Agrobacterium-Mediated Gene Transfer (Transformation) in Plants

Plant genetic transformation is a process of introducing foreign genes into plant cells to produce transgenic plants with desired traits. There are various methods for plant transformation, such as biolistics, electroporation, microinjection, and Agrobacterium-mediated transformation. Among these methods, Agrobacterium-mediated transformation is the most widely used and efficient technique for many plant species .

Agrobacterium is a soil-borne bacterium that causes crown gall or hairy root disease in many dicotyledonous plants. It has the ability to transfer a part of its plasmid DNA, called T-DNA (transferred DNA), into the plant genome during infection . The T-DNA carries genes that affect the plant`s hormonal balance and produce opines, which are nutrients for the bacterium. The T-DNA also integrates into the plant genome randomly and stably, allowing the expression of its genes in the plant cells .

Scientists have exploited this natural gene transfer system of Agrobacterium to create transgenic plants. By replacing the T-DNA with a gene of interest and a selectable marker gene, Agrobacterium can be used as a vector to deliver and integrate foreign genes into the plant genome . The gene of interest can be any gene that confers a desirable trait to the plant, such as resistance to pests, diseases, herbicides, or environmental stresses. The selectable marker gene can be a gene that confers resistance to antibiotics or herbicides, which can be used to select the transformed cells from the non-transformed ones .

The advantages of using Agrobacterium as a plant transformation tool are:

• It can transfer large DNA fragments (up to 30 kb) into the plant genome .
• It can integrate the foreign DNA into the plant genome with high efficiency and stability .
• It can generate transgenic plants with single or low copy number of the foreign gene, which reduces the risk of gene silencing and instability ^.
• It can transform a wide range of plant species, including dicots and monocots ^.
• It is relatively simple and inexpensive compared to other methods^.

Agrobacterium-mediated transformation has been used for various applications in plant biotechnology, such as producing recombinant proteins, antibodies, vaccines, pharmaceuticals, bioplastics, biofuels, and bioremediation agents in plants^ ^ . It has also been used to improve crop yield, quality, shelf-life, and biosynthesis by modifying plant genes involved in growth, development, metabolism, and stress responses^ ^.

Agrobacterium-mediated transformation is a powerful and versatile tool for plant genetic engineering. However, it also has some limitations and challenges, such as:

• It has a narrow host range and cannot infect some important crops like wheat and rice^ ^.
• It requires tissue culture and regeneration protocols for each plant species and genotype^ ^.
• It depends on various factors that affect the efficiency and specificity of T-DNA transfer and integration^ ^.
• It may cause unwanted mutations or rearrangements in the plant genome due to random integration of T-DNA^ ^.

These limitations and challenges can be overcome by optimizing the conditions and parameters of Agrobacterium-mediated transformation, such as selecting suitable Agrobacterium strains and plasmids, modifying T-DNA borders and sequences, choosing appropriate explants and wounding methods, adjusting cocultivation time and temperature, adding virulence inducers and inhibitors, using binary or co-integrate vectors, applying positive or negative selection schemes, screening for single-copy transformants, and verifying T-DNA integration sites^ ^.

In conclusion, Agrobacterium-mediated transformation is a popular plant transformation tool that utilizes the natural gene transfer mechanism of Agrobacterium to introduce foreign genes into plant cells. It has many advantages over other methods in terms of efficiency, stability, simplicity, and versatility. It has been widely used for various applications in plant biotechnology to produce transgenic plants with desired traits. However, it also has some limitations and challenges that require further improvement and innovation.

Agrobacterium-mediated gene transfer is a widely used technique for introducing foreign genes into plant cells and tissues. However, the efficiency and success of this technique depend on various factors that influence the interaction between the bacteria and the plant cells. Some of these factors are:

• Explant type and condition: The explant is the plant material that serves as the target for Agrobacterium infection. Different types of explants, such as leaves, stems, cotyledons, embryos, or callus, have different susceptibility and competence for Agrobacterium-mediated gene transfer. The explant type also determines the regeneration potential and the recovery of whole transgenic plants. The condition of the explant, such as its age, size, moisture content, and physiological state, also affects the transformation efficiency.

