Suspension Culture and Single Cell Cultures, Culturing methods, maintenance and application Generally, suspension culture is a one stop technology to produce secondary metabolites on a large scale in-vitro, irrespective of the climatic condition or nutrient availability (as required in field plants). In this presentation, we will see the importance of suspension culture, culturing methods and it's application (mostly with respect to plants) and also focus on what exactly is a single cell culture.

1. Suspension Culture and Single Cell
Cultures, Culturing methods,
maintenance and application

2. UNIVERSITY OF AGRICULTURALSCIENCES,BANGALORE
Presentation on
“Suspension Culture and Single Cell Cultures, Culturing
methods, maintenance and application ”
DEPARTMENTOF PLANTBIOTECHNOLOGY
PBT-606- Commercial Plant Tissue Culture
ANANYA
1ST PhD
PAMB0077

3. INTRODUCTION
• Plant Tissue Culture (PTC) is defined as a
collection of experimental methods of
growing plant cells, tissues and organs in an
artificially prepared nutrient medium static
or liquid, under aseptic conditions
• It is also referred to as micropropagation
• It was introduced by G. Haberlandt
• The basic key used in plant tissue culture is
the totipotency of plant cells, meaning that
each plant cell has the potential to
regenerate into a complete plant
• With this characteristic, plant tissue culture is
used to produce genetically identical plants
(clones) in the absence of fertilization,
pollination or seeds

4. Need of suspension cultures?

5. Cell suspension Culture
• The cell suspension culture also called as the plant cell
culture is a system for production of fine chemicals
• It can be defined as “The culture of tissue and cells
cultured in liquid nutrient medium, producing a
suspension of single cells and cell clumps“
• Cell suspension culture is the primary route for studying
plant cell secondary metabolism
• The cell suspension culture requires optimization of the cell
line, the cultivation media, and the bioreactor system.

6. • Friable calli are particularly useful for
the establishment of cell suspension
cultures, which are populations of
rapidly growing undifferentiated cells
grown in a liquid medium (Evans et
al.,2003 Chourey et al., 1985)
• Cell suspension cultures are useful
experimental systems due to the high
rates of cell multiplication, short life
cycle, and their ease of maintenance by
sub-culturing into fresh medium (Cai et
al., 1987)
• With generations of sub-cultures, single
cells slowly break off from cell clusters,
resulting in a finely suspended culture
whose growth parameters, such as cell
multiplication and viability, can be
assessed

7. 1) Isolation of single cell from plant organs:
• Most suitable material for isolation of single cell is the
leaf tissue; this is because the leaf has homogeneous
population of mesophyll cells. These are isodiametric and
loosely arranged and can be obtained easily. From such
intact plant organs like leaf tissues single cells can be
isolated by using two methods:
●Mechanical method
●Enzymatic method

8. Mechanical method:
Gnanam and Kulandaivelu (1969) developed
a process which has been used successfully
to isolate mesophyll cells (which are active in
photosynthesis and respiration) from mature
leaves of several species of dicots and
monocots.
The procedure involves mild maceration of 10
gram leaves in 40 ml of grinding medium (20
𝜇mol sucrose, 10 𝜇 mol MgCl2, 20 𝜇 mol
Tris HCL buffer with PH 7.8) with a mortar
and pestle. The homogenate is passed through
two layers of muslin cloth and the cells thus
released are washed by centrifugation at low
speed using the same medium.

9. Enzymatic method:
• First done by Takebe and his co-workers in 1968
• the leaf tissues are dissolved in a mixture of enzymes
pectinase, cellulase/ hemicellulase. Sometimes a mixture of
enzymes is taken to dissolve cellulose, hemicellulose and
middle lamella
• Mecerozyme can also be used to dissolve middle lamella. A
proper osmoticum is added before putting the tissues in the
solution of enzymes to prevent plasmolysis
• The middle lamella and the cell wall get dissolved and a
large number of metabolically active cells are obtained.

10. • Isolation of single cells by enzymatic method is more
convenient because a high yield of single cells is
obtained from spongy parenchyma with minimum
damage or injury to the cells. A Friable callus is
necessary for getting a fine cell suspension in a liquid
medium. However, it is difficult to obtain single cells of
cereals with this method because the mesophyll cells of
cereals are elongated with interlocking constructions
which prevent their isolation.
Bhojwani S.S and Razdan M.K.; 2006; Plant Tissue Culture : Theory and Practise, a Revised Edition

11. Isolation from cultured tissues :
• Freshly cut pieces of surface sterilized plant organs are
simply placed on suitable culture medium to initiate calluses.
The callus is transferred to a fresh medium to build a mass of
tissues. Repeated subcultures make the callus friable. A piece
of friable callus is then transferred in a continuously agitated
liquid medium. Agitation is done by placing the culture flask
on an Orbital Platform Shaker.
• Movement of the culture medium exerts mild pressure on
small pieces of tissue breaking them into free cells or small cell
aggregates.

