7 Plant Transformation
Faizo Kasule; Walter P. Suza; and Madan K. Bhattacharyya
Introduction
Plant transformation is cornerstone of modern biotechnology. It involves introducing foreign genes into plant cells to produce gene-edited or genetically modified plants with stable, integrated, and heritable traits. This process includes two main steps: (1) transient transformation, where foreign DNA is introduced into plant cells but not yet integrated into the genome, and (2) stable transformation, where the foreign DNA is integrated into the plant genome. Both steps are important in plant biotechnology, with the latter being essential for producing transgenic plants that pass desired traits to subsequent generations. Unlike traditional breeding methods that rely on crossing related species, plant transformation enables the transfer of a desired gene across species, genera, families, and kingdoms, which was previously impossible. In a matter of weeks, plant transformation produces millions of cells with regeneration capacity, which dramatically expanded the scope of plant genetic improvement, revolutionizing agriculture and plant science (Figure 1).
Through plant transformation, scientists have developed crops with enhanced resistance to abiotic stresses, including drought, salinity, and flooding, as well as biotic stresses such as pests and diseases. Additionally, the process has resulted in increased yields, improved nutritional quality, and enhanced pharmaceutical compounds in plants. Furthermore, scientists utilize plant transformation to study functional genomics and unravel the roles of specific genes in plant growth, development, and physiology.
History of plant transformation
The history of plant transformation traces back to early studies of plant totipotency and tissue culture (Figure 2). The notion that plant cells could regenerate into whole plants originated in the 18th century, when polyembryony was first observed in citrus seeds, which inspired the artificial regeneration of plants from somatic tissues. The first reports detailing in vitro cell culture date back to 1902, when Haberlandt proposed that isolated plant cells, under appropriate conditions, could grow and develop into whole plants, introducing the concept of totipotency. Although unsuccessful, his idea was later adopted when Gautheret (1934) achieved continuous in vitro culture of vascular plant tissues in the 1930s (Gautheret, 1939). By the 1950s, Straus and LaRue (1954) established the first maize callus cultures from immature endosperm, marking an early step toward cereal tissue culture (Straus & LaRue, 1954). Later, Steward et al. (1958) demonstrated somatic embryogenesis in carrots, confirming regeneration from single somatic cells (Steward et al., 1958).
In the 1970s, Green and Phillips established stable embryogenic maize callus cultures and identified genotype A188 for its high embryogenic potential (Green & Phillips, 1975). Around the same time, Agrobacterium tumefaciens, first identified in 1907 as the cause of crown gall disease, was shown to transfer a DNA segment (T-DNA) into plant genomes. This discovery revealed a natural mechanism for genetic transformation, and its subsequent development led to the stable transformation of transgenic plants, including tobacco, petunia, and sunflower, in the 1980s. However, despite its success in dicots, the transformation of monocots such as rice and maize remained elusive due to host incompatibility, which prompted the development of alternative DNA delivery methods.
Maize transformation was achieved by Armstrong and Green (1985) through friable Type II callus cultures from the inbred line A188, enabling efficient and stable plant regeneration. This system became a cornerstone of modern maize transformation. In the 1980s, Sanford and colleagues developed the biolistic gene gun, which enabled the creation of the first fertile transgenic maize (Klein et al., 1987).
By the 1990s, Agrobacterium-mediated transformation was successfully extended to rice and other maize genotypes, helping to bridge the gap between dicots and monocots. In the 2000s, transformation research using morphogenic regulators such as WUSCHEL (WUS) and BABY BOOM (Bbm) (Lowe et al., 2016), enabled the reprogramming of somatic cells into an embryonic state, thereby improving regeneration efficiency and expanding the range of transformable genotypes.
Building on these advances, the 2010s witnessed the integration of CRISPR/Cas genome editing into transformation pipelines, enabling the precise and efficient modification of plant genomes within regenerable tissues. Current innovations leverage the use of GROWTH-REGULATING FACTOR5, a chimeric fusion of GRF4 with its cofactor GRF-interacting Factor1 (GRF4-GIFI), Wuschel-related homeobox 5 (WOX5), and WOX2a, among others, to enhance transformation efficiency (Lee & Wang, 2023) (Figure 2).
Incorporation of superior genes
The goal of plant transformation is to introduce specific, well-characterized genes into plants to confer desirable traits, such as pest resistance, disease tolerance, nutritional enhancement, and stress adaptation (Figure 1). As mentioned earlier, unlike conventional breeding, which transfers thousands of genes at once, transformation enables the precise insertion of individual genes into plant genomes, including those from unrelated species. It also allows the use of genes from organisms that cannot be introduced naturally into a plant due to crossing barriers that prevent interspecific hybridization. These features make plant transformation a valuable tool for developing male-sterile parents, which are essential for producing hybrid cultivars. Beyond enhancing yields, improved nutritional quality, and enhanced pharmaceutical compounds, plant transformation also facilitates functional genomics studies to identify the roles of specific genes in plant growth and development.
Transformation system
A transformation system refers to the entire process and a set of tools used to introduce and integrate foreign DNA (transgene) into the plant genome, followed by regeneration of whole, fertile, and stable transgenic plants. A successful transformation system combines the following.
- A suitable explant (tissue that regenerates),
- An efficient DNA delivery method, and
- A reliable selection system to identify transformed cells
Choice of explant
In preparing plant tissue for transformation, the cells must be competent to integrate the construct into their genome. The transformed cells must also be competent for regeneration into a plant (see Plant Breeding Methods). However, not all tissues are competent for transformation and regeneration, and different plant species vary considerably in their competence for cell transformation and regeneration potential. Typically, only a fraction of cells or tissues can regenerate, and within that subset, only a small portion is capable of transformation.