• Explant wounding: Wounding of the explant is required to facilitate the entry of Agrobacterium into the plant cells and to induce the expression of virulence genes in the bacteria. The degree and method of wounding can vary depending on the explant type and the Agrobacterium strain. Some common methods of wounding include cutting, piercing, scraping, sonication, or particle bombardment. Wounding also triggers the release of phenolic compounds from the plant cells, which act as inducers of vir genes in Agrobacterium.

• Plant species and genotype: Agrobacterium-mediated gene transfer is more efficient in dicotyledonous plants than in monocotyledonous plants, due to the differences in cell wall composition and structure. However, some monocot species have been successfully transformed by using specific Agrobacterium strains or by modifying the culture conditions. Within the same plant species, different cultivars or ecotypes may also show different levels of susceptibility or resistance to Agrobacterium infection, due to genetic variations.

• Antibiotics: Antibiotics are used to eliminate or suppress the growth of Agrobacterium after the co-cultivation period, to prevent overgrowth of bacteria and interference with plant growth and development. The choice and concentration of antibiotics depend on the Agrobacterium strain and the plant species. Some commonly used antibiotics are carbenicillin, cefotaxime, rifampicin, or timentin.

• Plant growth regulators: Plant growth regulators (PGRs) are added to the culture medium to enhance the growth and differentiation of plant cells and tissues. The type and concentration of PGRs depend on the explant type and the plant species. Some commonly used PGRs are auxins (such as 2,4-D or NAA), cytokinins (such as BAP or zeatin), or abscisic acid (ABA).

• Light: Light affects various physiological processes in plants, such as photosynthesis, hormone levels, cell cycle progression, and stress responses. Light also influences the production and release of phenolic compounds from plant cells, which affect the vir gene induction in Agrobacterium. Different light regimes, such as light intensity, duration, quality, or photoperiod, may have different effects on Agrobacterium-mediated gene transfer.

• Temperature: Temperature affects both the bacterial and plant cell metabolism and activity. Temperature also affects the stability and expression of vir genes in Agrobacterium. The optimal temperature for Agrobacterium-mediated gene transfer varies depending on the plant species and the Agrobacterium strain. Generally, a temperature range of 19°C to 22°C is considered ideal for most plant species.

• Agrobacterium strains: Different strains of Agrobacterium have different abilities to infect and transfer genes into different plant species. The choice of Agrobacterium strain depends on several factors, such as the host range, plasmid compatibility, virulence level, T-DNA size and structure, marker genes, and selectable traits. Some commonly used Agrobacterium strains are LBA4404, EHA105, GV3101, or AGL1.

These factors need to be optimized for each plant species and each transformation experiment to achieve high efficiency and reproducibility of Agrobacterium-mediated gene transfer.

The principle of Agrobacterium-mediated gene transfer is based on the natural ability of the bacterium to transfer a segment of its DNA, called the T-DNA, into the genome of the host plant cell. The T-DNA is part of a large plasmid called the Ti (tumor-inducing) plasmid or the Ri (root-inducing) plasmid, depending on the type of Agrobacterium strain. The Ti/Ri plasmid also contains a region called the virulence (vir) region, which encodes several proteins that are essential for the T-DNA transfer process .

The mechanism of Agrobacterium-mediated gene transfer can be summarized as follows :

• The bacterium attaches to the wounded plant cell and senses the presence of phenolic compounds, such as acetosyringone, that are released by the injured plant tissue. These compounds activate a protein called VirA, which in turn activates another protein called VirG by phosphorylation. VirG then binds to the promoters of other vir genes and induces their expression.
• One of the vir genes, virD, encodes an endonuclease that nicks the T-DNA at its border sequences, generating a single-stranded copy of the T-DNA. This copy is covalently attached to a protein called VirD2 at its 5` end. Another vir gene, virE, encodes a protein that binds to the T-DNA and protects it from degradation. The T-DNA-VirD2-VirE complex is called the T-complex.
• The T-complex is then transferred across the bacterial and plant cell membranes by a type IV secretion system, which is composed of several VirB proteins. The type IV secretion system forms a channel-like structure that connects the two cells and allows the passage of the T-complex.
• Once inside the plant cell, the T-complex is targeted to the nucleus by nuclear localization signals present on VirD2 and VirE proteins. The T-DNA is then integrated into the plant genome by a process of illegitimate recombination, which is mediated by plant enzymes. The integration site is usually random and can occur in transcriptionally active or repetitive regions of the genome.
• The integrated T-DNA can then express its genes in the plant cell, resulting in various phenotypic changes depending on the nature of the genes. For example, some T-DNA genes can cause tumor formation or root proliferation by altering the hormonal balance in the plant cell. Other T-DNA genes can encode enzymes that synthesize opines, which are compounds that can be utilized by Agrobacterium as a source of carbon and nitrogen.

