Callus culture is the growth of undifferentiated cells from plant explants on artificial nutrient media. The history of callus culture began in the 1920s with experiments on carrot tissue. Key developments included the discovery of the plant hormone auxin in 1928 and the establishment of callus cultures using auxin in the 1930s-1940s. Callus cultures are initiated from explants placed on media containing auxin and cytokinin, then subcultured regularly. Callus appears as an unorganized mass of cells that can be used for experiments or regenerated into plantlets.

2. CONTENTS
History
Callus culture
Physical appearance of a callus
Culture establishment :media preparation, surface sterilization ,
inoculation and incubation conditions
Subculturing
Growth measurement of callus
Techniques to develop callus culture
References

3. HISTORY
1924 - Callus culture of carrots )- R.Blumenthat
and P.Meyer - Pathological implications ,compared callus
tumour growth.
1927 - L.Rehwald - Cultivation of callus from carrot slices.
P.Boysen- Jensen – growth promoting substances found in
plant shoot tip would diffuse across a wound covered with
gelatin.
1928 – Frits Went collected this growth substance form coleptile
tips in tiny blocks of Agar.

4. 1934 – F.Kogl , A. J. Hagen Smith and H.Erxleben – isolation and
chemical analyses of the substance -plant hormone or auxin- indole
-3-acetic acid (IAA)
1937- R.Gautheret – undifferentiated carrot tissues
1939 – R.J.Gautheret and Nobecourt, France, growth of callus from
carrot cambium when using auxin in the nutrient medium.
P.R.White (USA) – Excised root tips of tomatoes( )in continuous
culture.
1939 – White reported successful culture of tobbaco ( callus.

5. 1941 – J.van Overbeek, M.E. Conklin and Albert
F.Blakeslee- Coconut milk stimulated
callus formation in cultures of excised
embryos of jimson weed
( ).
1943 – White – A Handbook of Plant Tissue
Culture, accumulated knowledge of PTC.

6. CALLUS CULTURE
Callus : An unorganised mass of loosely arranged
parenchymatous cells which develop from parent
tissues due to proliferation of cells.
Angiosperms , gymnosperms , pteridophytes and
bryophytes.
Has the potentiality to produce normal roots and
embryoids-plantlets.
Callus culture: development of an unorganised mass of cells from an
explant on an artificial medium supplemented with suitable PGR when
provided with appropriate environment (i.e. Proper incubation conditions)

7. Physical Appearance Of A Callus
HARDNESS : Hard (due to lignification of cell walls), brittle or
sometimes soft
COLOUR : Dirty or off white , creamish to brown or light
green to dark green.
Degree of darker pattern varies from plant to plant and
mainly depends upon the quantity of polyphenols present in the
plant species.
Higher the polyphenol content darker the callus appear, i.e.
Brown coloration on the culture.

8. Development Of Aseptic Callus Cultures
EXPLANTS : Juvenile tissues, seedlings , young shoots , buds ,
root tips, developing embryos: fruits, floral parts, tubers and
bulbs.
CULTURE MEDIUM :
Media: MS
Hormonal balance: Auxin : Cytokinin = 1
Carbon source :sucrose (3% w/v )
ph = 5.6 -6.0, optimum 5.8
Gelling agent

9. Surface Sterilization
Wash throughly tween-20
under running tap ----- Antifungal (Bavistin; 0.04% )
water Antibacterial agents
(Streptomycin sulfate;0.0 4 % )
__UNDER_ROOM_ENV____________________
↓ 15-20 min
Double Distilled Water
↓ washing
70 % Ethanol treatment
↓ 1 min / dip
UNDER LAMINAR AIR FLOW HgCl2 treatment
(0.04 or 0.02 %)
↓ 4-5 min
Washing with Autoclaved Water

10. Inoculation and Incubation
Nodal explant Leaf segments, buds, root tips etc.
Cutting of the nodal ends that
comes in contact with the surface
sterilant with the sterile surgical
blade
Place the explant in vertical
position on medium supplemented
with appropriate PGR with the help
of forceps.
Segmentation of the explant
into2-3 parts i.e. Basal medium
and tip with sterile surgical blade
Place the explant with their abaxial
surface in contact with the
medium with the help of sterile
inoculating forceps.

11. Further development -Organogenesis
Nodal explant Bud segments
Rose (shoot proliferation) Lilly (shooting)

12. Incubation :
Incubation conditions : The Environment
Temperature :25 ± 2⁰ C
Light : 5000-10,000 lux m
Duration of incubation: 16 hr light
08 hr dark

13. How a piece of explant gets converted
into the callus ?
Produced from the outer layers of cortical cells
in a stem explant by repetitive division of cells
These deviding cells create pressure on the epidermis ---
rupturing of the epidermis exposing newly formed callus
Separation of callus and then subculturing it.
2-3 weeks to grow (10-15 days) but sometimes 4 weeks.

14. Callus culture of

15. SUBCULTURING OF THE CALLUS CULTURE
3-4 weeks
250-500mg approx. pieces -------- transferred to the fresh
media.
NEED:
1) Nutrition depletion
2) Accumulation of toxic substances
3) Drying of media
ADVANTAGES :
1)Maintains the state of viability of cells
2)Provides fresh instalment of media for further growth

17. DISADVANTAGES
1) Cells lose the power to regenerate to a plantlet.
2)Chromosomal abbrations : polyploidy and aneuploidy. Polyploid
cells appear to originate through endoreduplication( additional
rounds of DNA replication without intervening cell division) ;
aneuploid cells-anaphase irregularities
3)Non chromosomal changes : changes in metabolic pathways and
alteration in composition of media.
4)Selection of explant size highly dependent upon the type of
glassware is being used for culture purpose.

