Somatic hybridization and Protoplast Isolation

somatic hybridisation

1. Protoplast isolation and Somatic Hybridization Submitted by :- P.TEJASREE BAM-20-27 M.Sc 1 st Year Dept of GPBR DEPARTMENT OF GENETICS AND PLANT BREEDING Course No :- GP-509 Course Title :- BIOTECHNOLOGY FOR CROP IMPROVEMENT Assignment On :- PROTOPLAST ISOLATION AND SOMATIC HYBRIDIZATION Submitted to :- Dr. Lal Ahamed M. Associate Professor Dept of GPBR 1

2. Definition Development of hybrid plants through the fusion of somatic protoplasts of two different plant species/varieties is called somatic hybridization. This is a non conventional genetic procedure involving fusion between isolated protoplast under in vitro condition and subsequent development of their product ( heterokaryon ) to a hybrid plant. 2

3. History The term “Protoplast” was introduced in 1880 by Hanstein - A cell with its cell wall removed either mechanically or enzymatically is named as protoplast. First isolation of protoplast was achieved by Klercker in 1892 by using mechanical method. Küster in 1909 described the process of random fusion in mechanically isolated protoplasts. The real beginning in protoplast research was made by Cocking in 1960 who used enzymatic method for cell wall removal.****** Takebe et al (1971) were successful to achieve the regeneration of whole tobacco plant from protoplasts. Somatic hybridization (fusion of protoplasts) is relatively a new versatile technique to induce or promote genetic recombination in a variety of prokaryotic and eukaryotic cells ( Bhojwani S.S. et al 1977). 3

4. Need Of Somatic Hybridization** Crossing barriers among plant species and in organelle Species barriers encountered in sexual hybridization Transfer of genes from wild species into the genes of crop plants ** Tool for the modification and improvement of polygenic traits 4

5. Importance of Protoplast Isolation The term “Protoplast” refers to the spherical plasmolysed content of the plant cell enclosed by plasma membrane or naked cell without cell wall . Before culturing protoplast, it is important to isolate viable and uninjured protoplasts. Production of hybrid plants through the fusion of protoplasts of two different plant species/varieties is called Somatic Hybridization , and such hybrids are called Somatic Hybrids . Therefore, somatic hybridization can be resorted to only when the following two criteria are satisfied: i ) Isolation of protoplast in large quantity, and ii) Totipotency of the isolated protoplasts. 5

6. (q) Chromosome number of diploid 2n=2x=40. (r) Chromosome number of somatic hybrid (2n=4x=80). – Symmetric Hybrid*** 6

7. Source of Protoplasts Protoplast can be isolated from almost all plant parts: Roots, leaves, fruits, tubers, root nodules, endosperm, pollen mother cell, callus and suspension culture Spongy and palisade mesophyll tissue obtained from mature leaves of Nicotiana and Petunia . Anthers of Pelargonium (Abo El-Nil and Hilderbrandt , 1971) Callus of Gossypium hirsutum ( Bhojwani , Cocking, and Power, 1977) Crassulacean acid metabolism (CAM) plants ( Doddds , 1980) C3 and C4 plants (Kanai and Edwards, 1973) Solanum tuberosum ( Upadhya , 1975) 7

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10. Methods of Isolation of Protoplasts 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 uses these enzymes (cellulase, hemicellulase and pectinase) at pH 4.5-6.0 & temperature 25-30 C with incubation period of half an hour to 20 hrs. Credit of developing High Yield Protoplast Isolation Technique from higher plant protoplasts goes to Cocking (1960) MECHANICAL METHOD HISTORY: Klercher (1892) was the first to isolate protoplast from plasmolyzed cell of Stratiates aloides . The studies were later extended for protoplast isolation from tissues of Onion bulbs. PROCEDURE: Onion Scales were immersed in 1.0 M Sucrose until the protoplast shrunk away from their enclosing wall and then the plasmolysed tissue was cut into small strips . The protoplasts were released by Osmotic Swelling when these strips of the tissue were placed in Low Concentration Sucrose Solution . This method is suitable for isolation of protoplasts from higher plant tissue such as leaf, bulb scale, fruit epidermis, radish roots 10