In certain instances, tissues that are less competent can be induced to become competent by specific treatments (such as exposure to plant growth regulators or stress signals). A cell’s response to wounding can also influence its capacity for regeneration. In some dicot species, wounding triggers the formation of dedifferentiated cells around the wound site, which can then be induced to regenerate under appropriate conditions. However, monocot species respond differently to wounding, often resulting in cell death near the wound site. This cell death limits regeneration due to the lack of dedifferentiated cells. Consequently, transformation of many monocots relies on callus tissue or immature embryos, as embryogenic tissue can regenerate without undergoing dedifferentiation.
Explant or tissue types used for the transformation of plants
An explant refers to any part of a plant used as starting material for tissue culture. It is often confused with callus tissue, which consists of a mass of undifferentiated cells. Callus cells are totipotent, meaning a single cell can divide, differentiate into various specialized cell types, and ultimately regenerate into a whole plant.
Protoplasts from whole tissue or suspension cells
Protoplasts are plant cells that have had their cell walls removed using enzymatic or mechanical methods and are surrounded only by plasma membrane. Protoplasts are generally isolated from differentiated plant tissues and can regenerate new cell walls, forming callus with totipotent potential. These properties enable protoplasts to regenerate into whole plants. Additionally, the absence of cell walls, which normally resist foreign DNA uptake, enables protoplasts to readily take up foreign DNA for genetic transformation. Protoplasts have been utilized to study cellular processes, including hormone signaling, protein function, DNA transfection, and viral infection, among others. Crops such as potatoes and rice can be transformed from protoplasts.
Immature embryo
This explant is widely used for many crop transformations, such as maize, due to its high regenerative capacity and presence of many totipotent cells. Immature embryos are developmentally active and responsive to in vitro culture, making them ideal for inducing embryonic calli and regenerating gene-edited or genetically modified plants. In maize, for example, immature embryos can be harvested 8 to 20 days after pollination. Transformation efficiency using immature embryos is influenced by various factors, including genotype, embryo size, and hormone combinations, and must often be optimized for consistent results. Other crops transformed using immature embryos include Barley, wheat, sorghum, and rice.
Intact cells from suspension cultures
These offer a homogenous and rapidly growing source of explants for plant transformation. Intact cells from suspension cultures enable efficient metabolite production and analysis at the cellular level. Furthermore, their accelerated metabolic and biosynthetic activity, resulting from uniform exposure to nutrients and growth regulators, helps reduce time and labor compared to solid media. Common grape vine, Citrus spp, and rice are among the crops transformed using Intact cells from suspension cultures.
Leaf discs
These are widely used in Agrobacterium tumefaciens-mediated plant transformation due to several reasons, such as their accessibility and responsiveness. Tobacco and other model plants exhibit higher transformation efficiencies (TE) than recalcitrant species, such as potato and soybean, when using leaf discs. This is because TE varies depending on species, genotypes, and developmental stages of the explant. In addition, factors such as explant age and culture conditions can influence TE in leaf disc-based plant transformation.
Cotyledon sections
Embryonic leaves within a seed can regenerate into whole plants, especially in dicots. Juvenile cotyledon sections often regenerate at a higher rate than mature tissues, making them ideal for transformation. Crops transformed using cotyledon sections include tomatoes and cucurbit species, such as watermelons and cucumbers.
Embryogenic tissue
The ability of embryonic tissues to rapidly proliferate and produce multiple transformed lines makes them ideal explants for plant transformation. Embryogenic tissues originate from somatic cells that undergo dedifferentiation under specific hormonal conditions, allowing them to develop into whole plants through the process of somatic embryogenesis. Crops transformed using embryogenic tissues include Barley, wheat, maize, sorghum, and rice.
Seedling leaf whorls
Seedling leaf whorls are the central, tightly wrapped leaves at the shoot apex of young seedlings. Recently, leaf whorls have emerged as a promising explant for regenerating recalcitrant monocot genotypes in combination with morphogenic transcription factors BABY BOOM (Bbm) and WUSCHEL2 (Wus2). During the transformation process, three to four outer leaf sheaths are removed to expose the inner, greener, and softer leaf tissues that are more responsive to transformation and regeneration. Examples of crops that have been transformed using seedling leaf whorls include maize and sorghum, rice (both indica and japonica), teff, switchgrass, pearl millet, foxtail millet, barley, and rye
Introduction of DNA into the Cells of the Target Tissue
A requirement of a transformation system is that the DNA of interest must be able to integrate into the genome of target plant cells that can regenerate. It is also important that the process of integrating DNA into a cell does not render the cells unable to regenerate a new plant in tissue culture. Several methods are available to introduce DNA into the host. The method used depends on the host, its capacity to receive and incorporate the foreign DNA via the method, and the host’s ability to regenerate if transformation occurs in culture. The following methods can be used for plant transformation, with the choice depending on the recipient species and the type of transformation: (i) stable transformation or (ii) transient expression of genes.
- Direct DNA uptake—DNA cannot be taken directly into cells because of the cell wall; therefore, protoplasts are used. DNA is imported into the protoplasts with polyethylene glycol.
- Microinjection—Microcapillaries inject DNA directly into the host nucleus.
- Agrobacterium—mediated gene transfer—Agrobacterium tumefaciens or A. rhizogenes is used to deliver DNA into the recipient plant cells.
- Particle bombardment or biolistic transformation—a physical means of transfecting cells by bombarding tissue with high-velocity DNA-coated particles. This method can be applied to protoplasts, intact cells isolated from a suspension culture, or whole tissues or organs.
- Electroporation—DNA is electroporated into protoplasts.
