Somatic hybridisation is the fusion of protoplasts from two different plant species or varieties to create a somatic hybrid containing genomes of both parents. It bypasses normal sexual reproduction barriers — plants that cannot be crossed sexually can sometimes be hybridised somatically. Technique developed by Melchers and others in the 1970s. Most famous example: Pomato (potato + tomato). The technique is part of protoplast technology or protoplast fusion technology. While academic success has been demonstrated, practical agriculture use has been limited because the resulting hybrids often have fertility problems (odd ploidy, incompatible genomes).

Source material: young leaves, coleoptile cells, suspension cultures, or callus tissue. Two different plant varieties/species selected based on desired trait combination. Cells are mechanically separated from tissue or obtained from liquid suspension cultures. Surface sterilisation of plant material first (70% ethanol, sodium hypochlorite). Source cells from leaf mesophyll: cut leaf into small sections → incubate in enzyme solution. Purity is important — contamination with other cell types leads to heterogeneous protoplast population. In practice: most efficient sources are leaf mesophyll (contains well-separated cells) or suspension cultures (already in liquid).

Enzymatic digestion removes plant cell walls. Enzymes: cellulase (breaks β-1,4-glycosidic bonds in cellulose). Pectinase (breaks pectin in middle lamella). May also use hemicellulase, driselase. Conditions: isotonic solution (mannitol or sorbitol) to prevent osmotic lysis of protoplasts. Temperature: 25-28°C. Duration: 3-6 hours. Protoplasts are fragile — osmotic protection essential: if placed in hypertonic solution → shrink; hypotonic → burst. Isotonic medium (0.4-0.7 M mannitol) maintains protoplast integrity. After digestion: filter through sieves to remove undigested debris and tissue fragments. Centrifuge at low speed to pellet protoplasts.

After enzymatic digestion, protoplasts are isolated and purified. Purification: density gradient centrifugation or flotation on sucrose gradient. Washing: multiple washes in isotonic solution to remove enzymes. Assessment: viability tested by fluorescein diacetate (FDA) fluorescence (live protoplasts fluoresce green). Yield: typically 10⁶-10⁷ protoplasts per gram of leaf tissue. Protoplasts from two parents must be from genetically different backgrounds. Identification: one parent sometimes stained with different dye. Viability is critical — only healthy, intact protoplasts give successful fusion and regeneration.

Protoplast fusion — physical merging of two different protoplasts. Methods: PEG (polyethylene glycol)-mediated fusion: PEG is added to mixed protoplast suspension. PEG (MW 1500-6000) at concentration 10-50% → destabilises plasma membranes → protoplasts aggregate → wash out PEG → membranes reseal → fused protoplasts (homokaryon or heterokaryon). Electrofusion: alternating electric field (dielectrophoresis) aligns protoplasts → short DC pulse → membrane breakdown → fusion → membranes reseal. More controlled than PEG. Hybrid frequency: only ~5-20% of fusions are heterokaryons (fusion between two different protoplasts). Many homo-fusions (same type) also occur. Selection systems needed to identify true hybrids.

After fusion: hybrid protoplast → must regenerate cell wall and divide. Culture in: nutrient medium + plant hormones (auxin + cytokinin). Cell wall regeneration: within 24-48 hours. First division: 2-7 days. Colony formation (microcallus): 2-4 weeks. Callus growth: 4-8 weeks. Selection of hybrids: complementation selection (each parent carries mutation lethal in culture → only hybrid survives). Drug resistance markers. Isozyme analysis. Shoot regeneration: transfer to shoot-inducing medium (high cytokinin). Root regeneration: transfer to rooting medium (high auxin). Plantlet: transferred to soil. Result: somatic hybrid plant containing nuclear genome of both parents + cytoplasms of both (heteroplasmic for mitochondria and plastids — leads to 'cybrids' if only cytoplasm fused).

Major somatic hybrids produced: Pomato: potato (Solanum tuberosum) + tomato (Lycopersicon esculentum) by Melchers et al. (1978). Shows both potato and tomato features. Not commercially useful (sterile, weak). Tobacco hybrids: Nicotiana hybrids (closely related species) — most successful. Tomato + potato × eggplant. Citrus species hybrids: various rootstock improvement. Disease resistance transfer: tried to transfer virus resistance from wild species to cultivated. Limitations of somatic hybridisation: resulting plants often triploid/aneuploid → sterile. Incompatible genome combinations → poor performance. Specific gene transfer not possible (entire genomes mixed). Replaced in many applications by: Agrobacterium-mediated transformation (specific gene), CRISPR genome editing, conventional breeding with embryo rescue.

Somatic hybrid: contains complete nuclear genome of BOTH parents (complete genome fusion). All nuclear DNA from both plants present. May have mixed or one parent's organellar DNA. Cybrid (cytoplasmic hybrid): contains nuclear genome of ONE parent + cytoplasmic components (mitochondria, chloroplasts) of BOTH parents or the other parent. Produced by: protoplast fusion where one parent's nucleus is inactivated (UV or γ-irradiation) or by enucleation. Purpose of cybrids: transfer cytoplasmic male sterility (CMS) between plant varieties without changing the nuclear genome (CMS is mitochondrially encoded). Very useful in breeding F₁ hybrid varieties of crops. Also used to transfer herbicide tolerance encoded by plastid genes. The distinction is important: cybrid has one nucleus but mixed cytoplasm; somatic hybrid has mixed nucleus and cytoplasm.