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12. TYPES OF ENZYMATIC METHOD: There are two types of enzymatic method. Both methods have certain advantages and disadvantages (Evans and Cocking, 1977; Bajaj, 1977). Generally 50 mM CaCl 2 is added to increase the stability of released protoplasts (Rose, 1980). One Step Method (Direct/Mixed Method): In this method protoplasts are isolated from plant tissues directly by using two enzymes, cellulase and pectinase, simultaneously . Power and Cocking (1968) used this method for isolation of protoplasts. Two Step method (Sequential Method): This method was first used Takebe and others in 1968 in two steps. In this method, cells are first isolated from callus or tissues by using pectinase and to this cell suspension cellulase is added to digest the cell walls and release protoplasts 12

13. Purification of Protoplasts Commonly used methods include:- Filtration (For Removal of Debris):- Debris (undigested material) can be removed from protoplast suspension by filtering the preparation through a steel or nylon mes h of 100µ pore size . Sedimentation & Washing (For Removal of Enzymes):- Enzyme is removed by centrifuging protoplast suspension at 600 rpm for 5 minutes . The protoplasts settle to the bottom of the centrifuge tube while the supernatant is removed with the help of a pipette. The protoplasts are then resuspended in a Washing Medium containing an osmoticum only or osmoticum with nutrient medium or hydrated Calcium Chloride . The suspension is centrifuged again to settle the protoplasts and the washing medium is decanted. Traces of enzymes are removed by washing the protoplasts twice or thrice with the medium. 13

14. 3. Flotation (Separation of Protoplasts):- In this method intact protoplasts are separated from the broken debris by suspending the protoplast preparation in 20-40% Sucrose Solution and centrifuging at 350rpm for three (3) minutes . Intact protoplasts collect at the top of the sucrose solution and are carefully removed with a pipette (Gregory and Cocking, 1965; Power and Cocking, 1971; Evans et al., 1972). Schenk and Hildebrandt (1969) used Ficoll Solution. 4. Density Buffer Method: Larkin (1976) used this method for purification of protoplasts. In this method 0.5- 3.0 volumes of crude protoplast preparation after filtration through sterile muslin cloth is layered on LymphoPrep ( LymphoPrep ™ is a ready-made, sterile and endotoxin tested solution suitable for the purification of human mononuclear cells) in the centrifuge tube and then spun at 50-200 g for about 10 minutes . The protoplasts collect as a ring between the enzyme solution and lymphoprep and debris settle to the bottom (See Figure). 14

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17. Testing Viability of Protoplasts And Cell Wall Formation Cell Wall Formation Test: To the small volume of the protoplast suspension add equal volume of 0.1% Calcofluor solution , incubate for 5 minutes and then observe under fluorescent microscope. The cell wall will fluoresce and protoplast remain dark . Protoplast Viability Test: Fluorescein Diacetate (FDA) solution in acetone (5mg/l) is added to protoplast suspension to give a final concentration of 0.01% . After 5 minutes at room temperature the protoplasts are examined using fluorescent microscope. Only viable protoplasts can be seen . 17

18. Factors Affecting Protoplast Isolation And Its Viability As a thumb rule, low enzyme concentration at low temperature and high pH (5-8) for short incubation period prove to better than longer incubation periods with high enzyme concentration, high temperature and low pH value. Though the ionic salts when used with osmoticum degrade the enzymes but increase the stability of protoplasts . Protoplasts isolated in the presence of Ca++ or Mg++ showed a greater capacity for the cell wall regeneration as compared to protoplasts in the absence of these ions (Rose, 1980). 18

19. Methods of Protoplast Fusion 19

20. Methods of Protoplast Fusion Different methods of protoplast fusion are described by Bengochea and Dodds (1986). Protoplast fusion can be broadly classified into two categories: 1. Spontaneous fusion (fuse through their plasmodesmata) 2. Induced fusion (needs fusion inducing chemicals/ Fusogens ) a) Mechanical fusion b) Chemo fusion c) Electro fusion 20

21. Spontaneous Fusion Spontaneous Fusion Protoplast during isolation often fuse spontaneously and this phenomenon is called spontaneous fusion During the enzyme treatment, protoplast from adjoining cells fuse through their plasmodesmata to form multinucleate (2-40) protoplasts. 21

22. Induced Fusion Induced Fusion of freely isolated protoplasts from different sources with the help of fusion inducing chemicals agents is known as Induced Fusion. Normally isolated protoplast do not fuse with each other because the surface of isolated protoplast carries negative charges (-10mV to -30mV) around the outside of the plasma membrane. And thus there is a strong tendency in the protoplast to repel each other due to their same charges . So this type of fusion needs a fusion inducing chemicals ( Fusogens ) which actually reduce the electronegativity of the isolated protoplast and allow them to fuse with each other ( Narayanswamy S. 1994). 22

