TERMINOLOGY TERMINOLOGY USED IN TISSUE USED IN TISSUE

CULTURE CULTURE

AcclimatizationAcclimatizationIt is a process of gradual hardening of the cultured It is a process of gradual hardening of the cultured plants from laboratory to field so that the regenerated plants from laboratory to field so that the regenerated

plants should adjust to field conditions. plants should adjust to field conditions.

AndrogenesisAndrogenesis It is a process of regeneration of plants from pollen It is a process of regeneration of plants from pollen

grains. Such plants are haploids.grains. Such plants are haploids.

Auxenic or Aseptic cultureAuxenic or Aseptic cultureCultures devoid of foreign or undesired life forms are Cultures devoid of foreign or undesired life forms are

called auxenic culture. called auxenic culture.

Basal mediumBasal mediumMost of the media contain inorganic salts of major and minor Most of the media contain inorganic salts of major and minor

elements, vitamins and sucrose. A medium with these elements, vitamins and sucrose. A medium with these ingredients is called ingredients is called basal medium. basal medium.

CallusCallusMass of undifferentiated cells produced in tissue culture is called Mass of undifferentiated cells produced in tissue culture is called

callus. callus. The callus is highly vacuolated and unorganised cells. The callus is highly vacuolated and unorganised cells.

CloneCloneA clone is a group of plants produced from a single explant A clone is a group of plants produced from a single explant through asexual reproduction. All the members of a clone have through asexual reproduction. All the members of a clone have the same genotype as'" that of the parent. They are identical in the same genotype as'" that of the parent. They are identical in

genotype. Phenotypic differences within a clone are due to genotype. Phenotypic differences within a clone are due to external factors. external factors.

CryobiologyCryobiologyIt is a technique of preservation of plant tissues and It is a technique of preservation of plant tissues and

cells in liquid nitrogen at 196°C. This is highly cells in liquid nitrogen at 196°C. This is highly significant for storage of germplasm of those crops significant for storage of germplasm of those crops which do not produce seeds and reproduce only by which do not produce seeds and reproduce only by

vegetative means. Thus cryobiology is very useful in vegetative means. Thus cryobiology is very useful in crop improvement. crop improvement.

ExplantExplantSmall piece of viable tissues isolated from parent plant Small piece of viable tissues isolated from parent plant

is called is called explant .explant .

InoculationInoculationTransfer of explant to culture medium is called Transfer of explant to culture medium is called

inoculation.inoculation.

In vitroIn vitroCells/tissues removed from the intact organism and Cells/tissues removed from the intact organism and

grown in controlled condition in laboratory. grown in controlled condition in laboratory.

Micropropagation (clone propagation)Micropropagation (clone propagation)It is a process of production of clones similar to asexual It is a process of production of clones similar to asexual

reproduction. Plantlets are produced from shoot reproduction. Plantlets are produced from shoot tips/axillary buds on culture medium omitting callus tips/axillary buds on culture medium omitting callus

phase. Here, small amount of explant produce phase. Here, small amount of explant produce millions of clonal parts in a year directly. millions of clonal parts in a year directly.

OrganogenesisOrganogenesisProcess of differentiation of callus initially into embryo Process of differentiation of callus initially into embryo

like structure (embryoids) and then showing organ like structure (embryoids) and then showing organ like roots/shoots like roots/shoots

ProtoplastProtoplastAnimal, plant or fungal cell from which the Animal, plant or fungal cell from which the

entire cell wall has been removed. entire cell wall has been removed.

RegenerationRegenerationProduction of entire plant from explant is called Production of entire plant from explant is called

regeneration. regeneration.

Shoot tip cultureShoot tip cultureCulture of shoot tip (shoot apical meristem) Culture of shoot tip (shoot apical meristem)

along with one or more leaf primordia or along with one or more leaf primordia or mature leaves is called shoot tip culture. mature leaves is called shoot tip culture.

SomacloneSomaclone

Plants derived from somatic cell culture. Plants derived from somatic cell culture.

