Somatic hybridization and its applications

PRESENTED BY: Pawan NagarReg. no.: 04-2690-2015M.Sc.(Fruit Science)

SOMATIC HYBRIDIZATION

Protoplast also known as a naked plant cell refers to all the components of plant cell excluding the cell wall.

Development of hybrid plants through the fusion of somatic protoplasts of two different plant species/varieties is called somatic hybridization, and such hybrids are known as somatic hybrids.

Somatic hybridization:

Protoplast:

History Hanstein introduced the term ‘Protoplast’.

The isolation of protoplasts from was first achieved through by Klercker (1892) on plasmolysed cells.

Cooking (1960) for the first time isolated the protoplasts of plant tissues by using cell wall degrading enzymes viz., cellulase, hemicellulase, pectinase, and protease extracted from fungus Trichoderma viride and Myrothecium verrucaria.

First achievement in protoplast fusion by Power (1970)

Somatic hybridization technique

1. isolation of protoplast

2. Fusion of the protoplasts of desired species/varieties

3. Identification and selection of somatic hybrid cells

4. Protoplast culture and regeneration

1. Isolation of protoplast

A. Mechanical method B. Enzymatic method

A. Mechanical Method

Plant Tissue

Collection of protoplasm

Cells Plasmolysis

Microscope Observation of cells

Cutting cell wall with knife Release of protoplasm

Mechanical disruption Experimental cells are allowed to plasmolyse by

keeping them in hypertonic solution.

In plasmolysed state, cell wall is cut with a sharp knife.

Plasmolysed cell is transferred to hypotonic solution.

This results in the release of protoplast in outer solution through cut ends.

This method is suitable only for tissues with large cells in which evident plasmolysis occurs.

Limitation

Used for vacuolated cells like onion bulb scale, radish and beet root tissues

Low yield of protoplast

Laborious and tedious process

Low protoplast viability

B. Enzymatic MethodLeaf sterlization, removal of

epidermis

Plasmolysed cellsPlasmolysed cells

Pectinase +Cellulase

Pectinase

Protoplasm released Release of isolated cells

Cellulase

Isolated Protoplasm Protoplasm released

Surface sterilization of leaf sample

Rinsing in suitable plasmolyticum with distilled water

Peeling of off the lower epidermis towards margin with sharp forceps below the junction of a lateral vein and midrib.

Enzymatic treatment

Purification of isolated protoplasts.

Enzymatic protoplast isolation steps

Enzymatic dissolution Cell walls are dissolved by enzymes.

Such enzymes are extracted from fungi, bacteria Macerozyme, a pectinase enzyme, from Rhizopus fungus,

Driselase a mixture of cellulase and pectinase, from Trichoderma viride.

Pectinase breaks the tissues into cells by dissolving calcium pectate of middle lamella.

Hemicellulase and cellulase break down the cell wall.

Commercially available enzymes are "Pectolyase Y-23", Onozuka R-1O.

Protoplast using enzymes may be isolated by sequential method or mixed enzyme method.

In the first process two enzymes-pectinase and cellulase are used sequentially, while in the second process two enzymes are used simultaneously.

The enzyme mixture macerates the cells and simultaneously destroys their walls.

Sequential method is useful in isolating protoplasts from palisade layer. While mixture enzyme method is useful in isolating protoplasts from spongy parenchyma and upper epidermis.

Advantages

Used for variety of tissues and organs including leaves, petioles, fruits, roots, coleoptiles, hypocotyls, stem, shoot apices, embryo microspores

Mesophyll tissue - most suitable source

High yield of protoplast

Easy to perform

More protoplast viability

Protoplast purification Enzyme solutions are filtered with nylon mesh to remove

insoluble impurities.

Filtrate is centrifuged for 5 minutes at 700 rpm.

The protoplast forms pellet and goes at the bottom of contrifuge tube.

Supernatant is removed with Pasteur pipett.