Here, we discuss two commonly applied plant transformation procedures:
- Agrobacterium-mediated gene transfer
- Particle bombardment or biolistic transformation
Agrobacterium-mediated gene transfer
The genus Agrobacterium has been divided into several species based on the disease symptoms and host range. For example, A. tumefaciens causes crown gall disease , A. rhizogenes induces hairy root disease, A. rubi causes cane gall disease, and A. Vitis, which has been most recently proposed to cause galls on grapes and a few other plants. These soil-pathogenic bacteria are valued by scientists in plant biotechnology and genetic engineering for their ability to transfer DNA into plants via horizontal gene transfer. This unique ability has earned Agrobacterium the title of “nature’s genetic engineer”. Agrobacterium-mediated gene transfer leverages the bacterium’s capability to introduce a gene of interest into plant cells to create transgenic plants. Of the several species, A. tumefaciens is the most used species for generating stable transgenic plants, while A. rhizogenes produces transformed adventitious “hairy roots”, often applied in rapid functional analyses of root-expressed traits. In the following section, you will learn about the crown gall disease to some extent and the application of Agrobacterium in mediating stable plant transformation.
The Molecular Basis for the Crown Gall Disease
Agrobacterium tumefaciens infects wounded plant tissues by transferring a segment of its tumor-inducing (Ti) plasmid, a region called T-DNA, into the plant genome. Upon infection, this leads to crown gall disease, characterized by the formation of tumors on plant stems at or near the soil surface. This bacterium belongs to the family Rhizobiaceae, which includes Rhizobium, a genus involved in the formation of symbiotic nodules in leguminous plants. The Ti plasmid is exchanged between Agrobacterium species to facilitate the spread of this genetic element. A. tumefaciens tumors result from the rapid proliferation of plant cells after bacterial infection. Remarkably, tumor cells isolated from an infected plant can continue to grow in culture, even after the culture is cured of Agrobacterium, and in the absence of added external plant hormones, such as auxins and cytokinins. For many plant species, these hormones are normally needed to stimulate growth in culture, and the ratio of auxins to cytokinins determines whether roots, shoots, or undifferentiated tissue will form.
Agrobacterium-infected cells undergo natural transformation, resulting in undifferentiated growth and ultimately leading to the formation of tumors. The promoters driving these genes are constitutive and highly active in plant tissues, resulting in continuous, upregulated production of auxins and cytokinins. Examples of these genes are aux, cyt1; also called iaaM and iaaH, iptZ. Enzymes encoded by these genes convert existing plant compounds to indole-3-acetic acid (IAA) and isopentenyladenosine-5-monophosphate, naturally occurring auxin and cytokinin, respectively. As a result, infected plant cells are in a continuous state of undifferentiated proliferation, leading to tumor/gall formation. Opines are novel compounds derived from amino acids, rich in carbon and nitrogen. The most common opines are Octopine (carboxylarginine) and nopaline (dicarboxypropylarginine). Opines cannot be metabolized by plant cells or other microorganisms, but Agrobacteria can utilize them because the Ti plasmid contains additional genes for opine metabolism. Opine synthesis genes are located on the T-DNA that gets integrated into the plant genome, whereas opine metabolism genes remain in the Agrobacterium on the Ti plasmid. This creates a favorable environment for bacterial growth because plant cells are programmed to produce N- and C-rich compounds that can only be utilized by Agrobacterium.
Scientists harness this mechanism by replacing tumor-inducing genes with genes of interest to produce transgenic plants. The transformation process can be divided into four steps: T-DNA processing, T-DNA strand transfer, T-DNA integration, and transgene expression.
T-DNA processing and integration into the plant genome
The Ti plasmid, typically 200-800 kb in size, contains genes responsible for virulence, host specificity, T-DNA transfer, phytohormone synthesis, opine synthesis, and opine catabolism. A 25-bp border sequence flanks the T-DNA (Gelvin, 2003).
Agrobacterium-mediated transformation, a critical early step in the activation of the T-DNA transfer pathway, involves the induction of bacterial virulence (vir) genes through a signal transduction pathway (Figure 3). Wounded plant tissue produces phenolic signal molecules, such as acetosyringone, which are detected by sensors produced by Agrobacterium, specifically VirA and VirG proteins (Winans, 1992). VirA, a membrane-bound sensor kinase, is autophosphorylated upon detection of plant phenolics with the help of ChvE, a sugar-binding protein, and this in turn activates VirG by transferring the phosphate group. This causes the phosphorylated VirG to upregulate the expression of vir genes, initiating T-DNA processing and transfer cascade pathways.
The VirD1/VirD2 complex is involved in T-DNA processing from the Ti plasmid by nicking the bottom strand of the DNA at the 25bp border sequences that flank the T-DNA. However, VirD2 remains covalently attached to the 5’ end of the resultant single-stranded T-DNA strand, forming the core of the complex, which is transferable. The T-DNA strand is then escorted by VirD2 and later by VirE2, a single-stranded DNA-binding protein, to create the “T-complex”. VirE2 associates with the T-strand within the bacterium or upon entry into the plant cytoplasm to facilitate nuclear targeting and protection of the T-strand from nucleases. The export of T-DNA occurs via a type IV secretion system, composed of 11 VirB proteins and VirD4, which acts as a coupling protein that guides the T-complex to the VirB translocation channel. Some VirB proteins form the channel, while others, such as VirB2 and VirB5, form a T-pilus that anchors Agrobacterium to the plant cell surface and acts as a conduit for DNA/protein passage (Gelvin, 2017).
VirD2 possesses nuclear localization signal sequences that facilitate nuclear import and direct the T-complex to the plant nucleus. Studies have shown that VirD2 can direct small T-DNA segments into the plant nucleus independently, but for larger DNA molecules, VirE2 is required. These proteins together direct the T-strand to the plant nucleus for integration into the plant genome, facilitated by VirD2. Inside the nucleus, T-DNA integration occurs with the assistance of host DNA repair pathways, which are often activated by double-stranded breaks (DSBs) (Gelvin, 2003). VirD2 tethers the T-strand to the host’s chromatin by interacting with plant transcription factors and DNA repair components. Microhomology-mediated end joining (MMEJ), facilitated by the action of theta polymerase, also promotes the integration of T-DNA.
Read: Gelvin, S.B. (2003) for a detailed explanation of the molecular basis of Agrobacterium-mediated transformation and how T-DNA moves into plant cells.
