PROTOPLAST CULTURE first proposed by E.c clocking in 1960. The protoplast isolate by following by method—[A] Isolation of protoplast. [B]Fusion of protoplast.
(1) Isolation of protoplast:- the isolation of protoplast can be completed as following heading—
(i)By enzymatic method:- The enzymatic method is almost in variabling use how for the isolation of protoplast. Since it is large quantities of protoplast where cell are not broken & somatic srinking in minimum. However some machanical & enzymatic method are combined where cell are 1st seperate machenically & later use for isolation of protoplast through enzymatic treatment. The protoplast can be isolated from a variety of tissue including leaves ,root,in vitro shoot culture. Callus cells suspension are particularly leave ideal for isolation of protoplast in large quantity. However the most commonly used organ leaves which can be imploned for isolation of protoplast using following steps —
(1) Sterilization:-fully expended leaves are obtained from about 10weak old plant and are sterilize by 1st deeking then into 70% ethyl alcohol for about few minutes and then treating them with 21%soluyion of NaHcl for 20-30mints.
(2)Peeling of epidermis:-the lower epidermis of proof is carefully peeds of and the leaves are cut into small piaces.
(3) Enzymatic treatment:- The leaves are cut into small pieces ,place in petridisc containing enzymatic solvent . Now material is anculated 25⁰ for 6-8hour in dark protoplast are releaseby squizing the leaves pieces.
(4) Isolation of & cleaning of protoplast:- After isolation the enzymatic solution containing protoplast are felter the feltared is centrifuge at low speed (50-100round) finally a fresh protoplast suspension is obtain.
[B]Fussion of isolated protoplast:- The fusion of protoplast may be two types —
(1) Spontaneous fussion:- (i) Protoplast may be fused at the time enzymatic digration of the cell wall,(ii) Fussion Product are k/a Heterokaryons.(iii) Induce fussion— induce Fussion two types –
(i) CHEMICALLY:- Various chemicals are used to provide positive and negative charge in wide range poly ethylene glycol alcohol etc. Poly ethylene glycol is most suitable Fussion bcoz isthe replication high frequency of hetero low toxicity.
(ii) PHYSICALLY:- it is also known as electron Fussion the protoplast when induced in to a high frequency ulternative field between parrelar. The protoplast act as elicolor and are attracted to each other like this phenomenon is known as electro forces.
Importance of protoplast culture:-
Protoplast culture is an important technique used in plant biotechnology to study and manipulate plant cells. Protoplasts are plant cells that have had their cell walls removed, leaving only the plasma membrane and the cytoplasm. Protoplast culture allows researchers to study the behavior of plant cells in a controlled environment without the influence of the cell wall.
Here are some of the key importance of protoplast culture:
Genetic transformation:-Protoplast culture is used for genetic engineering of plants, allowing the introduction of foreign genes into the plant genome. Protoplasts can be transformed with foreign DNA using various methods such as electroporation, polyethylene glycol (PEG)-mediated transformation, and biolistic bombardment.
Cell fusion:-Protoplasts from different plant species or cultivars can be fused to create hybrid cells. This technique can be used to produce new plant varieties with desirable characteristics, such as disease resistance or improved yield.
Somatic hybridization:- Protoplasts from different plant species can also be fused to produce somatic hybrids. Somatic hybrids are valuable tools for studying the genetics of plants and for developing new crop varieties.
Secondary Metabolite Production:- Protoplast culture can be used to produce secondary metabolites such as alkaloids, flavonoids, and terpenoids. These metabolites have numerous industrial and medicinal applications.
Plant Regeneration:-Protoplast culture can also be used to regenerate plants from single cells. This technique is useful for the production of clonal plants with specific traits and for the rapid propagation of rare or endangered plant species.
Overall, protoplast culture is a powerful tool for plant biotechnology research and has numerous applications in plant breeding, genetic engineering, and plant secondary metabolite production.