23. Types of Induced Fusion The isolated protoplast can be induced to fuse by three ways; A) MECHANICAL FUSION: In this method the isolated protoplast are brought into intimate physical contact mechanically under microscope and using Micromanipulator or Perfusion Micropipette. B) CHEMO FUSION : Several chemicals have been used to induce protoplast fusion such as NaNO3 , Polyethylene Glycol (PEG) and Calcium ions (Ca++). Chemical fusogens cause the isolated protoplast to adhere (stick) each other and leads to tight agglutination followed by fusion of protoplast (Pasha C.R et al 2007; Jogdand S.N.2001). 23

24. NaNO3 Treatment:- Isolated protoplasts exposed to a mixture of 5.5% NaNO3 in 10% Sucrose Solution. Incubation carried out for 5 mins at 350C followed by centrifugation. Protoplast pellet kept in water bath at 300C for 30 mins during which fusion occurs. Induced fusion by NaNO3 was first demonstrated by Power et al (1970). Treatment With Calcium Ions (Ca++) At High pH :-This method involves spinning (centrifugation) the protoplasts in a Fusion Inducing Solution (0.05M CaCl2 , 0.4M mannitol at pH 10.5, Glycine-NaOH buffer) for 30 minutes at 50g, after which the tubes are placed in a water bath (37°C) for 40-50 minutes. This leads to fusion of 20-50% of the protoplasts. This method was developed by Keller and Melchers (1973) for fusing two different lines of tobacco protoplasts. The details of the protocol are described by Bhojwani and Razdan (1983). Polyethylene Glycol (PEG) Treatment :-Isolated protoplasts in culture medium (1ml) are mixed with equal volume (1ml) of 28-56% PEG (Mol. Wt. 1500-6000 dalton ) in a tube. Tube is shaken and then allowed to settle and settled protoplasts are washed several times with culture medium during which fusion occurs. 24

25. Electro Fusion Electro Fusion:- In this method an electric field of low strength (10Kv/m) gives rise to dielectrophoretic dipole generation within the protoplast suspension and a high strength of electric field (100Kv/m) for some micro seconds are applied this lead to fusion. 25

26. Fusion Products Fusion Products :- Fusion of cytoplasm of two protoplasts results in coalescence of cytoplasms . The nuclei of two protoplasts may or may not fuse together even after fusion of cytoplasms . Cells containing nonidentical nuclei are referred to as Heterokaryons or Heterokaryocytes (Mastrangelo, 1979). The fusion nuclei in a nucleate heterokaryon results in the formation of a true Hybrid Protoplast or Synkaryocyte ( Constabel , 1978). The fusion of two protoplasts from the same culture results in a Homokaryon . Frequently genetic information is lost from one of the two nuclei. If one nucleus completely disappears, the cytoplasms of the two parental protoplasts are still hybridized (see Figure) and the fusion product is known as “Cybrid” (Cytoplasmic Hybrid or Heteroplast ) 26

27. IDENTIFICATION AND SELECTION OF SOMATIC HYBRID CELLS Various protocols have been proposed and practiced for the effective selection of hybrids: Genetic Complementation: Complimentary selection of somatic hybrids on specific culture medium ( Melscher & Labib 1974; Smith et al.1976). In this case complementation or genetic or metabolic deficiencies of the two fusion partners are utilized to select the hybrid component. When protoplasts of two parents, (one parent bearing cytoplasmic albino trait and the other parent bearing green trait) each parent carrying a non-allelic genetic or metabolic defect are fused, it reconstitutes a viable hybrid cell, of wild type in which both defects are mutually abolished by complementation, and the hybrid cells are able to grow on minimal medium nonpermissive to the growth of the parental cells bearing green trait. Later, the calli of hybrid nature could be easily distinguished from the parental type tissue (albino trait) by their green color. The complementation selection can also be applied to dominant characters, such as dominant resistance to antibiotics, herbicides or amino acid analogues. 27

