For students, a details note on Agrobacterium Mediated Gene Transformation In Plants been prepared by the postgraduate research student Md Habibul Islam Safin from the University of Greenwich, School of Biotechnology.

Agrobacterium Mediated Gene Transformation In Plants

In the world of agriculture, Agrobacterium Mediated Gene Transformation In Plants is one of the ground breaking method for innovation and sustainable practices led by scientists to explore the enhance crop productivity and resilience. This cutting-edge technology holds the potential to revolutionize the way we cultivate crops, offering a promising avenue for genetic improvement and the development of more resilient and productive plant varieties (Biotech Crops or Genetically Modified Crops).

What Is Agrobacterium Mediated Plant Transformation?

Agrobacterium tumefaciens, a soil-borne bacterium, has become a powerful tool in genetic engineering. Its natural ability to transfer a segment of its DNA, known as the Ti (tumor-inducing) plasmid, into the host plant’s genome has been harnessed for introducing desirable traits into plants. The transformation process involves the integration of foreign genes into the plant’s DNA, leading to the expression of new traits or characteristics.

4 Steps in Agrobacterium Mediated Gene Transformation In Plants

1. Vector Construction:

Scientists design a binary vector containing the gene of interest flanked by specific DNA sequences recognized by Agrobacterium. This vector is then introduced into the bacterium.

Vector construction is a crucial step in Agrobacterium-mediated plant transformation, involving the design and assembly of a DNA construct that carries the gene of interest (GOI) and other necessary elements for successful integration into the plant genome. The vector, often referred to as a T-DNA (transfer DNA) binary vector, is then introduced into Agrobacterium tumefaciens for subsequent transfer into the target plant. Here is a detailed breakdown of the vector construction process:

Selection of Plasmid Backbone:

The vector construction begins with the selection of a plasmid backbone. Plasmids are circular DNA molecules that can replicate independently of the host organism’s chromosomal DNA. Commonly used plasmids in Agrobacterium-mediated transformation include pCAMBIA and pBIN series. The choice of plasmid backbone depends on factors such as the host plant, selectable markers, and regulatory elements required. When this plasmids are incorporated with tumor inducing genes, it’s called Ti-plasmid.

Incorporation of Selectable Marker Genes:

Selectable marker genes are crucial components of the vector, aiding in the identification and isolation of transformed plant cells. These markers are often genes that confer resistance to antibiotics or herbicides. Common selectable markers include the kanamycin resistance gene (NPTII), conferring resistance to kanamycin, and the phosphinothricin acetyltransferase gene (BAR), providing resistance to the herbicide glufosinate.

Promoter and Terminator Sequences:

The vector must contain regulatory elements for the expression of the gene of interest. A strong and plant-compatible promoter, such as the cauliflower mosaic virus (CaMV) 35S promoter, is often used to drive the expression of the GOI. Additionally, a terminator sequence is included to signal the end of gene transcription.

GOI and Fusion Proteins:

The gene of interest, which imparts the desired trait to the plant, is inserted into the vector. If necessary, fusion proteins or other modifications can be added to enhance the functionality or stability of the protein product. For example, tags like green fluorescent protein (GFP) may be added for visualization and tracking.

Introns and Enhancers:

Introns, non-coding DNA sequences within a gene, may be included to enhance gene expression. Enhancer elements can also be added to boost the activity of the promoter and ensure robust expression of the inserted gene in plant cells.

Selectable Marker for Agrobacterium:

In addition to the plant selectable marker, a separate selectable marker compatible with Agrobacterium is often included in the plasmid backbone. This marker ensures that only Agrobacterium cells carrying the T-DNA are selected during the transformation process.

Restriction Enzyme Sites and Cloning Strategy:

Specific restriction enzyme recognition sites are incorporated into the vector to facilitate the cloning of the various components. These sites enable the insertion of the selectable marker, promoter, terminator, and the gene of interest in a controlled and organized manner.

Verification of the Vector:

Before introducing the vector into Agrobacterium, its integrity and correctness are verified through restriction enzyme digestion, PCR (polymerase chain reaction), and sequencing. This ensures that the vector contains the intended genetic elements in the correct order and orientation.

Transformation of Agrobacterium:

The finalized vector is introduced into Agrobacterium tumefaciens using a suitable method such as heat shock or electroporation. Once transformed, the Agrobacterium cells are selected based on the presence of the Agrobacterium-selectable marker.

Curation of the Binary Vector:

The successfully transformed Agrobacterium cells containing the binary vector are cultured and the vector purified. The purified vector is then ready for use in the Agrobacterium-mediated plant transformation process.

In summary, the vector construction process is a meticulous and well-planned procedure that involves the thoughtful assembly of genetic elements to create a functional T-DNA binary vector. This vector serves as the vehicle for introducing desired traits into plants, paving the way for advancements in crop improvement and agricultural sustainability.