Somaclonal variationSomaclonal variation

Somaclonal variations are heritable variations for both Somaclonal variations are heritable variations for both qualitative and quantitative traits produced in plants qualitative and quantitative traits produced in plants

regenerated from cell culture and tissue cultures. regenerated from cell culture and tissue cultures. Variation caused during plant tissue culture is Variation caused during plant tissue culture is

called called somaclonal variation. somaclonal variation. Somaclonal variations Somaclonal variations arise as a result of chromosome structural changes, arise as a result of chromosome structural changes,

gene mutations, plasma gene mutations, gene gene mutations, plasma gene mutations, gene amplification, transposable elements etc. amplification, transposable elements etc.

Somatic embryogenesisSomatic embryogenesis

Process of production of embryo from somatic cells is Process of production of embryo from somatic cells is called called somatic embryogenesis somatic embryogenesis and such embryo is and such embryo is

called called embryoid.embryoid.

Stock plantStock plant

The plant from which explant is taken is called The plant from which explant is taken is called stock stock plant. plant.

Sub-cultureSub-culture

Callus produced and cultured again for production of Callus produced and cultured again for production of big mass of callus is called big mass of callus is called sub-culture.sub-culture.

Suspension cultureSuspension culture

A type of liquid culture in which cells or cell A type of liquid culture in which cells or cell aggregates grow and multiply. aggregates grow and multiply.

Transplant stageTransplant stage

Process of transfer of regenerated plants from Process of transfer of regenerated plants from test tube (i.e. in vitro) to the soil is called test tube (i.e. in vitro) to the soil is called

transplant stage.transplant stage.

Steps of Somatic HybridizationSteps of Somatic Hybridization

There are three steps of somatic hybridization:There are three steps of somatic hybridization:

(i) Isolation of protoplasts (i) Isolation of protoplasts

(ii) Fusion of protoplasts of desired species (ii) Fusion of protoplasts of desired species

(iii) Culture of hybrid protoplast to produce (iii) Culture of hybrid protoplast to produce whole plants.whole plants.

Isolation of protoplastsIsolation of protoplasts all plant parts but leaf mesophyll is the most all plant parts but leaf mesophyll is the most

preferred tissue. preferred tissue. after removal of cell wall.after removal of cell wall. Precautions are taken to remove cell wall Precautions are taken to remove cell wall

without damaging the cell cytoplasm and without damaging the cell cytoplasm and nuclear constituents. It is done by two nuclear constituents. It is done by two methods: methods:

Mechanical disruption Mechanical disruption of cell wall and of cell wall and Enzymatic dissolution Enzymatic dissolution of cell wall of cell wall

Mechanical disruptionMechanical disruption Experimental cells are allowed to plasmolyse by Experimental cells are allowed to plasmolyse by

keeping them in hypertonic solution. keeping them in hypertonic solution. In plasmolysed state, cell wall is cut with a sharp In plasmolysed state, cell wall is cut with a sharp

knife. knife. Plasmolysed cell is transferred to hypotonic solution. Plasmolysed cell is transferred to hypotonic solution. This results in the release of protoplast in outer This results in the release of protoplast in outer

solution through cut ends. solution through cut ends. This method is suitable only for tissues with large This method is suitable only for tissues with large

cells in which evident plasmolysis occurs. cells in which evident plasmolysis occurs. Damaged cells may release chemical which might Damaged cells may release chemical which might

affect culture of protoplast. affect culture of protoplast.

Enzymatic dissolutionEnzymatic dissolution

Cell walls are dissolved by enzymes. Cell walls are dissolved by enzymes. Such enzymes are extracted from fungi, bacteria and Such enzymes are extracted from fungi, bacteria and

Roman snail (Helix pomatia). Macerozyme, a pectinase Roman snail (Helix pomatia). Macerozyme, a pectinase enzyme is obtained from enzyme is obtained from Rhizopus Rhizopus fungus, Driselase. a fungus, Driselase. a mixture of cellulase and pectinase is obtained from mixture of cellulase and pectinase is obtained from Trichoderma viride. Trichoderma viride.

Pectinase breaks the tissues into cells by dissolving Pectinase breaks the tissues into cells by dissolving calcium pectate of middle lamella. 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-Commercially available enzymes are "Pectolyase Y-

23", Onozuka R-1O. Macerozyme. 23", Onozuka R-1O. Macerozyme.

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

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

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

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

Enzymatic protoplast isolation comprises of Enzymatic protoplast isolation comprises of several steps: several steps:

Surface sterilization of leaf sample Surface sterilization of leaf sample Rinsing in suitable plasmolyticum with Rinsing in suitable plasmolyticum with

distilled water distilled water Peeling of off the lower epidermis towards Peeling of off the lower epidermis towards

margin with sharp forceps below the junction margin with sharp forceps below the junction of a lateral vein and midrib. of a lateral vein and midrib.