12. Method of cell suspension culture
Bhojwani S.S and Razdan M.K.; 2006; Plant Tissue Culture : Theory and Practise, a Revised Edition

13. Types Of Cell Suspension Cultures
• There are two types of cellsuspension cultures :
A. Batch culture
B. Continuous culture
• Each of these cultures have its own advantage and all
types are being used in practice.

14. https://ib.bioninja.com.au/options/untitled/b1-microbiology-organisms/batch-versus-continuous.html
BATCH CULTURE Versus CONTINOUS CULTURE

15. A. Batch Culture
• Batch culture is a type of cell
suspension where the cell
material grows in a finite
volume of agitated liquid
medium
• These cultures are maintained
continuously by sub culturing
• Batch cultures are most
commonly maintained in
conical flasks incubated on
orbital platform shakers at the
speed of 80- 120 rpm
• It is a closed system, with no
additions or removal of
nutrient and waste products
during the period of
incubation

16. Types of Batch Culture
1. Slow rotating cultures :
o Single cells and cell aggregates are grown in a specially designed flask, the
nipple flask.
o Each nipple flask possesses eight nipple like projections, having a capacity
of 250ml
o They are loaded in a circular manner on the large flat disc of vertical shaker
o When the flat disc rotates at a speed of 1-2rpm, the cells within each
nipple of the flask are alternatively bathed in the culture medium and
exposed to air.

17. 2. Shaker cultures :
• very and effective
system
• single cells and cell
aggregates in fixed
volume of liquid
medium are placed in
conical flasks
• These flasks are then
mounted with the help
of clips on a
horizontal large square
plate of an orbital
platform shaker
• The square plate
moves in acircular
motion at the speed of
60-180 rpm.

18. 3. Spinning Cultures:
• In this culture
system, large bottles
are used, usually
bottles with the
capacity of 10L
• Large volumes of
cell suspension is
cultured in 10L
bottles, with the
bottles spinning in a
spinner at 120 rpm at
an angle of 45°

19. 4. Stirred Culture :
o This system is used for large scale batch culture.
o In this method, the large culture vessel (round-
bottom flask) is not rotated but the cell
suspension inside the vessel is kept dispersed
continuously by bubbling sterile air through the
culture medium
o Internal magnetic stirrer is used to agitate the
culture medium safely
o The magnetic stirrer revolves at 200-600 rpm.

21. B. Continuous Culture
• In continuous culture system, the old liquid
medium is replaced continuously by the fresh
liquid medium to stabilize the physiological
states of the growing cells
• In this system, nutrient depletion does not
occur due to the continuous flow of nutrients
and the cells always remain in the steady
growth phase
• Continuous culture is further divided into
two types
1. In closed type, the used medium is replaced
with the fresh medium, hence, the cells from
used medium are mechanically retrieved and
added back to the culture and thus, the cell
biomass keeps increasing

22. 2. In open type, both the cells and used medium are replaced
with fresh medium thus maintaining culture at constant and
submaximal growth rate
• Open continuous cell suspension culture is of two types :
i. Chemostat :
o Culture vessels are usually cylindrical or circular in shape and
possess inlet and outlet pores for aeration and the introduction
and removal of cells and medium
o Such a system are maintained in a steady state
o Thus in a steady state condition the density, growth rate,
chemical composition and metabolic activity of the cells all
remain constant
o Such continuous cultures are ideal for studying growth
kinetics and the regulation of metabolic activity in higher
plants

24. ii. Turbidostats :
o A turbidostat is a continuous
culturing method where the
turbidity of the culture is held
constant by manipulating the rate
at which medium is fed.
o the cells are allowed to grow upto
a certain turbidity, when the
predetermined volume of culture
is replaced by fresh culture
o The turbidity is measured by the
changes of optical density of
medium
o An automatic monitoring unit is
connected with the culture vessel
and such unit adjusts the medium
flow in such a way as to maintain
the optical density or PH at chosen,
present level.

25. Growth kinetics
1. Initial lag dependent on
dilution
2. Exponential phase (dt
1-30 d)
3. Linear/deceleration
phase (declining
nutrients)
4. Stationary (nutrients
exhausted) Plant Cell Suspension typical Growth curve
16
14
12
10
8
6
4
2
0
0 2 4 6 8 10 12 14 16 18 20 22
time (d)
Dry
weight
(g/l)

26. Growth and subculture of suspension culture :
During the incubation period the biomass of the suspension culture
increases due to cell division and cell enlargement. Due to continuous
cell division and enlargement some factors may get exhausted or there
may be accumulation of toxic substances in the medium. This is the
time when the cell suspension is transferred to a fresh liquid medium
of the same composition. The normal incubation time of a stock
culture is 21 to 28 days after which it is subcultured. The
incubation time may vary by ±3 days. The incubation period from
culture initiation to the stationary phase can be determined by:
a.Initial Cell density
b.Duration of lag phase
c.Growth rate of cell line

27. A typical growth curve consists of following stages
a) Lag phase
b) Acceleration phase
c) Log or exponential phase
d) Deceleration phase
e) Stationary phase
f) Death phase
Growth Curve

28. The biomass growth in batch culture follows a fixed pattern :
Fig : Graph showing different growth phase in a Batch culture
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.

