Protoplast culture: Isolation, purification, culture and fusion; Cybrids and hybrids

Somatic Hybridization:- The process of obtaining hybrid plants by fusion of protoplasts of two different species or varieties, is called somatic hybridization. It has 4 main steps -

1. Protoplast Isolation

2. Protoplast Fusion

3. Selection of Hybrid Cells

4. Culturing of Hybrid Cells

1. Protoplast Isolation:- It has 2 main steps -

a. Sterilization of Leaf

b. Enzyme Treatment

a. Sterilization of Leaf:- Soak the leaf in 70% ethanol for 1 minute. After this, keep this leaf in 2% NaOCl solution for 20 to 30 minutes. Now wash this leaf with distilled water for 3 minutes.

b. Enzyme Treatment:- Now this sterilized leaf is treated with 2 enzymes, pectinase and cellulase, respectively. The pectinase enzyme disintegrates the middle lamella. Cellulase enzymes decompose the cell wall. As a result protoplasts are obtained. To increase osmotic concentration, add 500 - 800 ml / L of sorbitol or mannitol.

2. Protoplast Fusion:- For this, 2 different protoplast samples are mixed together. Now Fusogen is added to this mixture which induces protoplast fusion.

One of the following factors can be used as Fusogen -

i. PEG (Poly Ethylene Glycol)

ii. High pH + Ca²⁺

iii. NaNO₃

iv. Electric Pulse

At the end of this step, 3 types of products are obtained -

i. Homokaryon

ii. Heterokaryon

iii. Unfused protoplasts

3. Selection of Hybrid Cells:- It has several methods -

i. Visual markers

ii. Staining of protoplast with fluorescent dyes

iii. Culture, Regeneration and Identification

iv. Red anthocynin marker and green protoplast marker:-

Tobacco (green)  X   Carrot (red)

v. Selective markers:-

Grape   X    Soybean

4. Culturing of Hybrid Cells:-

Ø  Isolated hybrid protoplast cells are proliferated on solid MS-medium in patridishes.

Ø  Osmaticum:- It is a substance that acts to increase the osmotic concentration. It is added to both enzyme mixture and culture medium. The protoplast requires osmotic protection during isolation and culture because the cell wall has not yet been formed. One of the following can be used as osmaticum -

i. Sorbitol

ii. Manitol

iii. Glucose

iv. Sucrose

Ø  Cover the patridish with a lid and seal it with paraffin wax so that the medium does not dry at 40ºC.

Ø  Now transfer this patridish to the incubator where the temperature is 25 - 28ºC and the light intensity is 2300 lux.

Ø  Cell wall formation starts after a few hours. The first cell division occurs after 2–7 days.

Ø  Multicellular groups are formed after 2–3 weeks.

Ø  Now these cellular groups are transferred to a medium free of osmaticum so that callus or embryo can develop.

Ø  Further differentiation leads to organogenesis and development of hybrid plants.

Types of Hybrids:- There are 3 types of cellular hybrids -

1. Symmetric Hybrids

2. Asymmetric Hybrids

3. Cybrids

1. Symmetric Hybrids:- When the genomes of both species are kept in the cells during mitosis of the hybrid cell, the plants thus developed are called symmetric hybrids.

Example:- Pomato (Potato X Tomato)

2. Asymmetric Hybrids:- When the genome of a single species is extinct from cells during mitosis, the plants thus developed are called asymmetric hybrids.

Example:- Tobacco X Carrot

3. Cybrids:- When nucleic and cytoplasm from one species and only cytoplasm from another species come during somatic hybrids, the hybrids thus formed are called cybrids.