Course: GEB 302

(Plant Cell and Tissue Culture)

Lecture: 03
Somatic hybridization and cybridization
Date : 22 November 2020
By Md Motiur Rahman
Questions:
1. What is plant protoplast?
2. What do you know about somatic hybridisation and Cybridisation?
3. Write down the methods of protoplast isolation.
4. Describe different processes of protoplast fusion.
Plasmodesmata (singular, plasmodesma)
microscopic channels of plants facilitating
transport and communication between
individual cells. It is microscopic channels
which traverse the cell walls of plant cells
enabling transport and communication
between them.

Somatic hybridization involving the following three stages :

1. Protoplast isolation
2. Protoplast fusion
3. Development of regenerated fertile plants from the fusion product
Isolation of Protoplast Leaf

Red onion
I. Protoplast Isolation
Protoplasts are isolated by
Mechanical Method
Enzymatic Method
1. Mechanical Method
Klercker in 1892 pioneered the isolation of protoplast by mechanical methods,
for which a small piece of epidermis from a plant is selected. The tissues/cells
are subjected to plasmolysis. This causes protoplasts to shrink away from the
cell walls. The tissue is then dissected to release the protoplasts.
Disadvantages
It is restricted to certain tissues which have large vacuolated cells.
Yield of protoplasts is generally very low.
The method is tedious and laborious.
Viability of protoplasts is low because of the presence of substances released
by damaged cells.
2. Enzymatic Method
Cocking in 1960 demonstrated the possibility of enzymatic isolation of protoplasts from higher
plants. Mechanical isolation method of protoplast are no more practically used. Protoplasts are
routinely isolated by treating tissues with the mixture of cell wall degrading enzymes in
solution, which contain osmotic stabilizer. The method involves the following steps.
Sterilization of leaves
Peeling off lower epidermis
Incubation in enzyme solution
Isolation and cleaning of the protoplast
The enzymes work to digest the cell wall, which has three primary components :
Cellulose, hemicelluloses and pectin substances.
The 1st two are the components of the primary and secondary structure of cell wall, while
pectin is a component of middle lamella that joins the cells.

Pectinase mainly degrades the middle lamella, while cellulase and hemicellulase are required
to digest the cellulosic and hemicellulose components of the cell wall, respectively.
It is important to combine one middle lamella dissolving and one cell wall digesting enzymes in
proper composition to achieve maximum protoplasts release from one gm material.
Following enzymes are used:
Macerozymes R-10, Cellulase- Onuzuka R-10, Hemicellulase, Pectinase, Drieselase, etc.

A combination of these enzymes in a concentration of 0.5 – 2 % is used. In many cases only

macerozyme and cellulose are sufficient to obtain protoplasts in significant number.
Protoplast fusion:
The following points highlight the two methods of protoplast fusion. They are:
(1) Spontaneous Fusion and (2) Induced Fusion.
(1) Spontaneous Fusion: Protoplasts during isolation often fuse spontaneously and this
phenomenon is called spontaneous fusion.
2. Induced Fusion:
Fusion of freely isolated protoplasts from different sources with the help of fusion inducing
chemical agents is known as induced fusion.
The isolated plant protoplasts can be induced to fuse by three ways:
Mechanical Fusion:
In this process, the isolated protoplasts are brought into intimate physical contact
mechanically under microscope using micromanipulator and perfusion micropipette.
Chemo-Fusion:
Several chemicals have been used to induce protoplast fusion. Sodium nitrate (NaN03),
polyethylene glycol (PEG), Calcium ions (Ca2+), Polyvinyl alcohol, etc. are the most commonly
used protoplast fusion-inducing agents which are commonly known as chemical fusogens.
Generally, chemo-fusion techniques are followed in most of induced fusion experiments.
Electro-Fusion:
Recently, mild electrical stimulation is being used to fuse protoplasts. This technique is known
as electro-fusion of protoplasts. Two glass capillary microelectrodes are placed in contact with
the protoplasts. An electrical field of low strength (10 kv m-1) gives rise to di-electrophoretic
dipole generation within the protoplast suspension.
……..next slide
 Some chemo-fusion methods :
(i) Fusion induced by Sodium or Potassium Nitrate (NaN03/ KN03)
(ii) Fusion induced by Calcium ions at high pH (Ca2+),
(iii) Fusion induced by PEG:
(iv) Fusion induced by Other Chemicals (like Polyvinyl alcohol)

Micromanipulator

Perfusion Micropipette
The somatic hybrid between Petunia parodii (2n =
14) and P. parviflora (2n = 18) is an aneuploid with
31 chromosomes. The missing chromosome was a
product of interchange between a large, satellited P.
parodii chromosome and a medium sized
chromosome from P. parviflora.
 Cybridization: The process of protoplast fusion
resulting in the development of cybrid is called as
Cybridization.

Hybridization :
The fusion between isolated somatic protoplasts under in vitro conditions and
subsequent development of their product to a hybrid plant is known as somatic
hybridization.
 Assignment
A somatic hybrid of Petunia parodii (2n = 14) and P.
parviflora (2n = 18) is an aneuploid with 31
chromosomes. Explain the matter of the missing
chromosome on the basis of the research article as
referred: White, J., and Rees, H. (1985). The
chromosome cytology of a somatic hybrid petunia.
Heredity, 55(1), 53-59.

Due date : 27 Nov 2020

1. What do you know about tissue culture?
2. Sketch out different techniques of plant tissue culture with their merits and
demerits.
3. Write down the application of plant tissue culture.
4. What are plant growth regulators?
5. Describe the different types of PGRs.
Or Describe the role and importance of the following PGRs:
Auxin, Cytokinin, Gibberellins, Ethylene, Abscisic acid

Material provided:
Plant Tissue Culture : An Introductory Text
Sant Saran Bhojwani
Prem Kumar Dantu