28. 2. Mechanical isolation by visual means and knowledge of identification of somatic hybrids. Schieder and Kohn (1986 ) used the scheme in which the parental protoplasts and heterokaryons were allowed to develop calli in cultures. The morphological differences in the resultant three types of calli permitted identification of the hybrid tissue, which could then be selected out to regenerate somatic hybrid plants. Individual heterokaryons can be identified visually under a light microscope, be isolated mechanically by means of Drummond pipette, and can be cloned in microdrop cultures. This approach suffers from the fact that it requires special culture media for each particular hybrid cell type to divide and form clusters. This is also called the fishing method . 3. Morphology :- of the plant after regeneration. Somatic hybrids in most of the cases show characters intermediate between the two parents such as, shape of leaves, pigmentation of corolla, plant height, root morphology and other vegetative and floral characters. The method is not much accurate as tissue culture conditions may also alter some morphological characters or the hybrid may show entirely new traits not shown by any of the parents. 28

29. Pigmentation 4. Pigmentation:-( Morpho-Physiological Basis of Calli): The whole mixture of the protoplasts are cultured after fusion treatment and the resulting calli or regenerants are screened for their hybrid characteristics. Occasionally the hybrid calli outgrow the parental cell colonies and are identified by their intermediate morphology, i.e. green with purple coloured cells . However, the process is labour intensive and requires glasshouse facilities. It is limited to certain combinations showing differences in their regeneration potential under specific culture conditions. 29

30. 5. Cytoplasmic Markers Fluorochromes like FITC ( fluoroscein isothiocyanate) and RITC (Rhodamine isothiocyanate) are used for labelling of parental cells Manual isolation requires that the two parental type protoplasts have distinct morphological markers and are easily distinguishable. For example, green vacuolated, mesophyll protoplasts from one parent and richly cytoplasmic, non-green protoplasts from cultured cells of another parent. The dual fluorescence method also helps easy identification of fusion products . In this case, the protoplast labeled green by treatment with fluorescein diacetate (FDA, 1-20 mgl-1) are fused with protoplasts emitting a red fluorescence, either from chlorophyll autofluorescence or from exogenously applied rhodamine isothiocyanate (10-20 mgl-1). The labeling can be achieved by adding the compound into the enzyme mixture. This can be applied even for morphologically indistinguishable protoplasts from two parents 30

31. 6. Nuclear staining: Heterokaryon is stained by carbol-fuschin , aceto-carmine or aceto-orcein stain. 7. Phytotoxins: (soybean resistant to Hm T toxin whereas Zea mays sensitive to it) 8. Molecular analysis: Specific restriction pattern of nuclear, mitochondrial and chloroplast DNA characterizes the plastomes of hybrids and cybrids. Molecular markers such as RFLP, RAPD, and ISSR can be employed to detect variation and similarity in banding pattern of fused protoplasts to verify hybrid and cybrid. 9. Specific amino acid : ( Conavalin present in soybean but not in sweet clover, alfalafa ) 10. Auxin autotrophy : (Nicotiana glauca and Nicotiana langsdorffi ) 31

32. 11. Isozyme Analysis: Multiple molecular forms of same enzyme which catalyses similar or identical reactions are known as “Isozymes”. Electrophoresis is performed to study banding pattern as a check for hybridity. If the two parents exhibit different band patterns for a specific isozyme the putative hybrid can be easily verified. The isozymes commonly used for hybrid identification include, acid phosphatase, esterase, peroxidase. 12. Chromosomal Analysis (Cytological Analysis): Chromosome counting of the hybrid is an easier and reliable method to ensure hybridity as it also provides the information of ploidy level. Cytologically the chromosome count of the hybrid should be sum of number of chromosomes from both the parents. Besides number of chromosomes, the size and structure of chromosomes can also be monitored. However, the approach is not applicable to all species, particularly where fusion involves closely related species or where the chromosomes are very small. Moreover, sometimes the somaclonal variations may also give rise to different chromosome number 32