Use of A. Tumefaciens in Plant Transformation
The steps in Agrobacterium-mediated plant transformation were described in Plant Breeding Methods. The process, however, is complex and relies on precise interactions between Agrobacterium virulence (Vir) genes and plant cellular components. A key event is the transfer of T-DNA from the bacterium into the plant genome, which is mediated by a type IV secretion system and guided by Vir proteins. Understanding the molecular mechanisms of T-DNA processing, transfer, and the plant’s response is essential for enhancing the efficiency of Agrobacterium in biotechnology applications.
In the early days of genetic engineering, scientists became interested in Agrobacterium’s ability to insert its DNA into plant cells. They subsequently modified the Agrobacterium by removing its ability to cause disease but retaining its ability to transfer foreign DNA into plant cells. The transferred DNA (T-DNA) has a left (L) and a right (R) border sequence, and all DNA in between these two borders is transferred to the plant genomes (Figure 4).
The Ti plasmid has been engineered to enhance its usefulness for plant transformation. For instance, genes responsible for altered plant hormone production (oncogenes) and other genes (such as those involved in opine synthesis) that are unnecessary for DNA transfer have been eliminated, thereby preventing aberrant plant growth and metabolism. A binary T-DNA plasmid vector can be constructed to contain the left and right T-DNA borders and restriction endonuclease sites between the borders to enable foreign DNA (any transgene you want to transfer into the plant genomes) to be cloned into the T-DNA region. A plant selectable marker (often an antibiotic resistance gene or herbicide tolerance gene) is also placed in the T-DNA. The right border initially enters the plant cells, and the subsequent selection of T-DNA molecules using a selectable marker adjacent to the right border results in partial integration of the T-DNA molecules. Placing the selectable marker next to the left border can enhance the complete integration of T-DNA and the expression of transgenes (Bhattacharyya et al., 1994).
The binary plasmid also has an origin of replication that enables its replication in both Escherichia coli and Agrobacterium. This principle enables the cloning of foreign genes into the T-DNA molecule in E. coli using a binary plasmid, which is significantly smaller than the Ti plasmid and, therefore, easier to manipulate. The binary vector does not contain the virulence genes necessary for infection and transfer of T-DNA. The binary plasmid is introduced into an Agrobacterium strain that carries a Ti plasmid, lacking tumor-inducing genes as well as the left and right border sequences. It is called a disarmed Ti plasmid, but it contains vir genes, which allow the Agrobacterium cells to infect plant cells and transfer T-DNA from the binary plasmid. An assembly of the DNA elements used for transformation is called a construct (see Plant Breeding Methods). A construct for T-DNA transformation must include the T-DNA border sequences, a marker for selection, such as a gene for herbicide resistance, and one or more genes intended for transfer into the recipient genome. Agrobacterium-mediated transformation offers high efficiency and throughput, reliably generating single-copy DNA insertion events.
As stated earlier in the chapter, only a fraction of the cells competent for regeneration are transformed. The use of selectable markers enables the selection of rare, transformed cells. Therefore, after Agrobacterium infection, the recipient cells carrying the inserted T-DNA continue to grow, whereas the antibiotics or herbicides used in selection kill non-transformed cells.
Use of A. Rhizogenes in Plant Transformation
A. rhizogenes is another species that also induces abnormal plant growth, in this case, adventitious roots on shoots of dicotyledonous plants. Like A. tumefaciens, A. rhizogenes infects plants through wound sites, followed by the transfer, integration, and expression of T-DNA from the root-inducing (Ri) plasmid. The expression of T-DNA from the Ri plasmid results in the formation of fine fibrous structures referred to as hairy roots. Hairy roots are formed due to changes in the cells’ physiology, rendering them more sensitive to auxins. Hairy roots are useful for producing high-value metabolites in medicinal plants and for phytoremediation studies (Suza et al., 2008).
A. rhizogenes is particularly useful for transforming legume species that are difficult to transform by the A. tumefaciens method (Collier et al., 2005). The transgenic roots enable the study of the function of genes required for root-specific processes (Kereszt et al., 2007). In A. rhizogenes-mediated transformation, the T-DNA from the binary vector is co-transferred with the T-DNA from the Ri plasmid into the host genome. Not all hairy roots may contain the transgene, so the hairy root phenotype is often used to select transformants in the absence of a selectable marker. However, molecular confirmation of transgene presence in individual hairy roots is typically done using PCR.
Read: Ex vitro composite plants: an inexpensive, rapid method for root biology Agrobacterium rhizogenes-mediated transformation of soybean to study root biology
Particle Bombardment or Biolistic Transformation
Particle bombardment, also known as biolistics, is a versatile and powerful method for plant transformation that often overcomes the limitations associated with Agrobacterium-mediated approaches. Unlike Agrobacterium, biolistics methods have no biological host constraints, allowing the transformation of a wide range of plant species and tissue types, including those recalcitrant to Agrobacterium infection. Biolistic transformation utilizes high-velocity gold or tungsten particles to deliver DNA molecules into living cells, resulting in stable transformation. The method can be applied to protoplasts, intact cells from a cell suspension culture, or tissues or organs, and is the most effective approach for organelle transformation, such as in chloroplasts and mitochondria. The method also supports the delivery of non-DNA-based Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) reagents, for example, RNA and ribonucleoproteins (RNPs). These reduce off-target effects and often lead to the production of transgene-free edited plants (Chapter 8), which is essential and valuable in precision plant breeding and regulatory approval (Thorpe et al., 2025).
How biolistics particle delivery works:
Microparticles of tungsten or gold coated with DNA are accelerated toward the target tissues. The particles penetrate the cell wall and carry DNA into the cells and even into the nuclei. The original biolistic instruments used gunpowder explosions to accelerate DNA-coated particles into target cells. Other instruments discharge sudden gas pressure to accelerate the particles (Figure 5).
