Transcript terminology used in tissue culture

TERMINOLOGY
USED IN TISSUE
CULTURE
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.
Auxenic or Aseptic culture
Cultures devoid of foreign or undesired life forms are
called auxenic culture.
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.
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.
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
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.
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.
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.
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.
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.
Isolation of protoplasts



all plant parts but leaf mesophyll is the most
preferred tissue.
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:


Mechanical disruption of cell wall and
Enzymatic dissolution of cell wall
Mechanical disruption






Experimental cells are allowed to plasmolyse by
keeping them in hypertonic solution.
In plasmolysed state, cell wall is cut with a sharp
knife.
Plasmolysed cell is transferred to hypotonic solution.
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.
Damaged cells may release chemical which might
affect culture of protoplast.
Enzymatic dissolution

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.
Pectinase breaks the tissues into cells by dissolving
calcium pectate of middle lamella.
Hemicellulase and cellulase break down the cell wall.
Commercially available enzymes are "Pectolyase Y23", Onozuka R-1O. Macerozyme.




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.
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.
Enzymatic protoplast isolation comprises of
several steps:
 Surface sterilization of leaf sample
 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.
 Enzymatic treatment
 Purification of isolated protoplasts.
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).
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.
Protoplast purification







Enzyme solutions are filtered with nylon mesh to
remove insoluble impurities.
Filtrate is centrifuged for 5 minutes at 700 rpm.
The protoplast forms pellet and goes at the bottom of
contrifuge tube.
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 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
Culture of Protoplast









hanging drop culture technique.
Two protoplasts isolated from genetically different plant cells are fused.
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 is intraspecific and rare.
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.
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

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.
Regeneration of Plants:




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.
This favours embryogenesis after 3-4 weeks.
Embryoids develop into seedlings and eventually into
mature plants.
Significance

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.
Application of tissue culture








Crop improvement
Horticulture
Synthetic seeds production
Forestry
Propagation of rare plants
Production of secondary metabolite
Shortening of breeding cycle
Production of disease-free plants