Isolation and Fusion of Protoplast

Isolation and Fusion of Protoplast

Isolation and Fusion of Protoplast

Objective

At the end of the lecture, the student will be able to:

• Discuss the methods of protoplast isolation, culture and fusion

Isolation and Fusion of Protoplast

Isolation of Protoplast

General Introduction:

Protoplast is a cell without cell wall/rigid cell wall/cells from which cell wall has been removed

Isolation and fusion of protoplast is one of the significant development in tissue culture field

Recent development is regeneration of whole plant  from protoplasts

Mechanical method of Isolation of Protoplast:

• Crude method

• Cells are placed in a plasmolyticum solution

• Cells are then cut with a fine knife, there by protoplasts are released

• Used only for cells with large vacuoles like onion bulb, radish etc

• Not suitable for cells with small vacuoles

• Poor yield of protoplasts

Enzymatic method of Isolation of Protoplast:

• Widely used method

• Used for variety of tissues and organs including leaves, roots, stems, petioles, fruits, shoots etc

• Mesophyll tissue – most suitable source

• High yield of protoplast

• Easy to perform

• More protoplast viability

i. Sterilization of leaves

Fully expanded leaves

70% alcohol for 1 min

Sodium hypochlorite, 10-30 min

Wash with sterile distilled water thrice

ii. Peeling of epidermis

It is done using forceps

Lower epidermis is peeled off

Leaves are cut into small segments

Mesophyll protoplasts are released from segments

Epidermal protoplasts from epidermis

iii. Enzymatic treatment

• Cell wall degrading enzymes are used

a. Direct (one-step method)

Leaf segments are incubated with enzyme mixture A (0.5% macerozyme + 2% cellulase in 13% mannitol, pH 5.4), 15-18 hrs

Filtered using nylon mesh in order to remove cell debris /cell organelles

Centrifuged at 100 g for 1 min, Protoplast forms a pellet and settled

Washed with 13% mannitol and 20% sucrose

Centrifuged again at 200g for 5 min

Protoplasts float and can be pipeted out

b. Sequential method (Two step method)

Leaf segments are infiltrated with enzyme mixture A (0.5% macerozyme + 0.3% potassium dextran, pH 5.8), 15 min

Replaced by fresh enzyme mixture and heated on water bath for 1 hr

Filtered through nylon mesh and centrifuged at 100g for 1 min, protoplasts form pellets

Cells are mixed with enzyme mixture B (2% cellulase in 13% mannitol) incubated at 60 0C for 90 min

Filtered and centrifuged at 100 g for 1 min, repeat the same procedure as in direct method

iv. Purification

a. Sedimentation and washing

Protoplasts are subjected to slow centrifugation at 50 g for 5 min

Allows protoplast to sediment and then washed

b. Flotation method

Gradients are used

Subjected to centrifugation, protoplasts float

A solution of mannitol, sorbitol and sucrose (0.3- 0.6 M) used as gradients

Protoplast Culture

Isolated protoplasts can be cultured in a suitable nutrient medium to regenerate cell wall and callus

Optimal culture conditions are

          – Optimal auxin to cytokinin ratio

          – Maintenance of osmotic pressure

          – Temp – 20-28 0C, pH – 5.5-5.9

          – NAA and BAP

Cell wall regenerated within 5-7 days followed by microscopic colonies

Formation of cell wall – stain with calciflour white stain- green fluorescence

Spontaneous fusion:

Occurs naturally

During isolation, protoplasts lying in close proximity undergo fusion themselves  à Homokaryons or homokaryocytes

Mechanically can also be induced by bringing protoplasts close by using micro manipulator

Induced fusion:

Induced artificially – fusogens

Induced by using certain chemicals like

       Sodium nitrate

      – Calcium ions

      – PEG

a. Treatment with sodium nitrate

Isolated protoplasts are suspended in 10% NaNO3

Incubated at 35 0C for 30 min

To obtain higher frequency, protoplasts can be centrifuged and subjected to 2 more cycles

b. Treatment with Ca++

Isolated protoplasts are suspended in 0.1 M Calcium chloride in 0.4 M mannitol, pH 10.5

Centrifuged at 50 g for 30 min, incubated on water bath for 30 min  

c. Treatment with PEG

I Method:  

When protoplasts are available in sufficient quantity, 1 ml of medium containing protoplast is mixed with 1 ml of 55% PEG, incubated at 35 0C for 30 min

II Method:

When available in small quantity, 4-8 µl is taken in petridish and allowed to settle and added 2-3 µl of PEG, incubated at 35 0C for 30 min  

Electro fusion:

Mild electrical current is used

Two glass micro electrode are placed in contact with protoplasts

Apply of electric field of low strength leads to pearl chain arrangement of protoplasts

Subsequent application of high strength leads to fusion

Electro fusion, Electrofusion,

Summary

Protoplast – Cell without a cell wall

Isolation of protoplast – Mechanical method and enzymatic method

Enzymatic method – Direct method and sequential method

Cell wall degrading enzymes – Cellulase, pectinase, macerozyme etc

Purification by washing and sedimentation method and flotation method

Protoplast culture – regeneration of cell wall on a suitable media

Protoplast fusion – Spontaneous and induced fusion

                   Dietary Supplement and Nutraceuticals 

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