protoplast fusion protoplast culture and somaclonal variation.pptx

This presentation covers the basics of protoplast fusion, protoplast culture, and somaclonal variation. Learn how these biotechnological techniques are used to create hybrid plants and generate genetic diversity. Ideal for students and anyone interested in plant science and agriculture.

1. Protoplast fusion, protoplast culture, and somaclonal variation Presented by : Janak Singh Rawal and Puspa RC 8 th Semester GAASC, IAAS, Baitadi, Nepal

2. Protoplast The protoplast of a living plant or bacterial cell whose cell wall has been removed Protoplasts: Have their cell wall entirely removed Spheroplasts: Have their cell wall only partially removed Protoplasts are isolated cells whose cell wall is removed and are bounded by plasmalemma Protoplasts can be the cells of plants, fungi, or bacteria. These are also called 'naked' cells Protoplasm is the complex, semifluid, translucent substance that constitutes the living matter of plant and animal cells Cellulase enzymes digest the cellulose in plant cell walls while pectinase enzymes break down the pectin holding cells together Once the cell wall has been removed the resulting protoplast is spherical in shape

3. Protoplast are the cells from which cell walls are removed and the cytoplasmic membrane is the outermost layer in such cells History Term protoplast introduced in 1880 by Hanstein The first isolation of protoplast was achieved by Klercker in 1892 by using the mechanical method A real beginning in protoplast research was made by Cocking in 1960 who used an enzymatic method for cell wall removal Rakabe et.al (1971) were successful in achieving the regeneration of the whole tobacco plant from protoplasts

4. Protoplasm

5. Isolation of Protoplast Protoplast can be isolated from almost all plant parts i.e. roots, leaves, fruits, tubers, root nodules, endosperm, pollen mother cell, etc. Protoplasts are isolated from cells by two methods 1. Enzymatic method 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 use these enzymes (cellulase, hemicellulase, and pectinase) at pH 4.5-6.0 & temperature 25-30 o C with an incubation period of half an hour to 20 hrs 2. Mechanical method A small piece of epidermis from a plant is selected The cells are subjected to plasmolysis which results in the shrinking of protoplast away from cell walls The tissue is dissected to release the protoplast

6. Figure: Mechanical method Figure: Enzymatic method

7. Purification of protoplast 1. Sedimentation & washing: In this method, the crude protoplast suspension is centrifuged at low speed (50-100g for 5 min) The intact protoplasts form a pellet and supernatant containing cell debris can be pipetted off The pellet is gently resuspended in fresh culture media plus mannitol and rewashed This process is repeated two or three times to get a relatively clean protoplast preparation 2. Flotation: A concentrated solution of mannitol, Sorbitol, and sucrose (0.3-0.6M) can be used as a gradient, and crude protoplasts suspension may be centrifuged in this gradient at an appropriate speed Protoplasts being lighter (low density) than other cell debris allow the protoplasts to float and the cell debris to sediment Protoplasts can be pipetted off from the top of the tube after centrifugation

8. Protoplast Fusion Protoplast fusion is a physical phenomenon. During fusion, two or more protoplasts come in contact and adhere with one another either spontaneously or in the presence of fusion-inducing chemicals Somatic fusion, also called protoplast fusion, is a type of genetic modification in plants by which two distinct species of plants are fused to form a new hybrid plant with the characteristics of both, a somatic hybrid The basic principle of protoplasm culture is the aseptic isolation of a large number of intact living protoplast removing their cell wall and culturing them on a suitable nutrient medium for their growth and development

9. Figure: Process of protoplasm fusion

10. 1. Spontaneous fusion: Protoplasts, during isolation, often fuse spontaneously Simple physical contact is sufficient to bring about the spontaneous fusion among similar parental protoplasts Protoplasts from adjoining cells fuse through their plasmodesmata to form a multinucleate protoplast Spontaneous fusion is strictly intraspecific and gives rise to homokaryon Methods of protoplasm fusion

11. 2. Induced fusion It is the method of fusion of freely isolated protoplasts from different sources with the help of fusion-inducing chemical agents Normally, isolated protoplasts do not fuse because the surface of the isolated protoplast carries a negative charge Thus, there is a strong tendency for protoplasts to repel one another due to their same charges. So this type of fusion needs a fusion-inducing chemical agent, which reduces the electronegativity of the isolated protoplasts and allows them to fuse. Induced fusion is a highly important and valuable technique because the protoplast from widely different and sexually incompatible plants can be fused by this procedure This technique can combine different genotypes and the main objectives of somatic hybridization are mainly based on induced protoplast fusion.

