Somatic hybridization

1. Somatic Hybridization
OR
Somatic Cell Fusion
OR
Protoplast Fusion
Amandeep Singh
Assistant Professor
Department of Biotechnology
GSSDGS Khalsa College
Patiala

2. Conventional Hybridization
Sexual Hybridization (to improve characteristics)
Limitation: Done between within a plant species or
very closely related plant species

3. Somatic Hybridization
In-vitro fusion of isolated protoplasts to form a
hybrid cell and its subsequent development to
form a hybrid plant.

4. Steps:
1. Fusion of protoplasts
2. Selection of hybrid cells
3. Identification of hybrid cells

5. 1. Fusion of protoplasts
• Spontaneous fusion (Homokaryon)
• Mechanical fusion
• Induced fusion (By fusion-inducing
agents/fusogens)

6. • Treatment with sodium nitrate (NaNo3)
5.5% NaNO3+ 10% sucrose
35⁰C, 5 minutes
Centrifugation 2000 rpm, 5 minutes
Protoplasts
Protoplasts Pellet
Protoplasts Fusion
Waterbath 30⁰C, 30 minutes
• High pH and Ca2+ ion treatment
0.4 M mannitol+ 0.05M CaCl2
37⁰C, 30-40 minutes
Protoplasts
Protoplasts Fusion

7. • Polyethylene glycol (PEG) treatment
Equal volume 28%-56% PEG
Protoplasts
Protoplasts Pellet
Advantages:
•High frequency of heterokaryon formation
•Low toxicity to cells
•Reduced formation of bi-nucleate heterokaryon
•Non-specific (so can be used for wide range of plants)

8. Electro-fusion
Protoplasts
Protoplasts Fusion
Electric field

9. Mechanism of Fusion
• Agglutination (Adhesion)
By PEG and high pH and Ca2+ ions
• Plasma membrane fusion
High pH and Ca2+ ions (neutralize surface charges of protoplasts, hence
cause fusion)
High temperature (intermingling of lipid molecules in membrane, thus
cause fusion)
PEG (cause agglutination, form clumps, formation of tight adhesion, thus
fusion)
• Formation of heterokaryon

10. Selection of Hybrid Cells
• Biochemical methods
• Visual methods
• Cytometric methods

11. Biochemical methods
• Drug Sensitivity • Auxotrophic mutants
NR nia-: Nitrate reductase apoenzyme deficient
NR cnx-: Nitrate reductase lacking molybdenum co-factor
Species A
(Petunia hybrida)
Sensitive to actinomycin D
Species B
(Petunia parodii)
Resistant to actinomycin D
Protoplasts Protoplasts
Fusion
Small colonies No
further division
Large colonies
Resistant to actinomycin D
Further growth
Callus
SOMATIC HYBRID PLANT
Culture medium supplemented with Actinomycin D
Large colonies Small colonies

12. Visual methods

13. Cytometric methods
Flow Cytometry
FACS (Fluorescent Activated Cell Sorting)