Enzymatic Method of Isolation of Protoplast,Enzymatic Treatment,Direct ...
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Protoplast Culture, Somatic
Hybrdization and Somatic
Cybridization
Mechanical Method of Isolation
of Protoplast
Enzymatic Method of
Isolation of Protoplast
Advantages of Enzymatic
Method of Isolation of
Protoplasts
Direct One Step Method
Sequential Two Step Method
Protoplast Culture and
Regneration of Plants
Isolation of Subprotoplasts
Protoplast Fusion and Somatic
Hybridization
Spontaneous Protoplast Fusion
Induced Protoplast Fusion
Somatic Hybridization and
Cybridization
Somatic Hybrids for Gene
Transfer
Asymmetry in Somatic Hybrids
for Gene Transfer
Somatic Hybrids for
Cytoplasmic Male Sterility
Cytoplasmic Hybrids OR Cybrids
Gene Modification OR
Transformation of Protoplasts
Protocol for Isolation of
Protoplasts From Leaf Cells by
simultaneous Method
Protocol for Isolation of
Protoplasts from Mesophyll
Cells of Cereals
Protocol for Isolation of
Protoplasts from Root Nodules
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Isolation of Protoplast
Commercially Available
Enzymatic Method of Isolation of Protoplast - The Enzymes used for Protoplast
enzymatic method is almost invariably used now for the Isolation
isolation of protoplasts, since it gives large quantities of
Culture of Protoplasts
protoplasts, where cells are not broken and osmotic
shrinkage is minimum. However, sometimes mechanical
and enzymatic methods are combined, where cells are
first separated mechanically and later used for isolation
of protoplasts through enzymatic treatment.
The protoplasts can be isolated from a variety of tissues
including leaves, roots, in vitro shoot cultures, callus, Culture Media
cell suspension and pollen. Young cell suspensions are Methods of Culture
particularly ideal for isolation of protoplasts in large Fusion of Protoplasts
quantities. However, the most commonly used organs
Regeneration of Protoplast
are leaves which can be employed for isolation of
protoplasts using the following steps: Selection of Somatic Hybrids
and Cybrids
Biochemical Selection of
Somatic Hybrids
(i) sterilization of leaves,
(ii) peeling off the epidermis, Complementary Selection of
(iii) enzymatic treatment and Somatic Hybridization
(iv) isolation and cleaning of protoplasts. Visual Selection of Somatic
Hybrids
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
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
Assessment of Somatic Hybrids
and Cybrids
The lower epidermis of the sterilized leaves is carefully Application of Somatic
peeled off and the stripped leaves are cut into small Hybridization and Cybridization
pieces. The peeling operation is easy, if the water supply
Purification of Isolated
is limited before excision or if leaves are allowed to Protoplasts
become flaccid after sterilization. Mesophyll protoplasts
Viability and Plating Density of
can be obtained from these peeled leaf segments, while
Protoplasts
those for epidermis are obtained from the peeled
epidermis. Selection of Fused Protoplasts
Chromosome Status of Somatic
In cereals, where it is difficult to peel off the epidermis, Hybrids
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
Fusion
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
Brassicaceae
methods:
(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.
6/1/2011 12:22 AM
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