The PDS-1000/He system (Figure 5) accelerates nucleic acid-coated gold or tungsten microparticles (0.6–1.6 μm) to velocities necessary to penetrate cells, tissues, or organelles (Figure 6). The system utilizes a burst of high-pressure helium gas to accelerate a plastic macrocarrier disk, which carries microparticles, toward the target cells. The helium pressure used to propel the macrocarrier is determined by the choice of rupture disk, a plastic seal designed to burst at a specific pressure. A stopping screen retains the macrocarrier while allowing the microparticles to pass through and penetrate the target cells. To increase the efficiency of the process, the chamber may be evacuated to subatmospheric pressures, reducing the frictional drag on the microparticles as they travel toward the target cells.
However, the biolistic transformation process has some associated challenges, like tissue damage, lower efficiency, and the risk of multiple or fragmented gene insertions. Despite these challenges, the method remains an indispensable tool for plant transformation, especially for species or applications where other transformation methods are inadequate.
A new biolistic enhancement that utilizes a flow guiding barrel (FGB) addresses the limitations associated with gas and particle flow. FGB significantly improves stable transformation in maize by over 10-fold and boosts transient expression and genome editing across crop species, addressing key delivery barriers. This method enables the delivery of DNA, RNA, and proteins, regardless of tissue type, genotype, or crop. This breakthrough makes biolistic genome editing more reliable and broadly applicable across plant species.
Requirements for a successful plant transformation
For the process of plant transformation to be successful, several essential components must be in place, including the facilities (tissue culture facility, greenhouse, and molecular laboratory), transformation protocols and systems, gene selection, and construct designs. Regeneration systems and Molecular Analysis of transgenics., divided into three major units, is required for a
Laboratory Facilities and Infrastructure
A specialized infrastructure divided into three major units is required for successful plant transformation. These include a tissue culture facility, a molecular laboratory, and a greenhouse.
Plant tissue culture (PTC) facility
Plant transformation relies on tissue culture for regenerating whole plants from explants. Typically, a standard PTC facility should have the following equipment: tissue culture incubators, laminar airflow hoods, multiple temperature-controlled shaking incubators, a –80°C freezer, multiple dissecting microscopes, a fluorescence microscope, a Bio-Rad PDS 1000/HE biolistic particle delivery system, a Bio-Rad Gene Pulser, and other necessary equipment for tissue culture. Additionally, the PTC facility should be equipped with autoclaves for sterilizing media, waste, and tools, a media preparation area featuring pH meters and a weighing balance, as well as a supply of lab consumables. This facility enables explant sterilization and establishes a transformation system under sterile conditions.
Molecular Biology Laboratory
This facility is important for DNA extraction, cloning, plasmid construction, PCR validation, gel electrophoresis, and bacterial transformation, among other applications. This facility should also have sterile conditions to prevent contamination during bacterial transformation.
Greenhouse Facility
After regenerating healthy and robust-looking plantlets, these can be transferred to greenhouse conditions under biosafety regulations. For proper acclimatization, temperature, humidity, and light must be regulated.
Crop Bioengineering Laboratory (CBL), Iowa State University
The CBL offers plant tissue culture and transformation services for maize and soybeans, utilizing a 1,352 ft2 lab space and a 4,100 ft2 greenhouse. Key equipment includes tissue culture incubators, laminar flow hoods, microscopes, and biolistic and Agrobacterium transformation systems.
Services:
Maize: Agrobacterium-mediated transformation of B104 immature embryos.
Soybean: Agrobacterium-mediated transformation of Williams 82 half-seeds.
DNA Construct Validation & Cloning: Transform plasmids into Agrobacterium, verify constructs, and assemble vectors for overexpression or CRISPR editing. For more information, read about the CBL and the services it offers.
Gene selection and construct design
For successful plant transformation, a well-designed gene construct is fundamental. Construct design involves careful selection of the gene of interest, regulatory elements, selectable markers, and vector backbone to maximize transgene expression and stability.
Gene of interest (GOI)
The GOI design should be well-characterized and associated with a defined target trait, such as pest resistance, disease resistance, drought tolerance, or improved nutritional quality, among others. Efficient expression requires codon optimization to match host plant usage, especially when introducing bacterial or other foreign genes into plants.
Expression Cassette Components
The T-DNA regions typically include promoters, introns, untranslated regions, the GOI, and terminators (These are discussed in Chapter 1). Promoters can be constitutive to drive expression in all tissues or tissue-specific for targeted expressions. Inclusion of introns can enhance mRNA processing and increase transgene expression, while terminators ensure proper transcription termination.
Selectable markers and reporter genes
Selection markers identify transformed cells during plant tissue culture, while reporter genes provide visual confirmation of transgene expression and protein localization (Table 1).
| Category | Gene | Function / Use | Detection / Selection method | Reference |
|---|---|---|---|---|
| Selectable Marker Genes | nptII | Confers resistance to Neomycin, Kanamycin, Paramomycin, Ribostamycin, Butirosin, and Geneticin (G418) | Transformed cells survive on kanamycin-containing medium | (Bevan et al., 1983) |
| hpt (hptII) | Confers resistance to hygromycin | Survives on hygromycin-containing medium | (Waldron et al., 1985) | |
| bar/pat | Confers resistance to phosphinothricin (glufosinate herbicide) | Culturing on media with glufosinate | (Thompson et al., 1987) | |
| aadA | Provides resistance to streptomycin/spectinomycin | Growth on antibiotic medium | (Svab & Maliga, 1993) | |
| Reporter Genes | GUS (uidA) | Produces β-glucuronidase enzyme | Blue staining with X-Gluc substrate | (Jefferson et al., 1987) |
| GFP (Green Fluorescent Protein) | Emits green fluorescence; real-time visualization | Fluorescence microscopy | (Chalfie et al., 1994) | |
| RFP / mCherry | Red fluorescent protein for protein localization | Fluorescence microscopy (red channel) | (Shaner et al., 2004) | |
| Luciferase (luc) | Produces bioluminescence in the presence of luciferin | Light emission measured with a luminometer | (Ow et al., 1986) | |
| LacZ | β-galactosidase enzyme; blue/white screening | X-gal staining (less common in plants) | (Casadaban & Cohen, 1980) | |
| RUBY | Produces red betalain pigment, visible without a microscope | Visibly red tissues in transformed cells | (He et al., 2020) |
Selection of Transformed Cells
Because only a few cells that can be regenerated will be transformed, one must have a method to eliminate the non-transformed cells to identify the transformed ones. A variety of antibiotics have been used to kill cells that are not transformed, such as kanamycin, spectinomycin, and hygromycin. Genes conferring resistance to the appropriate antibiotics are included in the T-DNA for A. tumefaciens-mediated transformation, and the gene of interest is in a plasmid for biolistic transformation. Other common selectable markers include herbicide resistance genes.
