4 Jun 2024

Protoplast Fusion: A Gateway to Genetic Engineering in Plants

Anjali Singh, MS

As a content and community manager, I leverage my expertise in plant biotechnology, passion for tissue culture, and writing skills to create compelling articles, simplify intricate scientific concepts, and address your inquiries. As a dedicated science communicator, I strive to spark curiosity and foster a love for science in my audience.

Table of Contents

Introduction

If you’ve heard of Golden Rice or Bt Cotton, you already have been introduced to a genetic engineering product!

The concept of genetic engineering  emerged in 1950s with the discovery of double helix DNA and in vitro DNA synthesis. However, the proper techniques to manipulate genes didn’t arrive before 1970s. 

As of today, the genetic engineering technique has been around for over four decades. The plant sector has widely adopted this technique to improve plant varieties, such as plants with better disease resistance, fruit quality, improved yield, or stress tolerance.

For those of you who are still wondering what exactly “genetic engineering” is and how it improves the quality of plants, let’s get to the basics of the technique. 

What is the Genetic Engineering technique?

Genetic engineering is the process of altering or modifying the genes (or genetic makeup) of an organism using lab-based technologies. 

Imagine the plant's DNA as a vast instruction manual containing the blueprint for its entire existence. Crucially, this analogy highlights that genetic engineers can now isolate specific genes (sections of the manual) that code for desired traits, like disease resistance or improved yield.

What is Genetic Engineering technique?

Genetic engineering is the process of altering or modifying the genes (or genetic makeup) of an organism using lab-based technologies. 

Imagine the plant's DNA as a vast instruction manual, containing the blueprint for its entire existence. Genetic engineers can now isolate specific genes (sections of the manual) that code for desired traits, like disease resistance or improved yield.

Figure: A timeline of different events till the development of diverse genetic engineering tools for crop improvement. 

Researchers have developed numerous techniques for genetic engineering of plants, including Agrobacterium tumefaciens transformation technology, liposome-mediated transfection, virus-vector-mediated technology, biolistic technology, polyethylene glycol induction, pollen-tube pathway method, and protoplast fusion.

This article dives deeper into the protoplast fusion technique, explaining what it is and how scientists use it to develop improved plant varieties.

What Is Protoplast Fusion?

Plant genetic engineers use a technique called protoplast fusion to create hybrid cells. This technique fuses all the contents (including the nucleus and cytoplasm) of two plant cells into one.

This process is crucial for plant breeding and crop improvement. Here's how it works: Scientists isolate protoplasts, which are plant cells without cell walls, from two different plants. Then, they fuse these protoplasts into one. Finally, they select the desired somatic hybrid cells and use tissue culture techniques to regenerate them into whole hybrid plants.

Figure: An overview of the process of protoplast fusion. 

Steps of Protoplast Fusion

Here's a breakdown of the steps involved in isolating protoplasts from plants, along with important information about the process:

1. Preparation of Plant Tissue:

• Select healthy, young plant tissue like leaves, stems, or roots.
• Sterilize the tissue to prevent contamination.

2. Enzyme Treatment:

• Enzymes are used to digest the cell wall, allowing the protoplast (cell without a wall) to be released.
• Enzymes used for plant cells: Cellulase and pectinase (or macerozyme)

3. Protoplast Isolation:

• The enzyme-treated tissue is incubated in a solution with a concentration different from the cell contents (osmoticum). This causes the cells to shrink (plasmolyze), making enzyme digestion more efficient.
• The digested solution is then filtered or centrifuged to separate the protoplasts from cell debris and enzyme residues.

4. Protoplast Purification:

• Techniques like differential centrifugation or density gradient centrifugation are used to purify the protoplasts from unwanted materials further.

5. Protoplast Culture (Optional):

• Isolated protoplasts can be cultured in a specific medium to induce cell wall regeneration and division, potentially leading to the formation of whole plants.

Important points to remember:

• The specific enzymes, incubation conditions, and isolation techniques may vary depending on the plant species and tissue type.
• Protoplasts are fragile and have a short lifespan. Careful handling is crucial throughout the process.

Applications of The Protoplast Fusion Technique

Protoplast fusion has a variety of applications in plant biotechnology, including:

• Production of Useful Metabolites: Protoplast fusion can be used to create plants that produce higher levels of desired metabolites, such as enzymes, vitamins, and antioxidants. This is because the fusion can combine the metabolic pathways of the two parent plants.
• Target Site Mutagenesis: Protoplasts can be used to deliver gene editing tools to plant cells. Scientists can use this technique to create targeted mutations in genes, leading to improved plant traits like disease resistance or herbicide tolerance.
• Production of Herbicide-Resistant Plants: Protoplast fusion can combine genes for herbicide resistance from different plants to create plants that are resistant to a wider range of herbicides.
• Introduction and Establishment of Disease Resistance: Protoplast fusion can be used to transfer disease resistance genes from one plant species to another. This can help to create crops that are resistant to a wider range of diseases.
• Insect Pest Control: Protoplast fusion can be used to create plants that are resistant to insect pests. We can achieve this by combining genes for insect resistance from different plants.

Advantages and Disadvantages of Protoplast Fusion

Protoplast fusion, a technique in plant genetic engineering, offers a unique approach to creating novel plant varieties. Unlike traditional breeding methods limited by sexual compatibility, protoplast fusion allows scientists to fuse entire cells from different plant species, even those that can't reproduce naturally.

This fusion creates a hybrid cell with the combined genetic information (nuclear and cytoplasmic) of both parent plants. While challenges like creating fertile offspring exist, protoplast fusion offers several exciting applications:

• Overcoming Sexual Incompatibility: This technique bypasses limitations in traditional breeding by allowing the creation of hybrids between incompatible species. Furthermore, imagine combining disease resistance from a wild plant with the high yield of a domesticated variety – protoplast fusion can make this possible.
• Transferring Organellar Genomes: Protoplast fusion can also transfer genetic material from organelles like mitochondria, bypassing maternal inheritance. Furthermore, this opens doors for applications like introducing male sterility or creating plants with enhanced stress tolerance.

Examples of Successful Fusions:

• Brassica and Arabidopsis: This fusion resulted in stable genotypes with combined genetic material from both species.
• B. napus and Diplotaxis harra: This intergeneric fusion demonstrated gene exchange between the species.

These examples showcase the potential of protoplast fusion in creating novel plant varieties with desirable traits that might not be achievable through conventional breeding methods.

However, it's important to acknowledge the limitations:

• Technical Difficulty: The process requires specialized skills and equipment, making it a complex technique compared to others.
• Unpredictable Outcomes: Fusing entire genomes can lead to unstable or sterile offspring. We need extensive screening and testing to identify desirable outcomes.
• Ethical Considerations: Creating entirely new genetic combinations raises ethical concerns. Careful evaluation of potential risks and benefits is crucial before widespread adoption.

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