Protoplast fusion

Protoplast is a naked cell (without cell wall) surrounded by a plasma membrane. It can regenerate cell wall, grow and divide.
Spheroplast cells have their cell wall only partially removed.
Is fragile but can be cultured and grow into a whole plant.
Cells can originate from any type of tissue (Mesophyll tissue - most suitable source ).
Can be applied in somatic hybridization.
Can be applied in biotechnology and microbiology.
Somatic hybridization is the development of hybrid plants through the fusion of somatic protoplasts of two different plant species/ varieties.
Somatic Hybridization was firstly introduced by Carlson in Nicotiana
glauca.
In 1960, E.C Cocking contributed to the enzymatic isolation and culture of protoplast.

1. By Assist.prof Dr. Berciyal Golda . P VICAS P R O T OPLA S T FUSION

2. Introduction Protoplast is a naked cell (without cell wall) surrounded by a plasma membrane. It can regenerate cell wall, grow and divide. Spheroplast cells have their cell wall only partially removed. Is fragile but can be cultured and grow into a whole plant. Cells can originate from any type of tissue (Mesophyll tissue - most suitable source ). Can be applied in somatic hybridization. Can be applied in biotechnology and microbiology. Somatic hybridization is the development of hybrid plants through the fusion of somatic protoplasts of two different plant species/ varieties. Somatic Hybridization was firstly introduced by Carlson in Nicotiana glauca. In 1960, E.C Cocking contributed to the enzymatic isolation and culture of protoplast.

3. 1. Isolation of protoplast 2. Fusion of the protoplasts of desired species/varieties Or with a desired DNA 3. Identification and Selection of somatic hybrid cells 4. Culture of the hybrid cells 5. Regeneration of hybrid plants Steps of protoplast fusion

4. Protoplast isolation Refers to the separation of protoplast from plant tissue Important to isolate viable and uninjured protoplast as gently and as quickly as possible Involves two methods: Mechanical Enzymatic

5. Tissue is immersed in 1.0 M sucrose until protoplasm shrunk away from their enclosing cell wall (Plasmolysis). Plasmolysed tissue is cut with a sharp knife at such thickness that only cell walls are cut. Undamaged protoplast in strips are released by osmotic swelling when placed in a low concentration of sucrose solution

6. Used for vacuolated cells like onion bulb scale, radish and beet root tissues Low yield of protoplast Tedious process Low protoplast viability

7. Refers to the use of enzymes to dissolve the cell wall for releasing protoplasts. The plant cell wall is mainly composed of cellulose, hemicellulose and pectin which are respectively degraded by the enzymes cellulase, hemicellulase and pectinase. In plant cells we mainly uses these enzymes (cellulase, hemicellulase and pectinase). Advantages: Used for variety of tissues and organs such as fruits, roots, petioles, leaves… Osmotic shrinkage is minimum Cells remain intact and not injured High yield of protoplast Easy to perform More protoplast viability .

8. Leaf sterilization, removal of epidermis Plasmolysed cells Plasmolysed cells P e c ti nase +c ell u la se P e c ti nase Protoplasm released Release of isolated cells c e llulas e Protoplasm released Isolated Protoplasm

9. Procedure Incubation of leaf segments overnight in enzyme solution at pH 4.5-6.0 & temperature 25-30 C . Mixture is filtered and centrifuged Protoplast forms pellet Then washed with sorbitol and re-centrifuged Clean protoplasts float They are pipetted out

10. Purification of protoplast Protoplasts are purified by removing: Undigested material (debris) Bursts protoplasts Enzymes Debris are removed by filtering the preparation through a nylon mesh Enzymes are removed by centrifugation whereby the protoplasts settle to the bottom of the tube and the supernatant removed with the help of a pipette Intact protoplasts are separated from broken protoplasts through centrifugation and removed by a pipette as they are collected at the top of tube

11. Purification of protoplast

12. Protoplast Culture Isolated protoplast can be cultured in an appropriate medium to reform cell wall and generate callus Optimal culture conditions: Optimal density to the culture. Optimal auxin to cytokinin ratio, glucose and sucrose. Maintain osmoprotectant in the medium 4. Temperature: 20-28°C, pH: 5.5-5.9.

