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Isolation of Protoplast
Protoplast Culture, Somatic Commercially Available
Hybrdization and Somatic Enzymatic Method of Isolation of Protoplast - The Enzymes used for Protoplast
Cybridization enzymatic method is almost invariably used now for the Isolation
isolation of protoplasts, since it gives large quantities of
Mechanical Method of Isolation Culture of Protoplasts
of Protoplast protoplasts, where cells are not broken and osmotic
shrinkage is minimum. However, sometimes mechanical
Enzymatic Method of
and enzymatic methods are combined, where cells are
Isolation of Protoplast
first separated mechanically and later used for isolation
Advantages of Enzymatic
of protoplasts through enzymatic treatment.
Method of Isolation of
The protoplasts can be isolated from a variety of tissues
Direct One Step Method including leaves, roots, in vitro shoot cultures, callus, Culture Media
Sequential Two Step Method cell suspension and pollen. Young cell suspensions are Methods of Culture
Protoplast Culture and particularly ideal for isolation of protoplasts in large Fusion of Protoplasts
Regneration of Plants quantities. However, the most commonly used organs
Regeneration of Protoplast
are leaves which can be employed for isolation of
Isolation of Subprotoplasts
protoplasts using the following steps: Selection of Somatic Hybrids
Protoplast Fusion and Somatic and Cybrids
Biochemical Selection of
Spontaneous Protoplast Fusion Somatic Hybrids
(i) sterilization of leaves,
Induced Protoplast Fusion (ii) peeling off the epidermis, Complementary Selection of
(iii) enzymatic treatment and Somatic Hybridization
Somatic Hybridization and
Cybridization (iv) isolation and cleaning of protoplasts. Visual Selection of Somatic
Somatic Hybrids for Gene
Transfer Fully expanded leaves are obtained from about 10 weeks old plants (in tobacco) and are sterilized by first dipping Morphological Selection of
them into 70% ethyl alcohol for about a minute and then treating them with 2% solution of sodium hypochlorite Somatic Hybrids
Asymmetry in Somatic Hybrids
for Gene Transfer fur 20-30 minutes. The leaves are then rinsed three times with sterile distilled water and subsequent operations Flow Cytometry and Sorting
are carried out under aseptic conditions (under "laminar air flow chamber'). Selection of Somatic Hybrids
Somatic Hybrids for
Cytoplasmic Male Sterility Assessment of Somatic Hybrids
and Cybrids
Cytoplasmic Hybrids OR Cybrids
The lower epidermis of the sterilized leaves is carefully Application of Somatic
Gene Modification OR
peeled off and the stripped leaves are cut into small Hybridization and Cybridization
Transformation of Protoplasts
pieces. The peeling operation is easy, if the water supply
Purification of Isolated
Protocol for Isolation of is limited before excision or if leaves are allowed to Protoplasts
Protoplasts From Leaf Cells by become flaccid after sterilization. Mesophyll protoplasts
simultaneous Method Viability and Plating Density of
can be obtained from these peeled leaf segments, while
Protocol for Isolation of those for epidermis are obtained from the peeled
Protoplasts from Mesophyll epidermis. Selection of Fused Protoplasts
Cells of Cereals
Chromosome Status of Somatic
Protocol for Isolation of In cereals, where it is difficult to peel off the epidermis, Hybrids
Protoplasts from Root Nodules leaves are cut in long strips and used with enzyme List of Intergenic Hybrids
mixture. When the starting material is an in vitro shoot Produced through Protoplast
culture, the sterilization step can be omitted and leaves Fusion
can be cut into pieces under aseptic condition and used List of Interspecific Hybrids
directly for enzymatic treatment. Produced Through Protoplast
Intertribal Somatic Hybrids
From the peeled leaf segments, obtained as above, the protoplasts can be isolated using anyone of the two Produced within the Family

(i) direct (one step) method, in which treatment with macerozyme (or pectinase) and cellulase is done
simultaneously, or
(ii) sequential (two step) method, in which cells i are first isolated using macerozyme and cells are then treated
with cellulase to isolate protoplasts. In both these methods, peeled leaf segments are placed with their lower
surface downwards in a Petri dish containing the enzyme mixture.

The enzyme mixture in direct method consists of 0.5%

macerozyme + 2% cellulase in 13% sorbitol or
mannitol at pH 5.4, while in sequential (or two step)
method, two enzyme mixtures ('mixture A' and
'mixture. B'; see later) are used one after the other.

While the yield of protoplasts is higher in one step

method (since both palisade and spongy mesophyll
tissues are used in this method, while only palisade is
used in two step method), the protoplasts isolated by
two step method are better for culture studies.

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Enzymatic Method of Isolation of Protoplast,Enzymatic Treatment,Direct ... http://www.molecular-plant-biotechnology.info/protoplast-culture-somati...

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