PROTOPLASTS

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Mrs. Rani Ashok Asst. Prof. of Zoology Lady Doak College Madurai – 2 Email: eaarani@rediffmail.com Protoplasts

PROTOPLASTS:

PROTOPLASTS PLANT, BACTERIAL OR FUNGAL CELLS THAT HAD ITS CELL WALL REMOVED USING EITHER MECHANICAL OR ENZYMATIC MEANS

PROTOPLASTS:

PROTOPLASTS DERIVED FROM GREEK COMPLETE REMOVAL OF CELL WALL INCOMPLETE/ PARTIAL REMOVAL OF CELL WALL – SPHEROPLASTS CELL WALL REMOVED BY MECHANICAL/ ENZYMATIC METHODS COMPOSED OF A CELL’S NUCLEUS AND THE SURROUNDING PROTOPLASMIC MATERIALS

ISOLATION OF PROTOPLASTS:

ISOLATION OF PROTOPLASTS

Mechanical Method :

Mechanical Method Used for vacuolated cells like onion bulb scale, radish and beet root tissues Low yield of protoplast Laborious and tedious process Low protoplast viability

MECHANICAL ISOLATION:

MECHANICAL ISOLATION PLANT TISSUE OBSERVE UNDER MICROSCOPE CELL WALL CUTTING USING SCALPEL PLASMOLYSED TISSUE CELL PLASMOLYSIS RELEASE & COLLECTION OF PROTOPLASTS

Enzymatic Method:

Enzymatic Method Leaf sterlization, removal of epidermis Plasmolysed cells Plasmolysed cells Pectinase +cellulase Pectinase Protoplasm released Release of isolated cells cellulase Protoplasm released Isolated Protoplasm

Enzymatic Method:

Enzymatic Method Used for variety of tissues and organs including leaves, petioles, fruits, roots, coleoptiles, hypocotyls, stem, shoot apices, embryo microspores Mesophyll tissue - most suitable source High yield of protoplast Easy to perform More protoplast viability

PROTOPLASTS:

PROTOPLASTS TEM PICTURE OF TOBACCO PROTOPLAST

USES:

USES STUDY MEMBRANE BIOLOGY STUDY UPTAKE OF MACROMOLECULES AND VIRUSES DNA TRANSFORMATION TO PRODUCE GENETICALLY MODIFIED CELLS PLANT BREEDING THROUGH PROTOPLAST FUSION

SOMATIC HYBRIDIZATION:

SOMATIC HYBRIDIZATION

PowerPoint Presentation:

Development of hybrid plants through the fusion of somatic protoplasts of two different plant species/varieties is called somatic hybridization

Somatic hybridization technique:

Somatic hybridization technique 1. isolation of protoplast 2. Fusion of the protoplasts of desired species/varieties 3. Identification and Selection of somatic hybrid cells 4. Culture of the hybrid cells 5. Regeneration of hybrid plants

PowerPoint Presentation:

Protoplast Fusion (Fusion of protoplasts of two different genomes) Spontaneous Fusion 2. Induced Fusion Intraspecific Intergeneric Electrofusion Mechanical Fusion Chemofusion

Spontaneous Fusion:

Spontaneous Fusion Protoplast fuse spontaneously during isolation process mainly due to physical contact Intraspecific produce homokaryones Intergeneric have no importance

Induced Fusion :

Induced Fusion Chemofusion- fusion induced by chemicals Types of fusogens PEG NaNo 3 Ca 2+ ions Polyvinyl alcohal

Induced Fusion :

Induced Fusion Mechanical Fusion- Physical fusion of protoplasts under microscope by using micromanipulator and perfusion micropipette Electrofusion- Fusion induced by electrical stimulation Pearl chain of protoplasts is formed by low strength electric field (10kv m -1 ) Fusion of protoplasts of pearl chain is induced by the application of high strength electric field (100kv m -1 ) for few microseco

Identification and Selection of somatic hybrid cells:

Identification and Selection of somatic hybrid cells Hybrid identification- Based on difference between the parental cells and hybrid cell with respect to Pigmentation Cytoplasmic markers Fluorochromes like FITC (fluoroscein isothiocyanate) and RITC (Rhodamine isothiocyanate) are used for labelling of hybrid cells Presence of chloroplast Nuclear staining Heterokaryon is stained by carbol-fuschin, aceto-carmine or aceto-orcein stain

PowerPoint Presentation:

Hybrid Selection (Several markers are used ) Genetic complementation Phytotoxins Specific amino acid Auxin autotrophy Antibiotics Auxotrophic and metabolic mutants Chromosomal analysis Herbicides

Culture of the hybrid cells:

Culture of the hybrid cells Hybrid cells are cultured on suitable medium provided with the appropriate culture conditions.

Regeneration of hybrid plants :

Regeneration of hybrid plants Plants are induced to regenerate from hybrid calli These hybrid plants must be at least partially fertile, in addition to having some useful property, to be of any use in breeding schemes.

Advantages of somatic hybridization:

Advantages of somatic hybridization Production of novel interspecific and intergenic hybrid Pomato (Hybrid of potato and tomato) Production of fertile diploids and polypoids from sexually sterile haploids, triploids and aneuploids Transfer gene for disease resistance, abiotic stress resistance, herbicide resistance and many other quality characters

Advantages of somatic hybridization:

Advantages of somatic hybridization Production of heterozygous lines in the single species which cannot be propagated by vegetative means Studies on the fate of plasma genes Production of unique hybrids of nucleus and cytoplasm

Limitations of Somatic hybridization:

Limitations of Somatic hybridization Poor regeneration of hybrid plants Non-viability of fused products Not successful in all plants. Production of unfavorable hybrids Lack of an efficient method for selection of hybrids No confirmation of expression of particular trait in somatic hybrids

Thank You:

Thank You

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