Induction and maintenance of callus and cell suspension cultures.Callus Culture : ● Callus represents an unorganized or undifferentiated mass of cells. ● They are generally composed of parenchymatous cells and usually undergo division. ● When an explant is cultured in a medium supplemented with sufficient amount of auxins, it starts producing mass of cells from the surface of the explant. ● The concentration of auxins required for each type of explant will be different and is mainly dependent on the physiological state of the explant tissue. ● Callus cultures can be maintained for a very long time by intermittent sub-culturing to a fresh medium. ● The callus cultures can be manipulated for different purposes by changing the hormone concentrations in the media Usage of callus Culture : ● Rregeneration of plantlets. ● Preparation of single cells ● Suspension cultures. ● Protoplasts preparation ● Genetic transformation studies. In some instances, it is necessary to go through a callus phase prior to regeneration via somatic embryogenesis or organogenesis. ● Callus cultures are suitable for the generation of useful somaclonal variants. ● Callus cultures can be used for in vitro selection of cells and tissue variants Induction of callus Culture : ● Induction of cell division in the permanent tissue is highly dependent on the high auxin content (e.g., 2, 4-D) in the medium. ● The hormone requirement for callus induction may be auxin alone, cytokinin alone or auxin and cytokinin in different ratios. ● The type of growth regulator requirement and its concentration in the medium depends strongly on the genotype and endogenous hormone content of an explant Transformation of any kind of tissue explant into proliferating callus mass depends highly on the plant genotype, the source of origin of the explant and the physiological state of the tissue at culture. ● For the initiation of callus cultures tissues from young seedling or from juvenile part of the mature plant either grown in vivo or in vitro are generally taken, either steri lised (when grown in vivo) or cut aseptically (when grown in vitro) and inoculated aseptically on a nutrient medium provided with different combinations of exogenous growth substances Maintenance of Callus Culture: ● After callus induction, the callus is grown further on a new medium, which is referred to as sub-culturing. ● During subculturing the callus tissue shows sigmoid growth pattern on a particular media. ● The time interval for sub-culturing is calculated according to the growth pattern of the particular tissue type and genotype of the plant. ● When the callus growth reaches to the stationary phase of growth then it needs to be cultured on to a fresh medium. ○Maintenance of Callus Culture: ● After callus induction, the callus is grown further on a new medium, which is referred to as sub-culturing. ● During subculturing the callus tissue shows sigmoid growth pattern on a particular media. ● The time interval for sub-cultur

1. VSPGC KAIRANA ( SHAMLI )
Assignment on
Paper code-H4004
Induction and maintenance of callus and cell suspension culture
Paper Title--Plants Cell Tissue and Organ Culture.
Submitted To : Submitted By:
Dr Rakesh Kumar
HOD, BOTANY
VSPGC
KM Nabila
Msc (botany)
4th sem

2. Induction and maintenance of callus and
cell suspension cultures.

3. Callus Culture :
● Callus represents an unorganized or undifferentiated mass of cells.
● They are generally composed of parenchymatous cells and
usually undergo division.
● When an explant is cultured in a medium supplemented with
sufficient amount of auxins, it starts producing mass of cells from
the surface of the explant.
● The concentration of auxins required for each type of explant will
be different and is mainly dependent on the physiological state of
the explant tissue.
● Callus cultures can be maintained for a very long time by
intermittent sub-culturing to a fresh medium.
● The callus cultures can be manipulated for different purposes by
changing the hormone concentrations in the media.

4. Usage of callus Culture :
● Rregeneration of plantlets.
● Preparation of single cells
● Suspension cultures.
● Protoplasts preparation
● Genetic transformation studies.
...Callus Culture can be used for

5. ● In some instances, it is necessary to go through a callus phase prior to
regeneration via somatic embryogenesis or organogenesis.
● Callus cultures are suitable for the generation of useful somaclonal variants.
● Callus cultures can be used for in vitro selection of cells and tissue variants.

6. Induction of callus Culture :
● Induction of cell division in the permanent tissue is highly
dependent on the high auxin content (e.g., 2, 4-D) in the
medium.
● The hormone requirement for callus induction may be auxin
alone, cytokinin alone or auxin and cytokinin in different ratios.
● The type of growth regulator requirement and its concentration
in the medium depends strongly on the genotype and
endogenous hormone content of an explant.

7. ● Transformation of any kind of tissue explant into proliferating
callus mass depends highly on the plant genotype, the source
of origin of the explant and the physiological state of the tissue
at culture.
● For the initiation of callus cultures tissues from young seedling
or from juvenile part of the mature plant either grown in vivo or
in vitro are generally taken, either steri lised (when grown in
vivo) or cut aseptically (when grown in vitro) and inoculated
aseptically on a nutrient medium provided with different
combinations of exogenous growth substances.

8. ● If the callusing is difficult to induce or if juveline tissue is
needed then imma ture embryos or seedlings or parts of
these are used. Endogenous hormone level depends on
the type of explants and its original position in the plant
which has an important influ ence in processes of cell
division.

9. ● Callus initiation on an explant. ● Callus initiation of a growing callus
culture

10. Maintenance of Callus Culture:
● After callus induction, the callus is grown further on a new
medium, which is referred to as sub-culturing.
● During subculturing the callus tissue shows sigmoid growth
pattern on a particular media.
● The time interval for sub-culturing is calculated according to
the growth pattern of the particular tissue type and genotype
of the plant.
● When the callus growth reaches to the stationary phase of
growth then it needs to be cultured on to a fresh medium.
○

11. Callus growth can be monitored by :
i) Fresh weight measurement (most convenient)
(ii) Dry weight measurement (more accurate)
(iii) Mitotic index measurement (for cell divisional rate).