12. Types of Induced fusion may be divided into three types: 1. Mechanical fusion: In this process, the isolated protoplasts are brought into physical contact mechanically under a microscope using a micromanipulator and micropipette. 2. Chemo fusion: In this process, the isolated protoplast is fused by the use of some chemical agent such as sodium nitrate, polyvinyl alcohol, polyethylene glycol, calcium ion, etc. Chemical fusogens cause the isolated protoplasts to adhere to one another and lead to tight agglutination by fusion of protoplast. The adhesion of isolated protoplast takes place either due to the reduction of negative charges of protoplast or due to the attraction of protoplast by electrostatic forces caused by chemical fusogens.

13. Fusogens Fusogens are agents that induce the fusion of protoplasts from different organisms Freshly isolated protoplasts can be induced to undergo fusion, with the help of a range of fusogens. Examples of Fusogens:- Sendai Virus – a parainfluenza virus (earlier used fusogen) Polyethylene glycol (PEG)- The most commonly used. Sodium nitrate High Ca2+, high pH Polyvinyl alcohol

14. Many fusion experiments are performed with a polyethylene glycol PEG induces protoplast aggregation and subsequent fusion However, the concentration and molecular weight of PEG are important concerning fusion A solution of 37.5% w/v PEG of molecular weight 1,540 or 6,000 aggregates mesophyll and cultured cell protoplasts during a 45-minute incubation period at room temperature In some studies, high pH/Ca2+ and PEG methods have been combined. The PEG method has been modified slightly to fuse higher plant protoplast PEG is more effective when it is mixed with 10-15% dimethyl sulfoxide (DMSO) Addition of concanavalin A (Con A) to PEG increases protoplast fusion frequency Sea water has been used alone or in combination with PEG to fuse protoplasts Fusion Induced by PEG

15. Figure: production of somatic hybrid cell

16. 3. Electrofusion: When the protoplasts are placed in a culture vessel fitted with micro-electrodes and an electric shock is applied, protoplasts are induced to fuse This technique is simple, quick and efficient The cells formed do not show cytotoxic responses as in the case of fusogens (including PEG) Limitations: This method requires specialized and costly equipment Figure: Electrofusion

17. Importance of protoplast fusion Combines genomes of different genera and species, overcoming sexual incompatibility Transfers useful genes (disease resistance, nitrogen fixation, rapid growth, protein quality, frost hardiness, drought resistance) between species Widens genetic base for plant breeding Cybrids Production: Result from elimination of one parent's genome post-fusion Fusion product with hybridized cytoplasm, known as cybrid, cytoplasmic hybrid, or heteroplast

18. Viability of Protoplasts There are several methods to assess the protoplast viability Fluorescein diacetate (FDA) staining method –The dye accumulates inside viable protoplasts which can be detected by fluorescence microscopy Phen safranine stain – It is selectively taken up by dead protoplasts (turn red) while the viable cells remain unstained Measurement of cell wall formation- Calcofluor white (CFW) stain binds to the newly formed cell walls which emit fluorescence Oxygen uptake by protoplasts can be measured by an oxygen electrode Photosyntheti c activity of protoplasts. The ability of protoplasts to undergo continuous mitotic divisions (this is a direct measure)

19. 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 Temperature: 20-28°C pH: 5.5-5.9 0.25% Casein hydrolysate BAP and NAA 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

20. The first cell division may occur within 2 to 7 days of culture Resulting in small clumps of cells, also known as microcolonies, 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 The microcalli can be made to develop into somatic embryos, which are then converted into whole plants through germination

21. Methods of Culture 1. Droplet Culture Protoplasts are suspended in a liquid medium at a density of about 105/ml either in conical flasks or plastic Petri dishes The droplet culture technique consists of placing approximately 50 ml of droplets containing protoplasts in plastic Petri dishes The plastic Petri dishes are sealed and incubated at 25 degrees to 30 degrees at low light intensities or in the dark

22. Figure: Plating method 2. Plating Method Protoplasts are suspended in a liquid medium in a Petri dish at double the concentration and mixed gently but quickly with an equal volume of the medium containing double the agar concentration The Petri dishes are sealed and incubated upside down in continuous light at 23 degrees to 25 degrees

23. 3. Microculture Chambers This method requires the culturing of 30- 50 microliter of medium containing one or more protoplasts on a microscopic slide, which is enclosed by a cover glass resting on two other cover glasses placed on either side of the drop The cultures are sealed with sterile paraffin oil and incubated in light at 23 to 25 degrees Celsius

24. Figure . Banana protoplast culture on feeder layer system. 4. Feeder Layers Non-dividing but metabolically active, X-irradiated protoplasts embedded in nutrient agar support, the growth of protoplasts plated at very low densities above them Feeder layer or nurse culture are also used where the fast-growing protoplasts aid the recalcitrant species