33. Antibiotics 13. Antibiotics: Drug sensitivity technique was originally developed by Power et al (1976) for the selection of hybrids of Petunia sp. This method is useful for the selection hybrids of two plant species, if one of them is sensitive to a drug. Protoplasts of Petunia hybride (species A) can form macroscopic callus on MS medium, but are sensitive to (inhibited by) actinomycin D. Petunia parodii protoplasts (species B) form small colonies, but are resistant to actinomycin D. When these two species are fused, the fused protoplasts derive both the characters-formation of macroscopic colonies and resistance to actinomycin D on MS medium. This helps in the selection of hybrids (Fig. 44.6). The parental protoplasts of both the species fail to grow. Protoplasts of P. parodii form very small colonies while that of P. hybrida are inhibited by actinomycin D – ruled out . Ishige (1995) used transgenic resistance to kanamycin in one potato dihaploid and hygromycin in another to regenerate exclusively somatic hybrids that varied for ploidy. However, the restrictions of this system are the extensive pre-breeding required to introduce transgenic resistance into the protoplast fusion partner(s) and the limitation of the procedure only to transgenic plants Pandeya et al., (1986) adopted a similar procedure for somatic hybrids between Nicotiana sylvestris and N. knightiana . This selection system makes use of two dominant drug resistant cell lines. Selection of somatic hybrids following protoplast fusion of two such cells, lines is carried out on, media containing both drugs. 33

34. Drug sensitivity technique was originally developed by Power et al (1976) for the selection of hybrids of Petunia sp 34

35. CULTURE OF HYBRID CELLS Hybrid cells are cultured on suitable medium provided with the appropriate culture conditions. • Iscove's MDM with 25 mM Hepes . • DMEM/F-12 with 15 mM HEPES. • Alpha MEM with Nucleosides • RPMI 1640 Medium 35

36. REGENERATION OF HYBRID PLANTS Plants are induced to regenerate from hybrid calli . These hybrid plants must be at least partially fertile. CELL WALL REGENERATION: May be completed in two to several days. Although protoplast in culture generally start regenerating a cell wall within a few hours after isolation. Protoplast lose their characteristic spherical shape once the wall formation is complete. Regeneration of cell wall can be demonstrated using Calcalfluor White ST fluoresecent Stain or Tinapol solution . 1. Protoplast fusion and somatic hybrid plantlet regeneration of cvs . ‘Kensington Pride’ + ‘Haden’. (a) Young leaves of cv. ‘Haden’. (b) PEM induction on ovular nucellus of cv. ‘Kensington Pride’. (c) PEM suspension culture. (d) Isolated protoplasts stained with Fluorescein Diacetate (FDA) under UV. (e, f) PEG-induced binary protoplast fusion. (g) Early cell division after protoplast culture. (h) PEM formation. ( i ) Globular embryo production. (j) Heart and torpedo-shape embryo production. (k) Mature embryos. (l, m) Germination of embryos with elongated radicle. (n) Regenerated somatic hybrid plantlets. (o) Plantlet at early acclimatization stage. (p) Acclimatized plant after 5 months. 36

37. PEG-mediated protoplast transformation. (a) Tobacco leaf strips treated with protoplasting enzyme solution. Protoplasts prior to PEG treatment (b), and recovery without selection for 1 (c), 2 (d), or 3 (e) weeks after PEG treatment, with newly formed cells lighter green in color. For selection of transformants, 2-week-old PEG-treated protoplasts are typically embedded in agarose for a week prior to antibiotic selection for another 6 weeks. Putative resistant calli in solution (f) or in embedded gel (g, h) transferred to plates ( i ) for shoot formation (j, k). Shoots excised to root in tissue culture boxes (l) 37

38. 38 PEG-induced fusion of citrus embryogenic protoplasts with leaf protoplasts RAPD analysis Root-tip cell Rooted cutting of somatic hybrid rootstock Fruiting tree of somatic hybrid breeding parent somatic hybrid

39. Principle of Somatic Hybridization The principle of somatic hybridization is to combine the protoplasts of two species and "fuse" the two protoplasts to produce a heterokaryon (a cell containing genetically different nuclei). The process of fusion is carried out in the presence of calcium ions either at high pH (10.5) or with the aid of polyethylene glycol (PEG) at a concentration of 25070-30%. Often a high loss of viable cells occurs, depending upon the genus, species, age of plant materials, and laboratory procedures, the principle problem being microbial contamination. However, with cell concentrations (density) at 2 x 105 cells/milliliter, a number of heterokaryons are still expected (Power et al., 1989). Following removal of the fusion media, the cells are transferred to a new medium for growth, and the cells can be observed microscopically for fusion of the two species, viability, and for rate of growth. If the fused cells are viable, the first mitotic division can be observed within 4 days. If this event does not occur, death of the heterokaryon ensues. Because plants vary widely in their need for hormones, light, and temperature, every somatic hybrid cross must entail a thorough study of growth requirements for success. Quite often the new somatic hybrid will develop a callus tissue (undifferentiated tissue) which must be transferred to a suitable medium for bulb growth, root development, and subsequent growth (power et al., 1989). 39