By modifying the Ti/Ri plasmid and replacing some or all of the native T-DNA genes with foreign genes of interest, Agrobacterium can be used as a vector to create transgenic plants with desired traits . The foreign genes can be driven by plant-specific promoters and terminators to ensure their proper expression in the plant cell. Additionally, selectable marker genes can be included in the T-DNA to facilitate the identification and selection of transformed plant cells.

To perform Agrobacterium-mediated gene transfer, you will need the following materials and reagents:

Materials/ Equipment

• Sterile 50 ml plastic tubes
• Autoclave
• Controlled Tissue Culture Rooms at 25°C with 16/8 hr light/dark period
• Shaker Incubator
• Vacuum pump
• Laminar hood for tissue culture
• Glassware (Beakers, cylinders, Petri dishes, Duran bottles, and Flasks)
• Filter paper
• Parafilm
• Forceps and Scalpel
• Pipettes
• Centrifuge
• Spectrophotometer
• Tissue culture vessels
• Surgical blades

Reagents

• Explant (Stems, embryo, cotyledons, or other tissues)
• Agrobacterium strain
• 13% Sodium hypochlorite
• B5 Medium
• Agar
• Tryptone
• Yeast Extract
• Sodium Chloride
• 35% Hydrochloric acid
• Sterile distilled water
• 75% Ethanol
• Sucrose
• Abscisic Acid
• Rifampicin
• Kanamycin monosulfate
• Gellan gun powder
• PCR primer star Mix
• Carbenicillin disodium salt

Media preparation is an important step in Agrobacterium-mediated gene transfer, as different media are required for different stages of the process. The media should be prepared with sterile distilled water and autoclaved before use. The following are some of the common media used for Agrobacterium-mediated gene transfer:

• LB medium for Agrobacterium culture: This is a nutrient-rich medium that supports the growth of Agrobacterium strains. To prepare 1 liter of LB medium, dissolve 5 grams of yeast extract, 10 grams of tryptone, and 5 grams of sodium chloride in distilled water. Adjust the pH to 7.0 with hydrochloric acid or sodium hydroxide. To select for Agrobacterium strains carrying the desired plasmid, antibiotics such as rifampicin and kanamycin can be added to the LB medium at appropriate concentrations .

• Murashige and Skoog (MS) medium for seed germination: This is a basal medium that contains essential salts, vitamins, and organic compounds for plant growth. To prepare 1 liter of MS medium, dissolve 4.43 grams of MS basal medium powder and 3 grams of sucrose in distilled water. Add 2.5 grams of gellan gum powder as a solidifying agent and autoclave. Pour 25 ml of the medium into sterile Petri plates under laminar flow .

• Cocultivation medium: This is a medium that facilitates the interaction between Agrobacterium and plant cells during cocultivation. To prepare cocultivation medium, add plant growth regulators such as benzylaminopurine (BAP) and abscisic acid (ABA) to the MS basal medium at suitable concentrations. Pour 25 ml of the medium onto sterile Petri plates with a layer of filter paper under laminar flow .

• Shooting medium: This is a medium that promotes the formation of shoots from the transformed plant cells. To prepare shooting medium, add antibiotics such as kanamycin and carbenicillin to the cocultivation medium at appropriate concentrations to eliminate Agrobacterium and select for transgenic cells .

• Rooting medium: This is a medium that induces the development of roots from the regenerated shoots. To prepare rooting medium, add antibiotics such as kanamycin and carbenicillin to the MS basal medium at suitable concentrations to maintain selection pressure .