12. Habituation :
This means that the callus tissue is able to grow on a standard
maintenance medium or basal medium which is devoid of
growth hormone. This property of callus tissue is called as
habituation.

13. ● Generally, maintenance of callus tissue growth needs
growth hormones in order to grow as long as they are
maintained through serial subcultures. But it has been
observed also that the callus tissue in some plant species
may become habituated after prolonged culture.

14. Cell-Suspension Culture :
● Suspension culture is a type of culture in which single cells or small aggregates
of cells multiply suspended in agitated liquid medium. It is also referred to as
cell culture or cell suspension cultu

15. Establishment of cell suspension
culture
● By callus Culture
● Enzymatic method

16. By callus culture —
● Single-cell cultures and suspension cultures can be established from callus cultures
by transferring a piece of callus tissue into liquid medium and subjecting it to
continuous shaking.
● A piece of the callus is transferred to a liquid medium in a vessel such as an
Erlenmeyer flask and the vessel placed on a rotary or reciprocal shaker.
● The culture conditions depend on plant species and other factors, but in general, the
cells are cultivated at 100 rpm on a rotary shaker at 25°C. By subculturing for several
generations, a fine cell suspension culture containing small-cell aggregates and
single cells is established.

17. Enzymatic method—
● Enzymatic methods can also be adopted for establishing a fine cell-suspension
culture.
● This is based on the use of certain pectin digesting enzymes in the culture
medium, such as pectinase or macerozyme.
● These enzymes act on the pectin, which joins two adjacent cells in plant
tissues, so that the cells become independent and grow freely as single cells.

18. Types of cell suspension culture—
These cultures are maintained by continuously propagating a small amount of the
liquid of the inoculum in a moving liquid medium and transferring it into a fresh
medium, at regular intervals. Cell suspensions are generally grown in100 to 250 ml
flask. Each culture flask contains 20-75 ml of the culture media. The flask
containing suspension culture is allowed to stand still for a few seconds after which,
the large colonies settle down. The suspension is then taken from the upper part of
the culture with the help of a pipette or syringe and transferred to a fresh medium.
● Batch culture

19. The biomass growth in batch culture follows a fixed pattern :
When the cell number in a suspension culture is plotted against the time
of incubation, a growth curve is obtained. The curve reveals that initially
the culture passes through a log phage followed by a brief exponential
phase (the most fertile period for active cell division). The growth declines
after 3 to 4 generation. This denotes that the culture has entered a
stationary phase. Batch culture shows a constant change in cell growth
and metabolism so they are not considered to be an ideal system for the
study of cellular behaviour.

20. ● Graph showing different growth phase in a Batch culture

21. Continuous culture:
The large-scale culture grown under steady state for long periods by adding fresh medium and
draining out the used medium in a number of specially designed culture vessels are known as
continuous or mass culture. Continuous cultures are of two types:
● Close type
● Open type
In close type, the addition of fresh medium is balanced by the outflow of the old medium. The cells
passing through the outgoing medium are separated and reintroduced in the culture. With the
proceeding growth, the cell biomass continues to increase. In open type the inflow of the medium
is done by balancing harvest of an equal volume of the culture medium and cells. This allows the
indefinite maintenance of cultures at a constant and submaximal growth rate. Basically there are
two major types of (continuous) cultures. Example: chemostat and turbidostat.

22. Usage of cell suspension :
● It can be used for the bioproduction of certain important
phytochemicals or secondary metabolites.
● Inducing somatic embryogenesis.
● Preparation of artificial seeds.
● Production of somatic mutation.
● selection of mutants by screening the cells just like
microbial cultures.

23. Protocol:
1. Take 150/250 ml conical flask containing autoclaved 40/60 ml liquid medium .
2. Transfer 3-4 pieces of pre-established callus tissue (approx. wt. 1 gm. each) from
the culture tube using the spoon headed spat ula to conical flasks.
3. Flame the neck of conical flask, close the mouth of the flask with a piece of
allumini um foil or a cotton plug. Cover the closure with a piece of brown paper.
4. Place the flasks within the clamps of a ro tary shaker moving at the 80-120 rpm
(rev olution per minute).
5. After 7 days, pour the contents of each flask through the sterilized sieve pore
diameter -60µ- 100µ and collect the filtrate in a big sterilized container. The filtrate
contains only free cells and cell aggregates.

24. 6. Allow the filtrate to settle for 10-15 min. or centrifuge the filtrate at 500 to 1,000 rpm and
finally pour off the supernatant.
7. Re-suspend the residue cells in a requisite volume of fresh liquid medium and dispense
the cell suspension equally in several ster ilized flasks (150/250 ml). Place the flasks on
shaker and allow the free cells and cell aggregates to grow.
8. At the next subculture, repeat the previous steps but take only one-fifth of the residual
cells as the inoculum and dispense equally in flasks and again place them on shaker.
9. After 3-4 subcultures, transfer 10 ml of cell suspension from each flask into new flask
containing 30 ml fresh liquid medium.
10. To prepare a growth curve of cells in sus pension, transfer a definite number of cells
measured accurately by a haemocytometer to a definite volume of liquid medium and
incubates on shaker. Pipette out very little aliquot of cell suspension at short intervals of
time (1 or 2 days interval) and count the cell number. Plot the cell count data of a passage
on a graph paper and the curve will indicate the growth pattern of suspension culture.

25. Th
● Flow diagram illustrating the method of cell suspension culture and plant regeneration

26. THANKS

27. References ;
Biology discussion.com
INTRODUCTION TO BIOTECHNOLOGY AND GENETIC ENGINEERING—A.J.NAIR.