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41. For the development of stable inter-generic allohexaploid Brassica, we have used B. juncea cv. RLM-198 as the cultivated germplasm with Indian S. alba (Gene Bank Accession No. DRMR-2183) as a donor for the yellow seed color and resistance to S. sclerotiorum . The B. juncea cv. RLM-198 was selected as a fusion parent due to its excellent regeneration potential. The three experiments included in this study were conducted using the same fusion parents. The protoplast of hypocotyl cells of B. juncea cv. RLM-198 and mesophyll cells of S. alba were isolated using the protocol of Kirti and Chopra (1990), followed by protoplast fusion, regeneration, and acclimatization according to Kirti et al. (1995). A total of five plants (clones) from each event were transferred to pots filled with soil:solarite (1:1) under net house conditions at the Indian Agriculture Research Institute (IARI), New Delhi, India and maintained till harvesting. 41

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45. SYMMETRIC HYBRIDS--PRODUCTION OF NEW SPECIES Symmetric hybrids can be produced between species, which cannot be hybridized sexually. These hybrids can be readily used in breeding programs for transfer of useful genes to crops or may be useful as new species. The first symmetric somatic hybrid of Citrus was created by protoplast fusion of C. sinensis and Poncirus trifoliata ( Ohgawara et al., 1985) and the production of hybrid plants between two sexually incompatible Citrus genera was first reported in 1988, where C. sinensis L. Osb . cv. ‘Hamlin’ protoplasts were fused with Severinia disticha (Blanco) Swing protoplasts (Grosser et al., 1988). Merits 45

46. Family Brassicaceae Model family for somatic hybridization Hybridization have been performed in Brassica genus Resynthesis of Brassica napus, an intergeneric hybrid Intertribal hybrid are also produced e.g., Raphanus x B.napus 46

47. Family Fabaceae Regeneration of plants was initially troublesome Now achieved in several genera like Pisum,Trifolium,especially in Medicago Flowering hybrids b/w Medicago sativa & Medicago falcate Intergeneric fusion b/w Onobrychis viciifolia , Sanfoin & Alfalfa 47

48. Family Poaceae Somatic hybridization was initially difficult Now several intergenric and intrageneric have been produced e.g., Panicum maximum (+) Pennisetum americanum , Saccharum officinarum (+) P. americanum etc Much work has been done on Saccharum spp. 48

49. Family Solanaceae For disease & insect resistance in eggplant, Solanum melongena To restore the ploidy level and for viral resistance in potato In Tomato, several interspecific hybrid plants have been produced E.g., Tomato + Solanum tuberosum, Tomato + Nicotiana tabacum, Tomato + S. tuberosum etc 49

50. CYTOPLASMIC TRANSFERS ARE TIME SAVING : Cytoplasm transfers can be affected in one year, while backcrossing may take 15-16 years. Even where backcrossing is not applicable, cytoplasm transfers can be made using this approach. Somatic Hybrids for Cytoplasmic Male Sterility Methods have been developed to substitute the nucleus of one species into the cytoplasm of another species, whose mitochondria are inactivated. This type of substitution in some cases, led to generation of cytoplasmic male sterility. For this purpose, a report by Melchers (1992); and his coworkers, the two types of protoplasts, used for the production of, somatic hybrids, and were treated differently, as follows: mesophyll protoplasts of tomato (Lycopersicon esculentum) were treated with iodoacetamide (IDA) to inactivate mitochondria and mesophyll protoplasts of Solanum acaule (or S. tuberosum = potato) were irradiated with γ or x-rays to inactivate nuclei. (see figure) The protoplasts were mixed in 1: 1 ratio and induced to fuse using Ca2+ and PEG, leading to the production of heterologous hybrids. Among the fusion products, some hybrid tomato plants were indistinguishable from the original cultivars, with respect to morphology, physiology and chromosome number (2n=24), but exhibited various degrees of male sterility. The variation included (Melchers,1992) MITOCHONDRION-CHLOROPLAST FUSION (RECOMBINANT ORGANELLAR GENOMES) : Mitochondria of one species can be combined with chloroplasts of another species. This may be very important in some cases, and is not achievable by sexual means even between easily crossable species. Recombinant organellar genomes, especially of mitochondria, are generated in somatic hybrids and cybrids. Some of these recombinant genomes may possess useful features. 50