As mentioned earlier, certain plant species respond to wounding by producing dedifferentiated cells around the wound site, and these can be induced to regenerate under suitable conditions. Dedifferentiated cells at the periphery lead to the development of multiple transformants when the leaf discs are placed on a plate containing the appropriate antibiotics to select transformed cells. The leaf disk transformation-regeneration process is illustrated in Figure 7.
There are also transformation protocols that do not require tissue cultures. For example, the floral dip method is extensively applied in Arabidopsis, where flowers are immersed in a solution containing Agrobacterium that carries the Ti and binary plasmid constructs.
Early meristematic tissue of the embryo is transformed. The transformed seeds can be easily screened by growing them on media containing the appropriate selective compounds. This procedure is composed of the following steps:
- Select a parent plant at flowering.
- Dip flowers in a solution of Agrobacterium containing a binary plasmid.
- Grow infected plants to maturity and harvest seeds.
- Plant seeds on selective media, usually kanamycin. All seeds germinate, but only those that are transformed yield seedlings that grow vigorously. These are then transferred to the soil and grown to maturity for seed harvest. The collected seed (T2) is analogous to an F 2 segregating 1:2:1 for the transgene, assuming a single transgene in the original T1. Note that the T1 is hemizygous for the transgene, not heterozygous. Heterozygosity is when there are two different alleles at a single locus. The T1 is hemizygous because one chromosome carries the transgene, and the other homologous chromosome carries nothing (i.e., no transgene).
Advances in Plant Transformation
Plant transformation using ternary vector systems and auxotrophic Agrobacterium tumefaciens
Recent advances in plant transformation, especially using ternary vector systems and auxotrophic Agrobacterium tumefaciens strains, have enhanced transformation efficiency and biosafety in crop genetic engineering. The ternary vector system consists of four components: (1) an Agrobacterium genome, which includes C1, C2 chromosomes, plus pAT plasmid, (2) disarmed Ti plasmid, which lacks tumor-inducing genes, (3) a T-DNA binary vector carrying the gene of interest, and (4) an additional virulence (vir) gene helper plasmid, which boosts expression of virulence genes crucial for T-DNA transfer into plant cells (Aliu et al., 2024; Lee et al., 2023). This ternary vector system significantly enhances transformation efficiencies in recalcitrant crops, such as maize. The Wang Lab at Iowa State University has developed helper plasmids, pKL2299 (Addgene # 186332) and its enhanced version, pKL2299A (Addgene # 222105) (Figure 8), with an additional virA to boost Agrobacterium-mediated plant transformation by providing additional vir genes essential for T-DNA transfer (Aliu et al., 2024; Lee et al., 2023).
Auxotrophic A. tumefaciens strains are genetically modified bacteria that cannot synthesize essential nutrients such as thymidine, methionine, and cysteine, and thus depend on external supplementation of such nutrients for survival (Aliu et al., 2024; Prías-Blanco et al., 2022). Unlike traditional strains that may persist and damage plant tissues despite antibiotic treatments, auxotrophic strains can be easily removed by simply withdrawing the required nutrient, reducing the need for antibiotics and minimizing phytotoxicity. This makes these strains valuable in plant transformation, as their nutrient dependence helps control bacterial overgrowth during cocultivation, a significant challenge in plant tissue culture. For example, a common Agrobacterium strain, LBA44404, a thymidine auxotrophic mutant, engineered by deleting a thymidylate synthase gene thyA, requires external thymidine for growth, aiding the control of the bacterial overgrowth during plant transformation (Aliu et al., 2024). When combined, ternary vector systems and auxotrophic Agrobacterium strains offer a robust and reliable platform for enhancing and advancing plant genetic transformation and gene editing.
The concept of combined ternary vector systems and auxotrophic Agrobacterium strains has been applied to modify the quick corn protocol, shortening the B104 transformation timeline from 160 to approximately 50 days. The traditional B104 transformation protocols were lengthy and had low transformation frequencies. Therefore, the new protocol developed by Kang et al. (2022) shortened the regeneration time for B104 and increased the average transformation frequency to over 20%. This rapid and efficient protocol is suitable for routine genome editing using B104, without the use of morphogenic transcription factors Bbm and Wus2, as is the case for the Quick Corn method developed by Masters et al. (2020). Building on this background and research, in 2022, Iowa State University established the Crop Bioengineering Laboratory (CBL) to meet the growing demand for plant transformation services in the research community. Using the Kang et al. (2022) Protocol, combined with ternary vector and plant selection systems, CBL achieves B104 maize transformation in approximately two months (Figure 9).
Morphogene-Assisted Transformation (MAT): A genotype-flexible breakthrough in Plant Biotechnology
A new advancement in plant biotechnology that uses morphogenic transcription factors (MTFs) has greatly improved the efficiency and flexibility of genetic transformation. These morphogenic genes, which are key regulators of plant development, enhance both regeneration and the transformability of a wider range of genotypes, including those previously considered recalcitrant. The ectopic expression of MTFs induces somatic embryogenesis and shoot formation directly from explant tissues. This approach effectively overcomes genotype dependence, a long-standing challenge in monocot transformation.