The pellet at the base is suspended in 10 ml of MS medium plus mannitol and the process is repeated thrice.

The resultant protoplast is pure.

2. Protoplast Fusion

A. Spontaneous fusion B. Induced fusion

Intraspecific Intergeneric ElectrofusionMechanical fusionChemofusion

A. Spontaneous fusion

Protoplast fuse spontaneously during isolation process mainly due to physical contact

Intraspecific

Intergeneric

Intraspecific protoplast fusion Intraspecific protoplast fusion is the cross between the

same species This technique offers the only way of carrying out

crosses  and genetic analysis.

Interspecific protoplast fusion Interspecific protoplast fusion is the crosses between

two different species. Interspecific protoplast fusions are of much importance

in the area where new products are to be produced. Due to new genetic set up many noval secondary

metabolites such as, antibiotics may be produced.

B. Induced fusion fusion induced by chemicals

1. PEG2. NaNo3

3. Ca 2+ ions4. Polyvinyl alcohal

Physical fusion of protoplasts under microscope by using micromanipulator and perfusion micropipette.

Fusion induced by electrical stimulation Fusion of protoplasts of pearl chain is induced by the

application of high strength electric field (100kv m-1) for few microsec.

Chemofusion:

Mechanicalfusion:

Electrofusion:

Fig. 1: A schematic representation of the three most successful protoplast fusion strategies

Fig. 2: Two tobacco plant protoplast are fused to produce a cell that acquires some of the characteristics of both parents

3. Identification and Selection Hybrid identification- Based on difference between the

parental cells and hybrid cell with respect to i. Pigmentation

ii. Cytoplasmic markers Fluorochromes like FITC (fluoroscein

isothiocyanate) and RITC (Rhodamine isothiocyanate) are used for labelling of hybrid cells

iii. Presence of chloroplast

iv. Nuclear staining Heterokaryon is stained by carbol-fuschin,

aceto-carmine or aceto-orcein stain

v. Several markers are used Genetic complementation Phytotoxins Specific amino acid Auxin autotrophy Antibiotics Auxotrophic and metabolic mutants Chromosomal analysis Herbicides

4. Protoplast culture and regeneration Plants are induced to regenerate from hybrid calli. Hybrid cells are cultured on sterile and cooled down

nutrient medium in petri dishes. The plates are incubated at 25°C in a dim white light. The protoplasts regenerate a cell wall, undergo cell

division and form callus. The callus can also be subcultured.

Embryogenesis begins from callus when it is placed on nutrient medium lacking mannitol and auxin. The embryo develops into seedlings and finally mature plants.

These hybrid plants must be at least partially fertile, in addition to having some useful property, to be of any use in breeding schemes.

Advantages of somatic hybridization Production of novel interspecific and intergenic

hybrid e.g. Pomato (Hybrid of potato and tomato) Production of fertile diploids and polypoids from

sexually sterile haploids, triploids and aneuploids Transfer gene for disease resistance, abiotic stress

resistance, herbicide resistance and many other quality characters

Production of heterozygous lines in the single species Studies on the fate of plasma genes Production of unique hybrids of nucleus and

cytoplasm

Limitations of Somatic hybridization

Poor regeneration of hybrid plants Non-viability of fused products Not successful in all plants Production of unfavorable hybrids Lack of an efficient method for selection

of hybrids No confirmation of expression of

particular trait in somatic hybrids

Application of Somatic hybridization Protoplast fusion to create somatic hybrids "wide crosses" where embryo culture won't work

i. Citopsis gilletiana (wild) x Citrus sinensisii. citrus sexually incompatible spp.iii. wild relative has disease/nematode resistanceiv. somatic hybrid used as a rootstock

Solanum somatic hybridsi. S. tuberosum dihaploids fused with wild diploid S.

chacoense resulting somatic hybrid (4n) is backcrossed to S. tuberosum cultivars (also 4n) overcomes sterility due to ploidy differences between somatic and sexual hybrids

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