avenue, similar to microbial fermentation [1]. In recent years, AI and machine learning have emerged as transformative tools in plant synthetic biology, accelerating design cycles and improving precision. These computational methods can analyze large biological datasets to guide genome and circuit design, predict optimal gene edits, and streamline metabolic engineering. For example, deep learning models have been used to design synthetic promoters by learning from large libraries of promoter activity, enabling the creation of short, inducible promoters with predictable strengths [111]. Likewise, ML algorithms can integrate genomics, transcriptomics, proteomics, and metabolomics data to infer gene networks and optimize complex pathways in silico [112]. By forecasting the behavior of genetic circuits and enzymes before experiments, AI/ML greatly reduces trial and error, shortening the design–build–test loop. Experts have noted that this computational approach to plant engineering is already “an irreversible trend”, with remarkable potential for future crop breeding [26,113]. However, addressing the regulatory landscape and public perceptions remains crucial for the successful deployment of plant synthetic biology applications. Experts have emphasized the need for agile regulatory policies that can adapt to rapidly developing technologies. Open communication, transparency, and engaging with stakeholders, including the public, indigenous communities, and industry, are essential for building trust and facilitating acceptance [12,31].

9. Conclusions

The journey of plant synthetic biology, rooted in early genetic engineering, to its current status as a sophisticated and independent discipline, is a remarkable testament to scientific innovation and engineering ingenuity. Driven by the principles of design, build, test, and learn, the field has steadily advanced towards the ambitious goal of making biology easier to engineer, particularly within the complex and powerful systems of plants. At the heart of this progress lies the connection between advancements in plant transformation methods and the revolutionary capabilities of genome editing technologies. The integration of these technologies enables sophisticated synthetic biology applications in plants. This includes engineering plant biomass for sustainable biofuels and bioproducts, developing plant-based systems for producing pharmaceuticals and vaccines (pharming), and enhancing the nutritional content of staple crops through biofortification. By improving stress tolerance, environmental sensing in precision agriculture, and nutrient use efficiency in crops, SynBio has already contributed to sustainable agriculture. Despite the achievements, challenges remain. Improving the predictability and robustness of engineered traits, scaling up the design and testing processes, and navigating the complex regulatory and public perception landscapes are critical for the future. Further, synthetic biology offers solutions that are scalable and can address real-world problems, such as climate change and global food security. Overall, plant synthetic biology represents a powerful approach to harness the inherent capabilities of plants for the benefit of humanity and the planet.

Author Contributions

S.K.: conceptualization, writing, review, and editing; P.K.V.: editing; M.R.J.: supervision, conceptualization, writing, review, and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