The MTF breakthrough was spearheaded by scientists from Corteva Agriscience, and in 2016, they showed that co-expression of maize BABY BOOM (Bbm) and WUSCHEL2 (Wus2) genes produced high transformation frequencies in maize inbred lines that were previously non-transformable using traditional biolistic and Agrobacterium transformation protocols (Lowe et al., 2016). Ectopic expression of Bbm and Wus2 increased transformation frequencies in maize, sorghum, rice, among other crops (Lowe et al., 2016). Bbm, one of the AP2/ERF family of transcriptional factors, is involved in plant growth and development processes, particularly in embryogenesis and cell regeneration , while Wuschel encodes a homeodomain transcription factor that regulates stem cell fate in plants, particularly in the shoot apical meristem.
The MAT technology was expanded in 2023 by Corteva Agriscience, and this time, the team used seedling leaf whorls as a new explant for monocot transformation (Wang et al., 2023). Given the difficulty in isolating immature embryos and seed from some grasses like teff and small millets, the use of these seedling leaf whorls as alternative explants for Agrobacterium-mediated transformation employing Bbm and Wus2 has expanded the list of recalcitrant monocot crop species (Fig. 6), which are now transformable using this leaf transformation method, including maize, sorghum, rice, switch grass, pearl millet, teff, foxtail millet, barley, wheat, and rye (Wang et al., 2023). This leaf transformation method utilizes seedling leaf whorls as alternative explants, and MTFs provide a faster, more accessible, and cost-effective process, which eliminates the need to maintain a state-of-the-art greenhouse required for providing immature embryos (Azanu et al., 2025).
Traditional transformation of grasses like maize relies on immature embryos, which limits efficiency and depends on greenhouse-grown ears that are both costly and seasonally constrained. A breakthrough approach now uses seedling leaf-whorl explants together with the morphogenic genes WUSCHEL2 (Wus2) and BABY BOOM (Bbm) in an Agrobacterium-mediated vector system. This innovation significantly improves transformation and genome editing efficiency, providing a practical alternative to immature embryo–based methods. For more information, read: Leaf transformation for efficient random integration and targeted genome modification in maize and sorghum.
Regeneration of New Plants from Tissue Culture
The tissue selected for transformation is usually chosen for its strong regenerative capacity. The strategies and tissue culture techniques used to regenerate plants vary widely among species and depend greatly on the type of tissue used. In some cases, the process is as much an art as it is a science. Factors influencing regeneration include the composition of the culture medium, particularly the types and concentrations of hormones, as well as light, temperature, and incubation time. Auxins promote root formation, while cytokinins stimulate shoot development. Regenerated transgenic plants may require six months or longer to mature in soil, depending on the species. Moreover, reproducing a successful regeneration protocol in another laboratory can be challenging because subtle and often unrecognized factors affect the outcome.
Verification of the Success and Stability of Transformation
It is important to screen progeny from regenerated plants to verify that the transgene is integrated into the genome and genetically transmitted. The following methods are commonly used for transgene analysis:
Detection of DNA
Southern hybridization and quantitative PCR (see Chapter 6 on Polymerase Chain Reaction) can be used to detect the presence and determine the copy number of the transgene.
Detection of mRNA
Detection of the transgene is necessary but insufficient to guarantee the expression of the transgene. The integration of transgenes at specific regions of the genome suppresses their expression. For many transformations, there is a broad variation in the level of expression among independent transformants. Independent transformants are individuals transformed with the same construct, but they arose from independent transformation events. Thus, they are not clonal products of a single transformed cell; therefore, the T-DNA is likely to have integrated into different regions in the genome in independent transformants. Detection of mRNA is considered to determine the extent of transgene expression levels. Northern blot hybridization, RT-PCR, and qRT-PCR are commonly used methods for detecting mRNA.
Detection of Protein
For example, the mRNA product may be present but not translated efficiently if a mutation occurs during the cloning of the gene into a binary vector. Therefore, detecting the protein product of the gene is a crucial step in validating the success of transformation. A common method used to detect specific proteins is Western blotting. Western blotting utilizes gel electrophoresis to separate proteins based on their size or structure, followed by the transfer of these proteins onto a membrane, where they are detected using antibodies specific to the protein of interest.
Phenotype of Biochemistry
Depending on the function of the transgene, one might expect an altered phenotype. However, an altered phenotype may result from mutations in other genes during tissue culture. The process of T-DNA integration is random, and the transgene may insert within a functional gene, rendering it nonfunctional. A study of several independent transformants and/or co-segregation of transgenes with phenotypes ruled out such artifacts. Biochemical analysis, such as enzyme activity or metabolite detection, may also be used to test the expression of transgenes in transgenic plants.