18. Flow sheet (subculturing process)
Callus work station(glass plate , sterile)
cutting of the callus into small
pieces (250-500 mg approx)
fresh media having appropriate composition
and hormonal balance
OR
Agitation can be done (25-150 rpm)-------fresh media

19. Growth measurement of callus culture
Fresh weight or wet weight method:
Pre weighed (in wet condition) circular filter of nylon
fabric supported in a Hartley funnel
Cell washing to remove the medium
Draining under vacuum, reweighing .

20. Dry weight method
Collection of cells on pre weighed dry nylon filter paper
Drying of cells for 12 hours at 60⁰ C
Reweighing
Cell weight is expressed as per culture or as per 10⁶
cells

21. Calculating the Mitotic index
MI-ratio of nuclei undergoing mitosis(including prophase) to
total nuclei.
MI =>
No. Of nuclei in mitosis × 100
Total No. Of nuclei examined in the sample
A MI of 0.3 means that 30% of cells in the population are
observed in mitosis.

22. calculating the respiration rate
Utilization of carbon source and oxygen are related to metabolic
activity of cells.
More is the consumption of sugar and oxygen more active are
the cells.

23. Techniques/routes to culture callus
THE FILTER PAPER RAFT NURSE TECHNIQUE:
Muir et.al (1954)--- to culture single cells and friable calli of
tobacco marigold.
Cultivating individual cells on top of an actively growing
callus(of related sp) .
Use of micropipette or microspatula.
Placing 8× 8mm squares of filter paper on the nurse
tissue(callus), wetted with nutrients and liquid from the nurse
tissue
Colony dev-transferred to agar medium.

24. A number of recalcitrant species , notably monocotyledons such
as rice and maize.
Eg : Rice----- Lorium grass
THE MICROCHAMBER THECHNIQUE :
Jones et. Al (1960)
Replacement of nurse tissue with conditioned media(spent
media ; already supported the growth of a tissue, have certain
growth factors that promote/boast the growth of another plant
tissue)

25. Process
A drop of the medium carrying the cells of interest is
isolated from suspension
Placing on a sterile microscope slide and ringed with sterile
mineral oil (a drop on either side of the culture drop and
coverglass placed on each drop
Placing of third coverglass----- formation of microchamber
slide placed in petri plate and incubated
development of cell colonies-------fres media

26. PLATING TECHNIQUES
1) POUR PLATE : Each dilution is mixed with 15 ml of molten
agar medium. Temp-48-50 ⁰ C
Pouring in sterile petri plate
Incubation and the colonies develops

27. SPREAD PLATE TECHNIQUE:
Agar plate should be dried for 15 min at 55⁰ C
for 45 min at 37⁰ C.
Add 0.2 ml of portions from each dilution and spread evenly
on surface of media
Disadvantage: some cells are taken up by the glass rod so this will
decrease the count.

28. DIALYSIS TUBING TECHNIQUE:
Street and Steward (1969)
Mainly involved in cell suspension cultures
Used when cell concentration is less tahn the critical cell
density=> 9 -15 × 10³ cells/ml
Carried out in dialysis tube
A high cell density of nursing tissues of closly related
species which is more responsive is selected
Use of conditioned media, high conc of growth factors
Nurse cell grows well and the growth factors diffuses out and
provide nutrition for growth of cells of interested callus of low
density.

29. Cell Suspension Culture
Definition:
Suspension culture is a type of culture in which single cells or
small aggregates of cells mul‑tiply while suspended in agitated
liquid medium. It is also referred to as cell culture or cell
suspen‑sion culture

30. Principle:
Callus proliferates as an unorganised mass of cells. So it is
very difficult to follow many cellular events during its growth
and develop‑mental phases. To overcome such limitations of
callus culture, the cultivation of free cells as well as small cell
aggregates in a chemically de‑fined liquid medium as a
suspension was initi‑ated to study the morphological and
biochemi‑cal changes during their growth and developmen‑tal
phases.

31. To achieve an ideal cell suspension, most commonly a friable
callus is transferred to agitated liquid medium where it breaks
up and readily disperses. After eliminating the large cal‑lus
pieces, only single cells and small cell aggre‑gates are again
transferred to fresh medium and after two or three weeks a
suspension of actively growing cells is produced.

32. This suspension can then be propagated by regular sub-culture
of an aliquot to fresh medium. Ideally suspension cul‑ture
should consist of only single cells which are physiologically and
biochemically uniform. Al‑though this ideal culture has yet to be
achieved, but it can be achieved if it is possible to synchro‑nize
the process of cell division, enlargement and differentiation
within the cell population.

33. The culture of single cells and cell aggregates in mov‑ing liquid
medium can be handled as the culture of microbes. The
suspension culture eliminates many of the disadvantages
ascribed to the cal‑lus culture on agar medium. Movement of
the cells in relation to nutrient medium facilitates gaseous
exchange, removes any polarity of the cells due to gravity and
eliminates the nutrient gradients within the medium and at the
surface of the cells.

34. Protocol:
1. Take 150/250 ml conical flask containing autoclaved 40/60
ml liquid medium (Fig 4.1).

36. . 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.
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.

37. 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.

38. References
Plant Biotechnology by Purohit
Plant tissue culture by S.S.Bhojwani and M.K.Razdan
Introduction to Biotechnology by A.K.Panday and K.S. Bilgrami.
Biotechnology by B.D.Singh
Plant Biotechnology by K.G.Ramawat
Methods in Plant tissue culture by U.Kumar
Intenet:http://google.com
http://yahoo.com