We acknowledge the support from the Department of Plant and Soil Science at Texas Tech University for resources and infrastructure.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Schematic representation of synthetic biology-driven transformation and cellular biofactory engineering. Synthetic gene constructs or plasmids carrying transgenes are introduced into host cells using transformation methods. Engineered cells act as biofactories, producing high-value metabolites, biofuels, and transgenic plants with enhanced traits.
Figure 1. Schematic representation of synthetic biology-driven transformation and cellular biofactory engineering. Synthetic gene constructs or plasmids carrying transgenes are introduced into host cells using transformation methods. Engineered cells act as biofactories, producing high-value metabolites, biofuels, and transgenic plants with enhanced traits.
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Figure 2. The Design–Build–Test–Learn (DBTL) cycle in synthetic biology. This cycle shows a step-by-step process for creating and improving biological systems. It starts with design, where new genetic parts or pathways are designed. Design is followed by build, where these designs are turned into real DNA and put into living cells. Next, in the test phase, the modified organisms are checked to see if they behave as expected. Finally, the learn step involves looking closely at the results to understand what worked and what did not, helping to make better designs the next time. This ongoing process helps fine-tune biological systems faster and more effectively, producing desired metabolites or proteins. The conceptual map is structured around the Design–Build–Test–Learn (DBTL) cycle, and it is the central framework that guides the whole SynBio engineering system [18].
Figure 2. The Design–Build–Test–Learn (DBTL) cycle in synthetic biology. This cycle shows a step-by-step process for creating and improving biological systems. It starts with design, where new genetic parts or pathways are designed. Design is followed by build, where these designs are turned into real DNA and put into living cells. Next, in the test phase, the modified organisms are checked to see if they behave as expected. Finally, the learn step involves looking closely at the results to understand what worked and what did not, helping to make better designs the next time. This ongoing process helps fine-tune biological systems faster and more effectively, producing desired metabolites or proteins. The conceptual map is structured around the Design–Build–Test–Learn (DBTL) cycle, and it is the central framework that guides the whole SynBio engineering system [18].
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Figure 3. Schematic representation of various genome editing tools. (a) Zinc-finger domains bind to specific DNA sequences flanking a target site, with the FokI nucleases forming a dimer to induce a double-stranded break (DSB) in the DNA. (b) TAL effector domains recognize the target DNA sequences, positioning the FokI nuclease dimer to introduce a DSB between the binding sites. (c) Cas9 endonuclease, guided by a single guide RNA (sgRNA), binds to the complementary target DNA and introduces site-specific double-stranded cuts. (d) A base editor complex consisting of a Cas9 nickase (dCas9) and a deaminase enzyme, guided by gRNA, facilitates the conversion of a single base at a defined site without creating DSBs. (e) A prime editor containing a Cas9 nickase fused to reverse transcriptase uses a prime editing guide RNA (pegRNA) to direct targeted sequence modifications, including insertions, deletions, and base substitutions.
Figure 3. Schematic representation of various genome editing tools. (a) Zinc-finger domains bind to specific DNA sequences flanking a target site, with the FokI nucleases forming a dimer to induce a double-stranded break (DSB) in the DNA. (b) TAL effector domains recognize the target DNA sequences, positioning the FokI nuclease dimer to introduce a DSB between the binding sites. (c) Cas9 endonuclease, guided by a single guide RNA (sgRNA), binds to the complementary target DNA and introduces site-specific double-stranded cuts. (d) A base editor complex consisting of a Cas9 nickase (dCas9) and a deaminase enzyme, guided by gRNA, facilitates the conversion of a single base at a defined site without creating DSBs. (e) A prime editor containing a Cas9 nickase fused to reverse transcriptase uses a prime editing guide RNA (pegRNA) to direct targeted sequence modifications, including insertions, deletions, and base substitutions.
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Figure 4. Various methods of gene delivery using plant transformation. (a) Agrobacterium-mediated transformation—A foreign gene is inserted into a Ti plasmid and transferred into plant cells via Agrobacterium tumefaciens, leading to its stable integration into the plant genome [58]. (b) Gene-gun-mediated transformation—DNA-coated gold or tungsten particles are shot into plant cells, facilitating DNA integration and plant regeneration from the transformed cells [59]. (c) Electroporation transformation—High-voltage electric pulses create transient pores in protoplast membranes, allowing plasmid DNA to enter cells [60]. (d) Microinjection—Direct injection of a transgene into the nucleus of a plant protoplast using a fine microcapillary needle [61]. (e) Silicon carbide whiskers—DNA-coated whiskers are mixed with plant cells and vortexed, causing the physical penetration of the DNA into the cells through puncture sites [62]. (f) Pollen tube pathway—DNA is introduced into an ovule via the pollen tube, allowing for fertilization with genetically modified pollen [63]. (g) Nanoparticle delivery—DNA is bound to nanoparticles, which cross the plant cell walls and membranes, enabling genetic transformation [64]. (h) Viral-vector delivery—Recombinant viral DNA is used to infect plants, leading to the transient or stable expression of the introduced gene [65]. (i) Hairy root transformation—Ri plasmids carrying DNA along with Agrobacterium are inserted into a plant by making a wound. Roots are grown in the wound area after successful insertion [66].