Transformation application in different main crops
Plant transformation is preferred because it enables precise, efficient, and targeted improvement of crops, surpassing the capabilities of traditional breeding. Plant transformation technologies have been widely applied to enhance economically important traits across major crops (Table 2)
| Crop | Transformed Trait(s) | Application / Purpose | Reference |
|---|---|---|---|
| Maize | Bt insect resistance | Protect against insect pests | (Abbas, 2018) |
| Herbicide tolerance | Roundup resistance | (Brookes, 2022) | |
| Drought tolerance | Survive water stress | (Edge et al., 2017) | |
| Rice | Provitamin A (Golden Rice) | Nutritional enhancement | (Potrykus, 2001) |
| Bacterial blight resistance (Xa21) | Disease resistance | (Fiyaz et al., 2022) | |
| Drought tolerance | Climate adaptability | (Ravikumar et al., 2014) | |
| Wheat | Fusarium resistance | Disease reduction | (Mao et al., 2023) |
| Herbicide tolerance | Weed management | (Nakka et al., 2019) | |
| Salt/drought tolerance | Abiotic stress resilience | (Mao et al., 2023) | |
| Soybean | Herbicide tolerance (Roundup Ready) | Efficient weed management | (Lundry et al., 2008) |
| Insect resistance | Protect against insect pests | (Parrott et al., 1994) | |
| High oleic acid content | Improved oil quality | (Buhr et al., 2002) | |
| Cotton | Bt insect resistance | Reduce bollworm damage | (James, 2007) |
| Herbicide tolerance | Glyphosate resistance | (Kishore et al., 1992) | |
| Fiber quality improvement | Better textile quality | (Constable et al., 2015) | |
| Tomato | Delayed ripening | Shelf-life extension | (Gupta et al., 2013) |
| Virus resistance | Disease resistance | (Raj et al., 2005) | |
| Higher lycopene | Nutrition improvement | (Li et al., 2018) | |
| Potato | Late blight resistance | Reduce post-harvest losses | (Bubolz et al., 2022) |
| Vitamin B6 | Nutritional enhancement | (Bagri et al., 2018) | |
| Cold storage tolerance | Extend storage life | (Chen et al., 2008) | |
| Cassava | Virus resistance (CMD, CBSD) | Disease control | (Lin et al., 2019) |
| Biofortification (vitamin A) | Nutrition improvement | (Beyene et al., 2018) | |
| Fusarium wilt resistance | Protect yield | (Harding et al., 2025) | |
| Banana | Bacterial wilt resistance | Disease protection | (Tripathi et al., 2017) |
| Provitamin A biofortification | Nutrition improvement | (Paul et al., 2017) | |
| Striga resistance | Improve yield in Striga-infested areas | (Kaniganti et al., 2025) | |
| Sorghum | Drought tolerance | Adaptation to dry conditions | (Ferguson et al., 2024) |
| Cold tolerance | Enhanced cold stress tolerance | (Gierczik et al., 2019) | |
| Salt tolerance | Stress resilience | (Mian et al., 2011) | |
| Barley | Drought tolerance | Adaptation to dry conditions | (Feng et al., 2020) |
Examples of accomplishments in plant transformation
Table 3 highlights key achievements in plant transformation that have significantly influenced agriculture, food security, and biotechnology.
| Bioengineered Crop | Purpose |
|---|---|
| Alfalfa | Engineered mainly for herbicide tolerance (e.g., glyphosate-resistant). |
| Apple (ArcticTM varieties) | Engineered to reduce enzymatic browning by silencing PPO genes. |
| Canola | Modified for herbicide tolerance and high oleic acid profiles. |
| Corn (Maize) | Includes Bt insect-resistant and herbicide-tolerant varieties. |
| Cotton | Modified for insect resistance (Bt) and herbicide tolerance. Used for cottonseed oil. |
| Eggplant (BARI Bt Begun) | Bt eggplant grown mainly in Bangladesh to resist fruit and shoot borer pests. |
| Papaya | Engineered for resistance to Papaya Ringspot Virus (PRSV); saved Hawaii’s papaya industry. |
| Pineapple (Pink-flesh) | Engineered by inserting a tangerine gene to increase lycopene → pink flesh. |
| Potato | Non-browning, reduced bruising, low acrylamide, late blight-resistant (Innate® Potato). |
| Soybean | Herbicide tolerance (e.g., Roundup Ready); high oleic, disease-resistant varieties. |
| Squash (Summer squash) | Engineered with viral coat protein genes for resistance to CMV, WMV, and ZYMV. |
| Sugarbeet | Modified mainly for herbicide tolerance. |
| Sugarcane (Bt) | Engineered for insect resistance (mainly in Brazil). |
Concerns about GMOs
Genetically modified (GM) crops offer numerous benefits that can enhance agricultural productivity and food security. They can provide improved nutrition, increased resistance to pests and diseases, higher yields, and greater tolerance to environmental stresses such as drought and salinity. Advances in gene editing now allow for precise modifications, including the development of transgene-free plants, which can improve public acceptance and facilitate regulatory compliance.
Although concerns have been raised regarding potential health and environmental impacts, evidence indicates that, when properly regulated and responsibly managed, these risks are minimal compared to the benefits of GM crops. A major advancement addressing GMO concerns is the use of gene editing technologies, such as CRISPR/Cas9, which enable precise modifications of plant genomes without introducing foreign DNA. These transgene-free edited crops mimic natural mutations and are increasingly exempt from strict GMO regulations in many countries. As a result, they are more publicly acceptable while still offering advantages such as disease resistance, enhanced nutrition, and improved climate resilience.
Overall, while traditional GMOs sparked ethical and ecological debates, modern gene editing represents a more precise, safer, and publicly acceptable alternative. A detailed exploration of gene editing technologies, their regulatory status, and applications is presented in Chapter 8.
Chapter Summary
Plant transformation enables the introduction of new genes for desirable traits into existing crop varieties. With plant transformation, sexual compatibility becomes irrelevant, and the process of developing a new variety becomes faster because transformed cells expressing genes can be selected directly. Successful transformation requires a well-coordinated transformation system, which integrates three key components: a suitable explant (tissue that regenerates), an efficient DNA delivery method, and a reliable selection system to identify transformed cells. In monocots, embryonic tissues such as immature embryos, calli, or seedling leaf whorls are commonly used, while in dicots, leaf discs, cotyledon sections, and embryonic calli are preferred. Several methods are available to introduce DNA into the host. These methods include direct DNA uptake, microinjection, Agrobacterium-mediated gene transfer, and particle bombardment, also known as biolistic transformation. In most cases, only a few cells that can be regenerated are transformed, requiring the elimination of non-transformed cells using selectable markers to identify those that have undergone transformation. It is also important to screen progenies of regenerated plants to verify that the transgene is integrated into the genome and is genetically transmissible. The use of morphogenic regulators, such as BABY BOOM (Bbm) and WUSCHEL2 (Wus2), enhances cell competence and regeneration, thereby broadening the range of genotypes amenable to transformation. Additionally, optimizing culture conditions, hormone conditions, and wound responses is often necessary to maximize transformation efficiency.