Enzymatic treatment Enzymatic treatment Purification of isolated protoplasts. Purification of isolated protoplasts.

Enzymatic treatmentEnzymatic treatment

Plasmolysed excised leaf cells are transferred Plasmolysed excised leaf cells are transferred to petri dishes containing enzyme solution (3% to petri dishes containing enzyme solution (3% macerozyme and 2% Okozuka cellulase macerozyme and 2% Okozuka cellulase dissolved in 13% mannitol at pH 5.4). dissolved in 13% mannitol at pH 5.4).

Petri dishes are sealed with paraffin, wrapped Petri dishes are sealed with paraffin, wrapped in aluminium foil and incubated for 12-18 in aluminium foil and incubated for 12-18 hours at 25°C. hours at 25°C.

The leaf portions are then teased slowly with The leaf portions are then teased slowly with forceps to release protoplasts. forceps to release protoplasts.

Protoplast purificationProtoplast purification Enzyme solutions are filtered with nylon mesh to Enzyme solutions are filtered with nylon mesh to

remove insoluble impurities. 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 The protoplast forms pellet and goes at the bottom of

contrifuge tube. 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 The pellet at the base is suspended in 10 ml of MS

medium plus mannitol and the process is repeated medium plus mannitol and the process is repeated thrice. thrice.

The resultant protoplast is pure. The resultant protoplast is pure. Outlines of isolation, purification, culture and Outlines of isolation, purification, culture and

regeneration of protoplasts are shown in figure below. regeneration of protoplasts are shown in figure below.

Outlines of isolation, purification, culture and Outlines of isolation, purification, culture and regeneration of protoplastsregeneration of protoplasts

Culture of ProtoplastCulture 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. Interaction-of two protoplasts leads to formation of somatic hybrids (Fig.

25.7). 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 Physical and chemical agents which reduce charges on protoplasts

surfaces, are used for the purpose. surfaces, are used for the purpose. Physical method includes electrofusion and chemical method includes the Physical method includes electrofusion and chemical method includes the

use of polyethylene glycol followed by elution with Ca++ ions at high pH. use of polyethylene glycol followed by elution with Ca++ ions at high pH. PEG agglutinates protoplast while Ca++ allows coalescence and PEG agglutinates protoplast while Ca++ allows coalescence and

cytoplasmic mixing. cytoplasmic mixing. During aggregation the plasma membrane of the adjacent protoplasts are During aggregation the plasma membrane of the adjacent protoplasts are

tightly adpressed over a portion of protoplast surface tightly adpressed over a portion of protoplast surface

Regeneration of Cell Wall: Regeneration of Cell Wall: Protoplasts begins to form new cell wall when it is cultured Protoplasts begins to form new cell wall when it is cultured

in suitable nutrient medium. Cell wall is formed in few in suitable nutrient medium. Cell wall is formed in few days. days.

Regeneration of Plants: Regeneration of Plants: Somatic hybrids obtained are now grown in culture Somatic hybrids obtained are now grown in culture

solution. It develops callus. solution. It develops callus. Callus is now sub-cultured in culture solution Callus is now sub-cultured in culture solution

supplemented with mannitol and auxin. 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 Embryoids develop into seedlings and eventually into

mature plants. mature plants.

SignificanceSignificance

In taxa, in which conventional breeding is a In taxa, in which conventional breeding is a problem due to incompatibility, protoplast problem due to incompatibility, protoplast culture and somatic hybridization may play a culture and somatic hybridization may play a significant role. significant role.

The fusion of a somatic hybrid protoplast with The fusion of a somatic hybrid protoplast with that of one of its parents is called that of one of its parents is called somoJic somoJic back hybridization.back hybridization.

Application of tissue cultureApplication of tissue culture

Crop improvementCrop improvement HorticultureHorticulture Synthetic seeds productionSynthetic seeds production ForestryForestry Propagation of rare plantsPropagation of rare plants Production of secondary metaboliteProduction of secondary metabolite Shortening of breeding cycleShortening of breeding cycle Production of disease-free plantsProduction of disease-free plants