Figure 4. Various methods of gene delivery using plant transformation. (a) Agrobacterium-mediated transformation—A foreign gene is inserted into a Ti plasmid and transferred into plant cells via Agrobacterium tumefaciens, leading to its stable integration into the plant genome [58]. (b) Gene-gun-mediated transformation—DNA-coated gold or tungsten particles are shot into plant cells, facilitating DNA integration and plant regeneration from the transformed cells [59]. (c) Electroporation transformation—High-voltage electric pulses create transient pores in protoplast membranes, allowing plasmid DNA to enter cells [60]. (d) Microinjection—Direct injection of a transgene into the nucleus of a plant protoplast using a fine microcapillary needle [61]. (e) Silicon carbide whiskers—DNA-coated whiskers are mixed with plant cells and vortexed, causing the physical penetration of the DNA into the cells through puncture sites [62]. (f) Pollen tube pathway—DNA is introduced into an ovule via the pollen tube, allowing for fertilization with genetically modified pollen [63]. (g) Nanoparticle delivery—DNA is bound to nanoparticles, which cross the plant cell walls and membranes, enabling genetic transformation [64]. (h) Viral-vector delivery—Recombinant viral DNA is used to infect plants, leading to the transient or stable expression of the introduced gene [65]. (i) Hairy root transformation—Ri plasmids carrying DNA along with Agrobacterium are inserted into a plant by making a wound. Roots are grown in the wound area after successful insertion [66].
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Table 1. Different gene editing techniques.
Table 1. Different gene editing techniques.
FeatureZFNsTALENsCRISPR-Cas9
OriginArtificial, based on zinc-finger domains + FokI.TALE proteins from Xanthomonas + FokI.Bacterial adaptive immune system (Streptococcus pyogenes).
DNA Recognition MechanismZinc-finger domains recognize triplet nucleotides.TALE repeats recognize single nucleotides.sgRNA base pairs with DNA target.
Targeting DesignComplex, labor-intensive protein engineering required.Modular and easier than ZFNs.Simple; change sgRNA sequence.
Nuclease DomainFokI (requires dimerization).FokI (requires dimerization).Cas9 (cleaves directly as a single unit).
PAM RequirementNo strict PAM, but binding context-dependent.No strict PAM, but spacer length is critical.Yes, PAM required (e.g., NGG for SpCas9).
Multiplexing CapabilityLimited.Limited.High (multiple sgRNAs can guide one Cas9).
SpecificityHigh, but off-target risk exists.High; more predictable than ZFNs.Good, but off-targets can occur without careful design.
Delivery ChallengesSmaller size (~1 kb); easier to deliver.Larger size (~3 kb); more difficult for vectors.Medium; size ~4.2 kb for SpCas9; deliverable via viral/non-viral means.
Ease of UseLow (complex protein design).Moderate (modular design).High (RNA guided, programmable).
Applications in PlantsDemonstrated (e.g., maize, rice, and soybean).Widely used (e.g., Arabidopsis, tobacco, and rice).Broad adoption across plant species.
First DemonstratedLate 1990s–early 2000s
[36,37].		Around 2010 [38,39].2012 [40].
Table 2. Different methods of gene delivery in plant systems.
Table 2. Different methods of gene delivery in plant systems.
MethodTypeAdvantagesLimitationsSizeReference
Agrobacterium  MediatedIndirect (Biological)Low copy number; high efficiency in some species; stable integration.Genotype-specific; random integration.~150 kb[53]
Particle BombardmentDirect (Physical)Broad species range; useful for organelle transformation.Tissue damage; random integration; costly equipment.<10 kb[2,67]
ElectroporationDirect (Electrical)Simultaneous DNA delivery to many cells; no vector needed.Requires protoplasts; lower efficiency; potential cell damage.~10–20[2]
MicroinjectionDirect (Physical)Precise DNA delivery to specific cells or nuclei.Technically demanding; low efficiency.~50 kb[68]
Silicon Carbide WhiskersDirect (Physical)Simple, low-cost, no expensive equipment.Low efficiency; potential cell toxicity.Typically small, <10 kb[2,62,69]
Pollen Tube PathwayDirect (Biological)No tissue culture required; easy to perform.Very low efficiency; species limited.Typically small, <10 kb[68]
Nanoparticle MediatedDirect (Chemical/Physical)Low cytotoxicity; potential species versatility.Emerging field; mechanism not fully understood.5–30 kb[70]
In Planta MethodsIn planta (Biological)Bypasses tissue culture; potentially genotype independent.Still under development; species-specific performance.Typically small, <10 kb[71]
Viral Vector MediatedIndirect (Biological)High expression levels; transient delivery; useful for gene function studies.Limited to transient expression; germline exclusion.~10 kb[72,73,74]
Hairy-Root TransformationIndirect (Biological)High transformation efficiency; rapid high-density root growth; robust secondary metabolite production.Only roots; non-heritable.~150 kb[75]
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Kambampati, S.; Verma, P.K.; Janga, M.R. Plant Transformation and Genome Editing for Precise Synthetic Biology Applications. SynBio 2025, 3, 9. https://doi.org/10.3390/synbio3030009

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Kambampati S, Verma PK, Janga MR. Plant Transformation and Genome Editing for Precise Synthetic Biology Applications. SynBio. 2025; 3(3):9. https://doi.org/10.3390/synbio3030009

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Kambampati, Sharathchandra, Pankaj K. Verma, and Madhusudhana R. Janga. 2025. "Plant Transformation and Genome Editing for Precise Synthetic Biology Applications" SynBio 3, no. 3: 9. https://doi.org/10.3390/synbio3030009

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Kambampati, S., Verma, P. K., & Janga, M. R. (2025). Plant Transformation and Genome Editing for Precise Synthetic Biology Applications. SynBio, 3(3), 9. https://doi.org/10.3390/synbio3030009

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