1. TERMINOLOGY USED IN TISSUE CULTURE

2. Acclimatization It is a process of gradual hardening of the cultured plants from laboratory to field so that the regenerated plants should adjust to field conditions. Androgenesis It is a process of regeneration of plants from pollen grains. Such plants are haploids. It is a process of regeneration of plants from pollen grains. Such plants are haploids. Auxenic or Aseptic culture Cultures devoid of foreign or undesired life forms are called auxenic culture.

3. Basal medium Most of the media contain inorganic salts of major and minor elements, vitamins and sucrose. A medium with these ingredients is called basal medium. Callus Mass of undifferentiated cells produced in tissue culture is called callus. The callus is highly vacuolated and unorganised cells. Clone A clone is a group of plants produced from a single explant through asexual reproduction. All the members of a clone have the same genotype as'" that of the parent. They are identical in genotype. Phenotypic differences within a clone are due to external factors.

4. Cryobiology It is a technique of preservation of plant tissues and cells in liquid nitrogen at­196°C. This is highly significant for storage of germplasm of those crops which do not produce seeds and reproduce only by vegetative means. Thus cryobiology is very useful in crop improvement. Explant Small piece of viable tissues isolated from parent plant is called explant. Inoculation Transfer of explant to culture medium is called inoculation.

5. In vitro Cells/tissues removed from the intact organism and grown in controlled condition in laboratory. Micropropagation (clone propagation) It is a process of production of clones similar to asexual reproduction. Plantlets are produced from shoot tips/axillary buds on culture medium omitting callus phase. Here, small amount of explant produce millions of clonal parts in a year directly. Organogenesis Process of differentiation of callus initially into embryo like structure (embryoids) and then showing organ like roots/shoots

6. Protoplast Animal, plant or fungal cell from which the entire cell wall has been removed. Regeneration Production of entire plant from explant is called regeneration. Shoot tip culture Culture of shoot tip (shoot apical meristem) along with one or more leaf primordia or mature leaves is called shoot tip culture.

7. Somaclone Plants derived from somatic cell culture. Somaclonal variation Somaclonal variations are heritable variations for both qualitative and quantitative traits produced in plants regenerated from cell culture and tissue cultures. Variation caused during plant tissue culture is called somaclonal variation. Somaclonal variations arise as a result of chromosome structural changes, gene mutations, plasma gene mutations, gene amplification, transposable elements etc.

8. Somatic embryogenesis Process of production of embryo from somatic cells is called somatic embryogenesis and such embryo is called embryoid. Stock plant The plant from which explant is taken is called stock plant. Sub-culture Callus produced and cultured again for production of big mass of callus is called sub-culture.

9. Suspension culture A type of liquid culture in which cells or cell aggregates grow and multiply. Transplant stage Process of transfer of regenerated plants from test tube (i.e. in vitro) to the soil is called transplant stage.

10. Steps of Somatic Hybridization There are three steps of somatic hybridization: (i) Isolation of protoplasts (ii) Fusion of protoplasts of desired species (iii) Culture of hybrid protoplast to produce whole plants.

11. Isolation of protoplasts all plant parts but leaf mesophyll is the most preferred tissue. all plant parts but leaf mesophyll is the most preferred tissue. after removal of cell wall. after removal of cell wall. Precautions are taken to remove cell wall without damaging the cell cytoplasm and nuclear constituents. It is done by two methods: Precautions are taken to remove cell wall without damaging the cell cytoplasm and nuclear constituents. It is done by two methods: Mechanical disruption of cell wall and Mechanical disruption of cell wall and Enzymatic dissolution of cell wall Enzymatic dissolution of cell wall

12. Mechanical disruption Experimental cells are allowed to plasmolyse by keeping them in hypertonic solution. Experimental cells are allowed to plasmolyse by keeping them in hypertonic solution. In plasmolysed state, cell wall is cut with a sharp knife. In plasmolysed state, cell wall is cut with a sharp knife. Plasmolysed cell is transferred to hypotonic solution. Plasmolysed cell is transferred to hypotonic solution. This results in the release of protoplast in outer solution through cut ends. 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. This method is suitable only for tissues with large cells in which evident plasmolysis occurs. Damaged cells may release chemical which might affect culture of protoplast. Damaged cells may release chemical which might affect culture of protoplast.

13. Enzymatic dissolution Cell walls are dissolved by enzymes. Cell walls are dissolved by enzymes. Such enzymes are extracted from fungi, bacteria and Roman snail (Helix pomatia). Macerozyme, a pectinase enzyme is obtained from Rhizopus fungus, Driselase. a mixture of cellulase and pectinase is obtained from Trichoderma viride. Such enzymes are extracted from fungi, bacteria and Roman snail (Helix pomatia). Macerozyme, a pectinase enzyme is obtained from Rhizopus fungus, Driselase. a mixture of cellulase and pectinase is obtained from Trichoderma viride. Pectinase breaks the tissues into cells by dissolving calcium pectate of middle lamella. Pectinase breaks the tissues into cells by dissolving calcium pectate of middle lamella. Hemicellulase and cellulase break down the cell wall. Hemicellulase and cellulase break down the cell wall. Commercially available enzymes are "Pectolyase Y- 23", Onozuka R-1O. Macerozyme. Commercially available enzymes are "Pectolyase Y- 23", Onozuka R-1O. Macerozyme.

