This document discusses protoplast isolation, culture, and fusion. It defines protoplasts as plant, fungal, or bacterial cells without cell walls. Protoplasts can be isolated from plant tissue through either mechanical or enzymatic methods, with enzymatic isolation being more common. The document outlines steps for protoplast isolation, purification, and assessing viability. It also discusses protoplast culture techniques and methods for inducing protoplast fusion, including electrofusion and PEG treatment. Applications of protoplast techniques in plant research include genetic engineering and crop breeding.

1. Guided By:
DR. PRANATI NAYAK
Submitted By:
SHRRADDHA SUMAN
+3 FINAL YEAR
ROLL NO.: BS-17-534
AUTO ROLL NO.:5201NA17040

2.  INTRODUCTION
 ISOLATION OF PROTOPLAST
 PURIFICATION OF PROTOPLAST
 ISOLATION OF SUB-PROTOPLAST
 VIABILITY OF PROTOPLAST
 PROTOPLAST CULTURE
 PROTOPLAST FUSION
 CYBRIDIZATION &SOMATIC HYBRIDIZATION
 APPLICATION
 CONCLUSION

3.  Protoplast can be a plant, fungal or bacterial cell without cell
wall but are bounded by plasma membrane.
 The cell wall can be completely or partially removed by
mechanical or enzymatic means.
 Protoplast have the capacity to regenerate cell wall, grow &
divide.

5.  Isolation of protoplast refers to the
separation of protoplast from the plant
tissue.
 Isolation of protoplast involves to methods:
a. Mechanical Method
b. Enzymatic Method

7. Mechanical method for protoplast isolation is no more in
use because of the following limitations:
Low yield of protoplast
Low protoplast viability
Laborious and tedious process
Only used for vacuolated cells like onion bulb scale,
radish, beetroot, etc.

8.  The enzymes that can digest the cell walls
are required in this process of protoplast
isolation.
 It involves two methods:
a) Direct/Simultaneous/One Step Method
b) Sequential/Two Step Method

9. Leaf sterilization, removal of
epidermis
Plasmolysed
cells
Protoplasm
released
Plasmolysed
cells
Release of
isolated
cells
Protoplasm
released
Isolated
protoplasm
Pectinase
+cellulase
Pectinase
Cellulase

11. •Debris are removed by filtering through a nylon mesh.
•Enzymes are removed by centrifugation, where the protoplasts settle to
the bottom of the tube and the supernatant float and are removed with
the help of a pipette.
• Broken protoplast are separated through centrifugation and are
removed by a pipette as they are collected at the top of the tube.

13. Viability of protoplast can be checked by
following methods:
 Fluorescein diacetate (FDA)dissolved
in acetone is used at a conc. Of 0.01%
& intact viable protoplast only fluoresce
when observed under UV.
 Phenosafranine staining where the
dead protoplasts turn red and viable
cells remain unstained.
 Evans blue: intact viable protoplasts
are impermeable to Evans blue.
 Cyclosis can be a measure of viability.

15. Protoplast can be cultured in following
ways:
Hanging drop method
In the wells of microtitre plates
On the semisolid medium using agarose
plates.

19.  Protoplast fusion can be classified in to two
categories as shown below:

21. It mainly occurs due to physical contact.

22. It can mainly categorized in to:
 Electro fusion
 PEG treatment
 Treatment with Ca++ ions at high pH

25. This method involves
centrifugation of protoplast in a
fusion inducing solution for 30
minutes, after this the tubes are
placed in a water bath for 40-50
minutes.

26. SOMATIC HYBRIDIZATION
Protoplast of different species can be fused to
generate a hybrid and the process is called as
somatic hybridization.

28. Protoplast culture has found its uses and
applications in advanced fields of plant research
such as genetic engineering, crop breeding, etc.
Protoplast can be used to study:
 Metabolic studies including photosynthesis
 For DNA transformation
 For somatic hybridization
 Ingesting foreign material into cytoplasm
 Single cell synthesis
 In crop breeding

29. The isolation, culture and fusion of protoplast is an
interesting field in plant research. The development
of protoplast system has led to the increase in
adaptability of plants. Protoplast isolation and their
culture provide millions of single cells for a variety
of studies and production of transgenic plants.
Transfer of useful genes are possible by protoplast
fusion. This opportunity will undoubtedly lead to
production of genetic variation thereby widening the
genetic base of plant breeding.