Explain the techniques involved in somatic hybridization in crop plants.

What is Somatic Hybridization?

Somatic hybridization, also known as protoplast fusion, is a technique used in plant breeding to create new combinations of genes that are not possible through sexual reproduction. It involves the fusion of somatic cells (non-reproductive cells like leaf, root, or mesophyll cells) from two different plants. The resulting hybrid cell, called a somatic hybrid, can then be regenerated into a whole plant. This bypasses the sexual incompatibility barriers that often prevent successful breeding between distantly related species.

Techniques Involved in Somatic Hybridization

1. Protoplast Isolation

The first step in somatic hybridization is the isolation of protoplasts. Protoplasts are plant cells from which the cell walls have been removed using enzymes like cellulase and pectinase. Different tissue sources are used based on their ease of isolation and content of desirable genes.

• Leaf protoplasts: Commonly used due to their abundance and ease of isolation.
• Root protoplasts: Rich in secondary metabolites.
• Callus-derived protoplasts: Obtained from callus tissue induced on explants.

2. Fusion Methods

Several methods are employed to induce protoplast fusion:

a) Chemical Fusion

This is the earliest and simplest method. Protoplasts are incubated in a chemical solution, typically polyethylene glycol (PEG), which brings the membranes closer and facilitates fusion.

• PEG Concentration: Usually between 20-40% (w/v).
• Advantages: Simple and inexpensive.
• Disadvantages: Low fusion efficiency, random fusion.

b) Electrofusion

This method utilizes electrical pulses to induce membrane fusion. Protoplasts are arranged in a specific pattern and exposed to short, high-voltage pulses.

• Pulse Parameters: Voltage, duration, and number of pulses are critical for successful fusion.
• Advantages: Higher fusion efficiency, precise control over fusion.
• Disadvantages: Requires specialized equipment.

c) Mechanical Fusion

This involves physically bringing protoplasts into close contact, often using a vortex or micro-droplets.

• Advantages: Relatively simple.
• Disadvantages: Low efficiency, limited applicability.

d) Centrifugal Fusion

Protoplasts are subjected to high centrifugal forces, bringing them close enough to fuse.

• Advantages: Can be used for a large number of protoplasts simultaneously.
• Disadvantages: Requires specialized equipment, can damage protoplasts.

3. Regeneration and Selection

After fusion, the hybrid protoplasts are cultured on selective media containing nutrients and growth regulators. This promotes cell division and callus formation. The callus is then induced to regenerate into shoots and roots, ultimately forming a somatic hybrid plant. Selection markers, such as selectable genes or morphological traits, are often used to identify and isolate the desired hybrid plants.

Method	Mechanism	Efficiency	Cost	Equipment
Chemical Fusion (PEG)	PEG brings membranes close	Low	Low	None
Electrofusion	Electrical pulses induce fusion	High	High	Electrofusion apparatus
Mechanical Fusion	Physical contact	Low	Low	Vortex mixer

Applications and Significance

Somatic hybridization has several important applications in crop improvement:

• Overcoming Sexual Incompatibility: Creates hybrids between distantly related species that cannot be crossed sexually.
• Combining Desirable Traits: Combines traits that are linked or difficult to breed together through conventional methods.
• Disease Resistance: Introduces disease resistance genes from wild relatives into cultivated crops.
• Improved Yield and Quality: Develops new varieties with higher yield, improved nutritional content, and better quality.
A classic example of somatic hybridization is the 'TomTato', a plant that produces both tomatoes and potatoes simultaneously. It was created by fusing protoplasts from Solanum tuberosum (potato) and Solanum lycopersicum (tomato). This showcases the potential to combine traits from different species. Researchers in India have been exploring somatic hybridization between rice and wheat to combine desirable traits like disease resistance from wild rice varieties and improved grain quality from wheat. The challenge lies in regenerating fertile plants, but significant progress has been made in callus selection and plant regeneration protocols. While a fully fertile and commercially viable somatic hybrid rice-wheat plant hasn’t been achieved yet, the research provides valuable insights into overcoming the barriers to intergeneric somatic hybridization and has advanced the understanding of protoplast fusion techniques.