1. T0PIC: PROTOPLAST CULTURE
MATIULLAH:11-ARID Ph.D ,BOTANY(1st semester).

2. PROTOPLAST CULTURE
SANA ULLAH

3. Protoplast
A protoplast is a plant, bacterial or fungal cell that had its cell wall completely or partially removed using either mechanical or enzymatic means

4. Why to remove the protoplast
Large molecules can not pass through the cell wall.
Somatic hybridization and genetic manipulation can not be done. So we have to remove the cell wall.
After removing it we get protoplasts and spheroplast. The term protoplast refers to the spherical shape assumed by Gram-positive bacteria while spheroplast refers to the spherical shapeassumed by gram-negative bacteria . So this represent the presence of single membrane in case of protoplast and two membrane in case of spheroplast.

5. Isolation of protoplast

6. 1.Mechanical Method
Klercker was the first to isolate protoplasts from plasmolysed cell.
Scales were immersed in hypertonic solution until the protoplast shrunk away from their enclosing wall.
The plasmolysed tissue was cut such a way that the thickness that only the cell walls are cut without damaging the protoplasts.
The protoplasts were released by placing the cells in hypotonic solution.

8. 2.Enzymatic Method Proposed by Cocking .
Used for variety of tissues and organs including leaves, petioles, fruits, roots, coleoptiles, hypocotyls, stem, shoot apices, embryo microspores.
The enzymatic method could be used as a one step method (direct method), or as a two-step method (sequential method).

9. In the one step method, protoplasts are isolated directly from the tissue by using two enzymes, cellulase and pectinase, simultaneously.
While, in the two-step method, cells are first isolated from callus or tissue by using pectinase and to this cell suspension cellulase is added to digest the cell wall and release protoplasts.

10. Enzymatic Method 11 Pectinase Protoplast released Release of
Leaf sterilization,
Removal of epidermis
Plasmolysed
cells
Plasmolysed
cells
Pectinase +cellulase
Pectinase
Protoplast released
Release of
isolated cells
Protoplast
released
cellulase
Isolated
Protoplast 11

11. The successful culture of protoplasts requires a pure population of intact and viable protoplasts at a high yield. So the protoplasts require to be purified by removing the undigested material (debris), burst protoplasts and enzymes.

12. Protoplast Viability
Fluorescein diacetate (FDA) solution in acetone is added to protoplasts suspension and after 5 minutes at room temperature the protoplasts are examined using fluorescent microscope. Only viable protoplasts can be seen.

13. Viable protoplasts

14. Protoplast Culture
Isolated protoplasts can be cultured in an appropriate medium to reform cell wall and generate callus.
Protoplast can be cultured in the following ways.
Hanging drop method
Agar plating method

15. 1.Hanging drop method
In this method, a suspension of protoplasts is placed as 50 µl drops in plastic Petri plates, sealed and incubated in an inverted position at 25-30°C.
After cell wall regeneration and the initiation of cell division, fresh medium is added to make cell suspension.
The small size of drop helps in providing enough aeration to protoplasts.

16. 2.Agar plating method
On a semi- solid medium using agarose plates is an effective method as it provides a supporting matrix for the protoplasts.
It provides growth factors that stimulates wall synthesis and cell division.

17. Thank
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