25. Importance of Protoplast Culture Study of Osmotic behavior Help in crop improvement To develop a novel hybrid plant through protoplast fusion Study of Cell wall formation Organelle isolation Study of Morphogenesis Virus uptake and replication Study of photosynthesis Gene Transfer

26. Factors affecting protoplast culture 1. Plant species and varieties: 2. Plant age and organ: Age of donor plant and its developmental stage, Stages are germinating embryos, plantlets, leaves 3. Pre-culture conditions: Protoplast cultures are highly influenced by climatic factors and different cultures of seedlings yield protoplast having different responses when cultured 4. Pre-treatment to the tissue, before isolating protoplasts: Cold treatment, plasmolysis, and hormones increase the chance of recovery of viable protoplasts and their plating efficiency 5. Density: At higher densities, protoplasts compete with one another while at lower densities losses of metabolites from protoplasts are greater

27. Somaclonal Variation Soma - somatic cells Clonal – clones - generations Variation - changes Somaclonal variation – somaclones (variation in somaclones)

28. The somaclonal variation term was first coined by Larkin and Scowcroft (1981) According to Larkin and Scowcroft (1981), “Somaclonal variation is the genetic variability which is regenerated during tissue culture” or plant variants derived from any form of cell or tissue cultures However, the first report of morphological variation in plants regenerated from cell culture was made by Heinz and Mee in 1971 in sugarcane The plants derived from cell and tissue cultures are termed somaclones and the plants displaying variation as somaclonal variants Somaclonal variations may lead to desirable characteristics like increased pest resistance, fruit quality, stress resistance, nutritional quality improvement, yield improvement Genetic variations are inherited by the clones of the treated plant

29. Causes of Somaclonal Variation Physiological cause Genetic cause Biochemical cause

30. Causes of Somaclonal Variation 2. Biochemical Cause Lack of photosynthetic ability due to alteration in carbon metabolism Biosynthesis of starch via carotenoid pathway Nitrogen metabolism 1 . Physiological Cause Exposure of culture to plant growth regulators Culture media conditions 3. Genetic cause 3.1. Change in chromosome number Aneuploidy (gain or loss set of chromosomes) Polyploidy (organism with more than 2 chromosomes set) Monoploidy (organism with one chromosome set)

31. 3.2. Change in chromosome structure Deletion (a segment of the base is deleted) Inversion (a segment of chromosomes is reversed) Duplication (addition of chromosomes) Translocation (arms of chromosomes switched) Transversion Insertion Deletion 3.3. Plasma gene mutation 3.4. Changes in DNA 3.5. Change in protein

32. Based on the tissue from which variation originates Somaclonal variation can be divided into the following types Gametoclonal variation: variation observed among the plants regenerated from gametic cultures Androclonal variation : observed among the plants regenerated from anther (or) pollen culture Gynoclonal variation: from ovule (or) ovary culture Protoclonal variation : variation observed among the plants regenerated from protoplast cultures Calliclonal variation: variation observed among the plants regenerated from callus cultures Types of somaclonal variation

33. Factors Affecting Somaclonal Variation 1. Species and genotype : Vegetatively Propagated > sexually propagated 2. Ploidy level: positive correlation between ploidy level and variation e.g. barley 1%, wheat 40% 3. Source of explant: petiole is preferred to stem 4. Tissue culture technique: protoplast more, than others 5. Duration of cell cultur e: positive correlation

34. Origin of Somaclonal Variation Somaclonal variation may occur either for a single trait (or) more than one trait at a time. Variation may arrive due to any one (or) a combination of the following mechanisms Somaclonal variation may occur due to chromosomal abnormality in cultured cells and plants regenerated from them The chromosomal abnormality may be due to changes in chromosomal number (ploidy level) and chromosomal structure

35. Application 1. Isolation of regenerants resistant to diseases. Maize lines having Texas male sterile cytoplasm are susceptible to southern leaf blight caused by Helminthosporium maydis which produces a toxin that binds to mitochondria. Maize cells resistant to this toxin have been selected and plants regenerated from them were resistant to leaf blight caused by Helminthosporium maydis A tomato line resistant to bacterial wilt caused by Pseudomonas solanacearum was isolated 2. Isolation of variants resistant to abiotic stresses Cell lines resistant to chilling have been isolated in several cases E.g.: -Chillies, Nicotiana sylvestris 3. Development of varieties with improved seed quality A variety Ratan of Lathyrus which has low neurotoxin content has been developed through somaclonal variation

36. Thank you all References: Online notes, notes from research papers and Books by Google Search Engine