13. Culture of protoplasts Protoplasts cultured in suitable nutrient media first generate a new cell wall The formation of a complete cell with a wall is followed by an increase in size, number of cell organelles, and induction of cell division The first cell division may occur within 2 to 7 days of culture resulting in small clumps of cell, also known as micro colony, within 1 to 3 weeks From such clumps, there are two routes to generate a complete plant (depending on the species) Plants are regenerated through organogenesis from callus masses ( Micropropagation) The micro calli can be made to develop into somatic embryos ( somatic embryogenesis ), which are then converted into whole plant through germination

14. Importance of Protoplast Culture (without fusion) Gene Transfer Biological examinations Study of Osmotic behavior Study of Plasma lemma Study of Cell wall formation Organelle isolation Study of Morphogenesis Virus uptake and replication Study of photosynthesis

15. Factors affecting protoplast culture Plant species and varieties Small genetic difference leads to varying protoplast responses to culture conditions Plant age and organ Age of donor plant and its developmental stage (smaller better) Pre-culture conditions Climatic factors affect the yield of protoplast and response when cultured Pre-treatment to the tissue before isolating protoplasts Cold treatment, plasmolysis and hormone increases the chance of recovery of viable protoplasts and their plating efficiency Density It influences plating efficiency and surviving of protoplasts. At higher density, protoplasts compete with one another while at lower density losses of metabolites from protoplasts is more.

16. Protoplast Fusion (Somatic hybridization) Protoplast fusion techniques: Electrofusion Polyethene glycol - induced fusion (PEG) High Ca2+ , high pH NaNO3 treatment Mechanical fusion

17. FUSION PRODUCTS - THE HYBRIDS AND CYBRIDS . The nuclei of two protoplasts may or may not fuse together even after fusion of cytoplasms. The binucleate cells are known as heterocyte . When nuclei of two different sources are fused the cells are known as hybrid. Only cytoplasms fuse and genetic information from one of the two nuclei is lost is known as cybrid i.e. cytoplasmic hybrid. Some of the protoplasts of the same type may undergo fusion to produce homocytes each with 2-40 nuclei.

18. Electrical fusion If P r o t opl a s t s are p l aced in t o a sm a ll c u lture v es s el containing electrodes and a potential difference is applied, then the protoplasts will line up between the electrodes. If now an extremely short, electric shock is applied, protoplasts can be induced to fuse.

19. PEG (Polyethylene glycol) Fusion It has a high molecular weight about 1500-6000. Usually a PEG solution of about 28-50% is used for protoplast fusion. This polymer binds to the lipid membrane of cells and thus induces fusion Fusion takes place for 45 min in incubation .

20. Mechanical fusion In this the isolated protoplast are brought i nto inti m a t e contact mechanically. physical Under and using or perfusion microscope micr o mani pu lat o r micropipette.

21. Hybrid identification- Based on difference between the parental cells and hybrid cell with respect to Pigmentation Cytoplasmic markers Fluorochromes like FITC (fluoroscein isothiocyanate) and RITC (Rhodamine isothiocyanate) are used for labelling of hybrid cells Presence of chloroplast Nuclear staining Heterokaryon is stained by carbol-fuschin, aceto-carmine or aceto- orcein stain Regeneration Plants are induced to regenerate from hybrid calli. These hybrid plants must be at least partially fertile, in addition to having some useful property, to be of any use in breeding schemes.

22. Application of Protoplast Protoplasts can be used: In the production of Cybrid For Somatic Hybridization to overcome sexually incompatible species Ingesting “Foreign” material into cytoplasm For DNA transformation Used to study wall synthesis and decomposition Studied as Single Cell System

23. Production of Cybrid Cybrid contain nuclear and cytoplasmic genome of one parent and only the cytoplasmic genome of the second .

24. Ingesting “Foreign” material into cytoplasm Protoplast being wall-less show high pinocytic activity and can ingest biological active foreign bodies such as DNA, plasmids, bacteria , viruses etc. results into modified cells. Advantageous to plant breeder in getting more efficient crop varieties in near future.

25. Somatic hybridization Fusion of protoplast that facilitates the mixing of 2 whole genomes and could be exploited in crosses at: intergeneric, interkingdom and interspecific levels Somatic hybridization is used to produce hybrids from sexually incompatible species. This method could also be used to study selection procedures.

26. Advantages of Protoplast fusion It facilitates the mixing of two genomes and can be used in crosses at interspecific, intergeneric or even intraspecific levels To create new strains with desired properties and for strain improvement Mixing two genomes opens the door to gene transfer and a study of gene expression, stability of several traits and cell genetic changes

27. Disadvantages of Protoplast Fusion During the mechanical method of isolation of protoplasts: It yields a very small amount of protoplasts after a rather tedious procedure It is not suitable for isolating protoplasts from meristematic and less vacuolated cells During and subsequent to digestion of the cell wall, the protoplast becomes very sensitive to osmotic stress. Thus, cell wall and protoplast storage must be done in an isotonic solution to prevent rupture of the plasma membrane.

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