This presentation consists of information about protoplast , methods of isolation , reagents and their role

1. Submitted by ,
R. Yamuna,
2nd M.Sc. Biotechnology,
Annamalai University.
PROTOPLAST
ISOLATION AND
CULTURE

2. SYNOPSIS
i. What is a protoplast?
ii. What are the methods used ?
iii. Enzymatic method.
iv. Reagents and their role.
v. Procedure .
vi. Protoplast culture.

3. WHAT IS A PROTOPLAST ?
 Living part of the plant cell.
With cytoplasm and nucleus.
No cell wall.
Isolated from – roots , leaves,
tubers, root nodules, endosperm.
Convinent method – leaf.
Reason – mesophyll cells loosely
arranged.

4. WHAT ARE THE METHODS USED ?
 Mechanical method.
 Enzymatic method.
1.MECHANICAL METHOD – cutting / breaking the
cell wall.
2.ENZYMATIC METHOD – digestion of cell wall with
enzymes.

5. ENZYMATIC METHOD
Leaf
sterlization,
removal of
epidermis.
Plasmolyse
d cells
Release of
isolated
cells.
Isolated
protoplasm
Plasmolyse
d cells
NON SEQUNTIAL SEQUNTIAL
Protoplasm
released.
Protoplasm
released
Pectinase +
cellulase
Pectinase

6. REAGENTS AND THEIR ROLE
 70 % Ethanol – sterlization and washing
70% ethanol+30%water – easily penetrate into
cell . More polar – better osmosis.
 2% Sodium Hypochloride – disinfectant /
antimicrobial agent.
Mannitol – plasmolysis , hyperosmotic
environment . ( osmoticum )

7. Pectinase – breakdown protein – holding cells
together .
Cellulase – digest cellulose.
Sorbitol – plasmolysis , hyperosmotic
environment (osmoticum).
Sucrose – density gradient – centrifugation –
interface.

8. (cont.)
Potassium dextran sulphate – release DNA
from DNA histone, inhibits binding of RNA to
ribosomes.
Distilled water.
Centrifuge.
Micro pipittes.
Petri plates.

9. PROCEDURE
Surface sterlize (70%) and
Wash – 2% Na hypochloride
Peel the lower
epidermis – place in
petri dish – pectinase
Agitate – slowly . After 15-
20 minutes – replace the
enzyme mixture
0.5% macerozyme,0.3%
potassium dextran sulphate
in 13% mannitol

10. (cont.)
Incubate – 1 hr.
Filter the
isolated cells –
nylon membrane
Isolated
cells+enzyme
mixture
Centrifuge
100xg -1m
Centrifuge
100xg -1m
Decant the supernatant
and wash the pellet with
mannitol

11. Clean the
protoplast – 25%
sucrose
Sucrose+pellet+0
.4sorbitol+10mm
cacl2.
Sample - interface
Sample collected . Added to
salt solution – 0.2%w/v cacl2
and 2.5% Hcl
PROTOPLAST
Centrifuge at
low speed

12. PROTOPLAST CULTURE
 Nagata and takebe medium
CULTURE
TECHNIQUES

13. THANK YOU