The protocol or procedure for the Agrobacterium-mediated transformation might differ depending on the type of explants selected for the process. The following is a general protocol for Agrobacterium-mediated transformation using leaf discs as explants;

• Preparation of Agrobacterium culture: A single colony of Agrobacterium strain containing the desired plasmid vector is inoculated into 5 ml of LB medium supplemented with appropriate antibiotics and grown overnight at 28°C with shaking. The next day, 1 ml of the overnight culture is transferred to 50 ml of fresh LB medium with antibiotics and grown until the optical density at 600 nm reaches 0.6-0.8. The bacterial culture is then centrifuged at 4000 rpm for 15 minutes and resuspended in 10 ml of infiltration medium (MS liquid medium containing 100 µM acetosyringone and 5% sucrose). The bacterial suspension is incubated at room temperature for 2-3 hours before use.
• Preparation of leaf discs: Healthy and young leaves are collected from the plant species of interest and washed thoroughly with tap water. The leaves are then surface-sterilized by immersing them in 70% ethanol for 1 minute followed by 10% bleach solution for 10 minutes. The leaves are rinsed three times with sterile distilled water and blotted dry on sterile filter paper. Leaf discs of about 0.5-1 cm in diameter are cut from the leaves using a cork borer or a sterile scalpel and placed on MS solid medium containing appropriate plant growth regulators (PGRs) for pre-culture. The leaf discs are incubated in a growth chamber at 25°C with a 16/8 h light/dark cycle for 2-3 days.
• Inoculation of leaf discs: The pre-cultured leaf discs are transferred to sterile Petri dishes and immersed in the Agrobacterium suspension prepared earlier. The Petri dishes are placed under vacuum for 10-15 minutes to facilitate the infiltration of bacteria into the leaf tissues. The vacuum is then released slowly and the excess bacterial suspension is removed by blotting with sterile filter paper. The inoculated leaf discs are placed abaxial side up on MS solid medium containing PGRs and 100 µM acetosyringone for co-cultivation. The Petri dishes are sealed with Parafilm and incubated in the dark at 25°C for 2-3 days.
• Selection and regeneration of transgenic shoots: After co-cultivation, the leaf discs are rinsed briefly with sterile water containing 500 mg/l carbenicillin to remove the excess bacteria. The leaf discs are then transferred to MS solid medium containing PGRs, carbenicillin (500 mg/l), and a suitable selection agent (such as kanamycin or hygromycin) depending on the plasmid vector used. The Petri dishes are incubated in a growth chamber at 25°C with a 16/8 h light/dark cycle for 4-6 weeks. During this period, transgenic shoots will emerge from the leaf discs while non-transgenic tissues will be killed by the selection agent. The transgenic shoots are transferred to fresh MS medium containing PGRs, carbenicillin, and selection agent every two weeks until they reach a height of about 2-3 cm.
• Rooting and acclimatization of transgenic plants: The transgenic shoots are excised from the leaf discs and transferred to MS solid medium containing PGRs, carbenicillin, selection agent, and activated charcoal (0.5 g/l) to induce rooting. The Petri dishes are incubated in a growth chamber at 25°C with a 16/8 h light/dark cycle for 2-4 weeks until roots develop. The rooted transgenic plants are then carefully removed from the medium and washed gently with tap water to remove any traces of agar or charcoal. The plants are potted in soil or vermiculite and covered with transparent plastic bags to maintain high humidity. The plants are gradually acclimatized to ambient conditions by opening the bags slightly every day for a week and then removing them completely. The plants are watered regularly and grown in a greenhouse until they reach maturity.