2. Cocultivation

The prepared Agrobacterium carrying the desired genes is introduced to plant tissues. This can be achieved through various methods such as vacuum infiltration, leaf disc transformation, or flower-dipping, depending on the plant species.

3. Integration and Selection

The transferred genes integrate into the plant genome during cell division. Selective pressure, often in the form of antibiotics or herbicides linked to the introduced genes, helps identify and isolate transformed cells.

4. Regeneration

Transformed cells are cultured in a nutrient medium to encourage the development of whole plants. This stage is crucial for the successful regeneration of plants with the desired genetic modifications.

What is The Infection Process of Agrobacterium?

Agrobacterium tumefaciens is a bacterium that is known for its ability to transfer a piece of its genetic material, specifically the Ti (tumor-inducing) plasmid, into the DNA of plants. This process is called horizontal gene transfer and results in the formation of crown gall tumors in infected plants. The infection process of Agrobacterium can be broken down into several steps:

Recognition of Host Plant

Agrobacterium recognizes and attaches to specific plant cells at wound sites. Wounding is crucial for the infection process, and it can occur naturally through activities like insect feeding, or it can be induced artificially. Usually, chemotaxis like acetosyringone are the trigger which can be recognized by the bacteria. The can also activate the virulence gene in the Ti- plasmid.

Attachment and Agrobacterial Virulence (Vir) Proteins

Agrobacterium uses surface structures, such as pili and adhesins, to attach to the plant cell. The bacterium then injects virulence (Vir) proteins into the plant cells through a type IV secretion system. These Vir proteins play a crucial role in the subsequent steps of the infection process.

T-DNA Processing

The Ti plasmid of Agrobacterium carries a region known as the transfer DNA (T-DNA), which contains genes responsible for tumour formation and other functions. The Vir proteins process the T-DNA and prepare it for transfer to the plant cell. Single-strand DNA (T-DNA) with the endonuclease attached is transferred through a type IV secretion system into host cell where it is likely coated and protected from nucleases by a bacterial secreted protein to form the T-complex.

A nuclear localisation signal in the endonuclease guides the transferred strand (T-strand), into the nucleus where it is integrated randomly into the host chromosome.

T-DNA Transfer to Plant Cell

The processed T-DNA is transferred from Agrobacterium into the plant cell’s nucleus. The transfer is facilitated by the Vir proteins and occurs through a channel formed by the type IV secretion system.

Integration of T-DNA into Plant Genome

Once inside the plant cell nucleus, the T-DNA integrates into the plant genome. The integration is mediated by both plant and bacterial factors.

Tumour Formation and Genetic Changes

The integrated T-DNA carries genes that induce the plant cells to undergo uncontrolled growth, leading to the formation of a tumour, known as a crown gall. The T-DNA also carries genes that facilitate the synthesis of opines, which are compounds that Agrobacterium can use as a food source.

The ability of Agrobacterium to transfer genes into plants has been harnessed for genetic engineering purposes. Scientists use modified versions of Agrobacterium to introduce specific genes into plants for various purposes, such as improving crop traits or developing genetically modified organisms (GMOs).

What Are The Advantages of Agrobacterium-Mediated Plant Transformation?

Precise Gene Transfer

Agrobacterium-mediated transformation offers precise integration of genes into the plant genome, reducing the likelihood of unintended genetic alterations.

Wide Applicability

This method is applicable to a broad range of plant species, from major crops like rice and wheat to horticultural crops and ornamental plants.

Stable Integration

The integration of foreign genes is often stable over generations, providing a reliable and heritable trait expression.

Reduced Regulatory Concerns

Many countries have recognized Agrobacterium-mediated plant transformation as a natural and safer method compared to other genetic modification techniques, leading to fewer regulatory hurdles.

What Are The Applications of Agrobacterium-Mediated Plant Transformation?

Disease Resistance

Genes encoding resistance to pests, pathogens, or specific environmental stresses can be introduced into crops, enhancing their ability to withstand various challenges.

Improved Nutritional Content

Essential nutrients and vitamins can be introduced to crops, addressing nutritional deficiencies in the human diet.

Abiotic Stress Tolerance

Plants can be engineered to tolerate adverse environmental conditions such as drought, salinity, or extreme temperatures, ensuring stable yields in challenging climates.

Enhanced Crop Quality

Traits related to crop quality, including flavour, shelf life, and appearance, can be improved through Agrobacterium-mediated transformation.

Wrapping Up

I hope you all get an idea about the Agrobacterium Mediated Gene Transformation In Plants. Agrobacterium mediated plant transformation stands at the forefront of agricultural biotechnology, offering a precise and versatile method for enhancing crop traits. As researchers continue to unravel the intricacies of this transformative technology, the agricultural landscape holds the promise of more resilient, nutritious, and sustainable crop varieties. The ongoing exploration of Agrobacterium-mediated plant transformation underscores its potential to contribute significantly to global food security and sustainable agriculture in the years to come.

Prepared By

Md Habibul Islam Safin

MSc Biotechnology

University of Greenwich