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

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