Agrobacterium-mediated gene transfer is a powerful and widely used technique for genetic engineering of plants. It has many applications in various fields of plant biotechnology, such as:

• Production of recombinant proteins and vaccines: Agrobacterium-mediated transformation can be used to introduce genes encoding useful proteins, such as antibodies, enzymes, hormones, antigens, and vaccines, into plant cells. These proteins can be expressed in plant tissues or secreted into the culture medium for extraction and purification. For example, Agrobacterium-mediated transformation has been used to produce recombinant antibodies (plantibodies) and edible vaccines (plantigens) in plants .
• Crop improvement and trait engineering: Agrobacterium-mediated transformation can be used to modify the traits of crop plants for enhanced yield, quality, resistance, tolerance, or nutritional value. For instance, Agrobacterium-mediated transformation has been used to create insect-resistant crops by introducing genes encoding toxins such as Bt toxin. It has also been used to improve the shelf-life and biosynthesis of fruits and vegetables by modifying genes involved in ethylene production and ripening. Moreover, Agrobacterium-mediated transformation can be used to introduce genes for nutrient capture, nitrogen fixation, or stress tolerance into plants.
• Functional genomics and gene discovery: Agrobacterium-mediated transformation can be used to study the function and regulation of plant genes by creating transgenic plants with gene overexpression, knockdown, knockout, or reporter constructs. For example, Agrobacterium-mediated transformation has been used to generate transgenic Arabidopsis plants with various gene insertions or deletions for functional analysis. It has also been used to create transgenic plants with reporter genes such as GFP or GUS for visualization of gene expression patterns.
• Biomonitoring and bioremediation: Agrobacterium-mediated transformation can be used to create transgenic plants that can detect or degrade environmental pollutants or toxins. For example, Agrobacterium-mediated transformation has been used to generate transgenic plants that can sense heavy metals, herbicides, explosives, or pathogens by expressing specific biosensors or indicators. It has also been used to produce transgenic plants that can detoxify contaminated soil or water by expressing enzymes or transporters for degradation or sequestration of pollutants.

These are some of the major applications of Agrobacterium-mediated gene transfer in plant biotechnology. However, there are still many challenges and limitations associated with this technique, such as the narrow host range, the random integration of T-DNA, the labor-intensive and time-consuming procedures, and the biosafety and ethical issues. Therefore, further research and development are needed to improve the efficiency, specificity, and safety of Agrobacterium-mediated gene transfer for various purposes.

Even though Agrobacterium-mediated transformation has been advancing over the years with much success, there are some problems and limitations associated with this technique. Some of the commonly encountered limitations and problems with the technique are:

• The most important limitation associated with this technique is the narrow host range, as it is still limited to particular plant species. Some plants are naturally resistant or recalcitrant to Agrobacterium infection, while others require specific conditions or treatments to enhance their susceptibility.
• Even though a lot is known about the mechanism of T-DNA transfer in the bacteria, not much is known about the plant-encoded factors that affect the efficiency of this process. The integration of T-DNA into the plant genome is a complex and random process that depends on various factors such as chromatin structure, DNA repair mechanisms, and gene expression.
• The technique is labor-intensive as it requires the development of plant regeneration protocols and detailed time-consuming processes. Many of these processes are prone to in vitro variations, resulting in unfavorable results. The success of transformation also depends on the quality and quantity of explants, which can vary depending on the plant species and growth conditions.
• The success of transformation in the case of monocots depends on the use of embryos as the explants; however, these are only available for a short period of time during the year. Moreover, some monocots require pre-treatment with chemicals or physical agents to increase their competence for Agrobacterium-mediated transformation.
• Agrobacterium-mediated transformation cannot transfer large DNA molecules into more economically important plants, which indicates a possible introduction of a powerful vector system. The size and structure of T-DNA can affect its stability and expression in the plant cells.

These limitations suggest that there is still room for improvement and optimization of Agrobacterium-mediated transformation for different plant species and applications. Some possible strategies to overcome these limitations include:

• Expanding the host range by modifying the Agrobacterium strains or vectors, or by using alternative methods such as particle bombardment or protoplast transformation.
• Identifying and manipulating the plant factors that influence T-DNA integration and expression, such as recombination proteins, transcription factors, and epigenetic modifiers.
• Developing more efficient and standardized protocols for plant regeneration and selection, as well as reducing the risk of somatic variation and chimerism in transgenic plants.
• Exploring new sources and types of explants for monocot transformation, such as callus, leaf, or anther cultures.
• Improving the capacity and stability of T-DNA transfer by using binary vectors, co-transformation, or site-specific recombination systems.

By addressing these challenges, Agrobacterium-mediated transformation can become more versatile and reliable for plant genetic engineering and biotechnology.

We are Compiling this Section. Thanks for your understanding.