The term protoplast has been defined as that part of the plant cell which lies within the cell wall. It can be plasmolysed and isolated by removing the cell wall either mechanically or using enzymatic digestion technique. The protoplast is, therefore, a naked cell surrounded by the plasma membrane and is potentially capable of cell wall regeneration, growth and division. The protoplast is quite fragile but it too can be cultured and regenerated into a whole plant. The first demonstration of totipotency of protoplasts was by Takebe et al. who obtained tobacco plants from mesophyll protoplasts. The technology of plant protoplast has opened up new vistas and has awakened the interest of plant physiologists, plant pathologists, molecular biologists, and cytogeneticists. In protoplast technology, protoplasts are isolated either from any two genotypically different plants or from the somatic cells (diploid) and are experimentally fused to obtain parasexual hybrid protoplasts.

2. TOPIC - SOMATIC HYBRIDIZATION

3. CONTENTS
INTRODUCTION.
 SOMATIC HYBRIDIZATION TECHNIQUE.
PROTOPLAST ISOLATION , FUSION, IDENTIFICATION
OF SOMATIC HYBRIDS, CULTURE AND REGENERATION.
 CONCEPT OF CYBRIDS.
 ADVANTAGES AND DISADVANTAGES OF SOMATIC
HYBRIDIZATION.
CONCLUSION .

4. INTRODUCTION
The term protoplast has been defined as that part of the plant cell which lies within the
cell wall. It can be plasmolysed and isolated by removing the cell wall either
mechanically or using enzymatic digestion technique.
 The protoplast is, therefore, a naked cell surrounded by the plasma membrane and is
potentially capable of cell wall regeneration, growth and division.
 The protoplast is quite fragile but it too can be cultured and regenerated into a whole
plant. The first demonstration of totipotency of protoplasts was by Takebe et al. who
obtained tobacco plants from mesophyll protoplasts. The technology of plant protoplast
has opened up new vistas and has awakened the interest of plant physiologists, plant
pathologists, molecular biologists, and cytogeneticists.
 In protoplast technology, protoplasts are isolated either from any two genotypically
different plants or from the somatic cells (diploid) and are experimentally fused to
obtain parasexual hybrid protoplasts.

5. The hybrid protoplasts contain heteromorphic cytoplasm and
two fused parental nuclei.
The fused protoplast is grown in vitro with an aim to obtain a
hybrid plant. So, the in vitro fusion of plant protoplasts derived
either from somatic cell of the same plant or from two
genetically different plants is called somatic hybridization

24. IDENTIFICATION AND SELECTION OF HYBRID
CELLS
• Chlorophyll deficiency complementation
• Auxotroph complementation
• Complementation of resistance markers
• Use of metabolic inhibitors
• Use of visual characteristics- use of morphologically distinct
cells, fluorescent labelling, difference in the morphology of
callus.
• Compound selection systems

26. CULTURE TECHNIQUES
• AGAR CULTURE – Bergmann cell plating technique is followed for plating of
protoplasts. Temp. 45 degree Celsius.
• LIQUID CULTURE – generally preferred Because, it allows easy dilution and
transfer, protoplasts of some species in agarified media , osmotic pressure of
medium can be effectively reduced.
• LIQUID DROPLET METHOD - suspending protoplasts in culture media and
pipetting 100-200micro ml droplets into 60*15 ml plastic petri dishes.
• HANGING DROPLET METHOD – small drops(40-400micro ml) of protoplast
suspension are placed on inner side of lid of petri dish.
• FEEDER LAYER
• CO- CULTURING etc.,

32. CONCLUSION
• protoplast fusion and somatic hybridization have opened up a new avenue in
plant science. protoplast fusion provides a method of combining the different
genomes of different genera and species with the potential of overcoming
sexual incompatibility barrier between plants. besides this, studies of fusion
product can give information about compatibility or incompatibility of the nuclei
or cytoplasm. By protoplast fusion, it is possible to transfer some useful genes.
This opportunity will undoubtedly lead to the production of new genetic variation
and thereby widen the genetic base for plant breeding. More over in case of
vegetatively reproducing plants, the genetic variation can be induced through
protoplast fusion. Thus, in spite of having some limitations protoplast culture and
somatic hybridization have immense potentialities and prospect in future plant
biotechnology especially for the improvement of different plant species and
varieties having high economic values.