14. Protoplast using enzymes may be isolated by sequential method or mixed enzyme method. 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. 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. 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 palisade, spongy parenchyma and upper epidermis. Sequential method is useful in isolating protoplasts from palisade layer. while mixture enzyme method is useful in isolating protoplasts from palisade, spongy parenchyma and upper epidermis.

15. Enzymatic protoplast isolation comprises of several steps: Surface sterilization of leaf sample Surface sterilization of leaf sample Rinsing in suitable plasmolyticum with distilled water 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. Peeling of off the lower epidermis towards margin with sharp forceps below the junction of a lateral vein and midrib. Enzymatic treatment Enzymatic treatment Purification of isolated protoplasts. Purification of isolated protoplasts.

16. Enzymatic treatment Plasmolysed excised leaf cells are transferred to petri dishes containing enzyme solution (3% macerozyme and 2% Okozuka cellulase dissolved in 13% mannitol at pH 5.4). Plasmolysed excised leaf cells are transferred to petri dishes containing enzyme solution (3% macerozyme and 2% Okozuka cellulase dissolved in 13% mannitol at pH 5.4). Petri dishes are sealed with paraffin, wrapped in aluminium foil and incubated for 12-18 hours at 25°C. Petri dishes are sealed with paraffin, wrapped in aluminium foil and incubated for 12-18 hours at 25°C. The leaf portions are then teased slowly with forceps to release protoplasts. The leaf portions are then teased slowly with forceps to release protoplasts.

17. Protoplast purification Enzyme solutions are filtered with nylon mesh to remove insoluble impurities. Enzyme solutions are filtered with nylon mesh to remove insoluble impurities. Filtrate is centrifuged for 5 minutes at 700 rpm. Filtrate is centrifuged for 5 minutes at 700 rpm. The protoplast forms pellet and goes at the bottom of contrifuge tube. The protoplast forms pellet and goes at the bottom of contrifuge tube. Supernatant is removed with Pasteur pipett. 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 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. The resultant protoplast is pure. Outlines of isolation, purification, culture and regeneration of protoplasts are shown in figure below. Outlines of isolation, purification, culture and regeneration of protoplasts are shown in figure below.

18. Outlines of isolation, purification, culture and regeneration of protoplasts Outlines of isolation, purification, culture and regeneration of protoplasts

19. Culture of Protoplast hanging drop culture technique. hanging drop culture technique. Two protoplasts isolated from genetically different plant cells are fused. Two protoplasts isolated from genetically different plant cells are fused. Interaction-of two protoplasts leads to formation of somatic hybrids (Fig. 25.7). Interaction-of two protoplasts leads to formation of somatic hybrids (Fig. 25.7). Fusion of the protoplast may be spontaneous or induced. Fusion of the protoplast may be spontaneous or induced. Fusion of the protoplast is intraspecific and rare. Fusion of the protoplast is intraspecific and rare. Physical and chemical agents which reduce charges on protoplasts surfaces, are used for the purpose. Physical and chemical agents which reduce charges on protoplasts surfaces, are used for the purpose. Physical method includes electrofusion and chemical method includes the use of polyethylene glycol followed by elution with Ca++ ions at high pH. Physical method includes electrofusion and chemical method includes the use of polyethylene glycol followed by elution with Ca++ ions at high pH. PEG agglutinates protoplast while Ca++ allows coalescence and cytoplasmic mixing. PEG agglutinates protoplast while Ca++ allows coalescence and cytoplasmic mixing. During aggregation the plasma membrane of the adjacent protoplasts are tightly adpressed over a portion of protoplast surface During aggregation the plasma membrane of the adjacent protoplasts are tightly adpressed over a portion of protoplast surface

20. Regeneration of Cell Wall: Regeneration of Cell Wall: Protoplasts begins to form new cell wall when it is cultured in suitable nutrient medium. Cell wall is formed in few days. Protoplasts begins to form new cell wall when it is cultured in suitable nutrient medium. Cell wall is formed in few days. Regeneration of Plants: Regeneration of Plants: Somatic hybrids obtained are now grown in culture solution. It develops callus. Somatic hybrids obtained are now grown in culture solution. It develops callus. Callus is now sub-cultured in culture solution supplemented with mannitol and auxin. Callus is now sub-cultured in culture solution supplemented with mannitol and auxin. This favours embryogenesis after 3-4 weeks. This favours embryogenesis after 3-4 weeks. Embryoids develop into seedlings and eventually into mature plants. Embryoids develop into seedlings and eventually into mature plants.

21. Significance In taxa, in which conventional breeding is a problem due to incompatibility, protoplast culture and somatic hybridization may play a significant role. In taxa, in which conventional breeding is a problem due to incompatibility, protoplast culture and somatic hybridization may play a significant role. The fusion of a somatic hybrid protoplast with that of one of its parents is called somoJic back hybridization. The fusion of a somatic hybrid protoplast with that of one of its parents is called somoJic back hybridization.

22. Application of tissue culture Crop improvement Crop improvement Horticulture Horticulture Synthetic seeds production Synthetic seeds production Forestry Forestry Propagation of rare plants Propagation of rare plants Production of secondary metabolite Production of secondary metabolite Shortening of breeding cycle Shortening of breeding cycle Production of disease-free plants Production of disease-free plants