51. Melchers (1992); and his coworkers, the two types of protoplasts, used for the production of, somatic hybrids, and were treated differently with iodoacetamide (IDA) , γ or x-rays 51

52. Production Of Novel Interspecific And Intergenic Hybrid Pomato (Hybrid of potato and tomato) (Melchers,1978) Protoplasts arisen by fusion of tomato and potato protoplasts. The cytoplasmic part of the tomato (Lycopersicon esculentum var. cerasiforme mut. yellow green 6) is recognizable by the green chloroplasts present in the tomato mesophyll protoplasts. The cytoplasmic part of the potato (Solarium tuberosum, dihaploid stock HH 258) is colourless as the potato protoplasts were made from submersed cultured callus cells with proplastids . 52

53. Production Of Fertile Diploids And Polyploids From Sexually Sterile Haploids, Triploids And Aneuploids: Anssour et al. (2009) examined both allo - and auto-tetraploid Nicotiana species, and showed that there were substantial changes both in morphology and in the genome which are found in the hybrid species compared with their ancestral diploids. GENE TRANSFER Gene Transfer for disease resistance (e.g. TMV, Potato Virus X, Club Root Disease), abiotic stress resistance (cold tolerance gene in tomato), herbicide resistance and many other quality characters. Asymmetric hybridization is very promising as it allows partial genome transfer (Derks et al., 1992; Trick et al., 1994; Liu & Deng, 2002), which may be better tolerated than a whole-genome transfer ( Ramulu et al., 1996a,b). As in other agricultural species, trait introgression from ‘wild’ species of the genus Nicotiana has been used to improve the cropped species, and characters from at least 13 different species have been transferred into tobacco (Lewis, 2011). 53

54. PRODUCTION OF HETEROZYGOUS LINES : Production of heterozygous lines in the single species which cannot be propagated by vegetative means. FATE OF PLASMA GENES : Somatic cell fusion is useful in the study of cytoplasmic genes (plasma genes) and their activities and this information can be applied in plant-breeding experiments. PRODUCTION OF UNIQUE HYBRIDS OF NUCLEUS AND CYTOPLASM : Cybridization has made it possible to transfer cytoplasmic male sterility. 54

55. DEMERITS CEREALS & PULSES : Techniques for protoplast isolation, culture and fusion are not available for many important crop species like many cereals and pulses. CHROMOSOMAL ELIMINATION : In many cases, chromosome elimination occurs from somatic hybrids leading to asymmetric hybrids. Such hybrids may be useful, but there is no control on chromosome elimination. GENETIC INSTABILITY : Many somatic hybrids show genetic instability, which may be an inherent feature of some species combinations. ABNORMALITIES : Many somatic hybrids either do not regenerate or give rise to sterile regenerants. Such hybrids are useless for crop improvement. All interfamily somatic hybrids are genetically unstable and/or morphologically abnormal, while intergeneric and intertribal hybrids are genetically stable but produce abnormal and/or sterile plants or only teratomata . 55

56. POOR REGENERATION : Poor regeneration of hybrid plants NON-VIABILITY : Non-viability of fused products. INEFFICIENT SELECTION METHODS : There are limitations in the selection methods of hybrids, as many of them are not efficient. Not very successful in all plants. NO CONFIRMATION OF EXPRESSION OF PARTICULAR TRAIT : No confirmation of expression of particular trait in somatic hybrids. Techniques for protoplast isolation, culture and fusion are very complicated. 56

57. CONCLUSION Protoplast technology has various applications other than regeneration of complete plants and production of hybrids of sexually incompatible species. These techniques have been instrumental in generating basic scientific information on cell biology, plant incompatibility, membrane functions, cell organelle studies, cell wall regeneration, ultrastructure and molecular architecture of plant cells. These techniques are now being used for transfer of cytoplasmic male sterility . Protoplasts can take up macromolecules (nucleic acids and proteins), viruses, cell components like chromosomes and chloroplasts by phagocytosis . Somatic hybridization allows transfer of cytoplasmic organelle in a single generation and offer unique opportunities for combining mitochondria of one species and chloroplast of another species in a single hybrid. This technique in the future will be one of the most frequently used research tools for tissue culturists, molecular biologists, biochemical engineers and biotechnologists. 57

58. Thank you 58