29. 1. Cell counting - the steps to count the cells in suspension cultures are:
Add 1ml of culture to the 2 ml of chromic trioxide.
Heat it at 70 ℃ for 2-15 minutes.
Cool the solution and shake it vigorously for 10 minutes.
Count the cells in a hemocytometer.
Packed cell volume (PCV): Transfer a uniformly dispersed suspension to a
centrifuge tube and spin it for 5 minutes to calculate the PCV.
2. Cell fresh weight - to calculate the fresh weight, follow the given
procedure:
Fit a circular nylon fabric in Hartley funnel to collect the cells.
Wash the cells with water.
Weigh the cells.
3. Cell dry weight - to calculate the fresh weight, follow the given
procedure:
Fit a circular nylon fabric in Hartley funnel to collect the cells.
Dry the cells for 12 hours at 60 ℃.
Weigh the cells.
WHAT ARE THE METHODS TO ASSESS AND MEASURE THE GROWTH OF CELLS IN
SUSPENSION CULTURE?

30. • Fluorescein diacetate (FDA): FDA is added to a few drops of cell
culture and the cells are observed under fluorescent microscope.
FDA itself does not fluoresce. Once inside the cell, it is cleaved by
esterase and green glowing fluorescein is released. Fluorescein is
not freely permiable across the plasma membrane and it
accumulates in living (but not dead) cells
• Evans blue stain: It is taken up by dead cells and excluded by
living cells. Easily seen under light microscope.
• Phase-contrast microscopy: This method works on the principle of
observation of cytoplasm streaming and the presence of a healthy
nucleus in the suspension.
• Tetrazolium salt reduction: This method measures the respiratory
efficiency of the cells by reducing 2,3,5-triphenyl tetrazolium
chloride (TTC) to the red dye formazan.
Determining viability of cells in suspension culture

31. Significance of Cell Suspension Culture
• secondary metabolite production.
• ideal to study factors affecting growth and
differentiation and the role of compounds and
metabolites.
• have also found application in the field of somatic
embryogenesis
https://www.researchgate.net/publication/332290593_Callus_and_Cell_Suspension_Culture

32. Importance of cell suspension culture
Such systems are capable of contributingsignificant
information about cell physiology, biochemistry,
metabolic events, etc.
It is important to build up an understanding ofan
organ/embryoid formation starting from a single cell.
Mutagenesis studies maybe facilitated by cell
suspension culture to produce mutant cell clone from
which mutant plants can be raised.

34. Table 1: Alkaloids produced by in vitro culture techniques
Plant name Phytoconstituents/alkaloids Explant used/culture medium/conditions
Brucea javanica (L.)
Merra (Simaroubaceae)
Canthinone alkaloids Fruits/MS 2,4-D (1 mg/l), kinetin (0.1 mg/l),
sucrose (5%)/cultures maintained under illumination on
an orbital shaker with 2 cm stroke, temp -25 2°C
Catharanthus roseusa
(Apocynaceae)
Catharanthine, ajmalicine Leaf, stem/MS+NAA (2 mg/l), IAA (2 mg/l),
kinetin (0.1 mg/l), sucrose (3%)/incubated on rotary
shaker at 23 2°C in darkness
Catharanthus roseusb
(Apocynaceae)
Dimeric indole alkaloids:
Vindoline, vincristine, vinblastine
Petiole segments of seedlings (4-day old)
MS+NAA (0.1, 5, 10, 20 ppm), Kin (0.1,5,10,20ppm);
maintained in dark for first 2 weeks followed by 24 h
light period (fluorescent, cool white light, 7 W/m2) for
next 2 weeks at temp -35°C
Cereus peruvianusb
(Cactaceae)
Alkaloids Shoots/MS+tyrosine/incubated at temp -25 1°C
under dark conditions
Chonemorpha
grandiflorab
(Apocynaceae)
Camptothecin Inter-nodal segments of stem/MS 2,4-D
(4.52 M)/16/8 h (light/dark) photoperiod, temp -25°C
Leucojum aestivumb
(Amaryllidaceae)
Galanthamine Bulb, shoot/MS+BAP, NAA/cultured under illumination
provided by fluorescent tubes or under dark at 25°C
Nandina domesticab
(Berberidaceae)
Protoberberine alkaloids,
dehydrodiscretamine, berberine,
jatorrhizine
Fruits/MS 2,4-D (1.0 mg/l), kinetin (0.1 mg/l)/
constant illumination under fluorescent light, temp -
25 2°C
Ahmad et al., 2012

35. Nicotiana rusticab
(Solanaceae)
Alkaloids, nicotine Seeds and aseptically germinated seedlings there
from/LS agar 2,4-D (1 M), kinetin (1 M)/placed at
30°C in dark
Papaver bracteatumb
(Solanaceae)
Thebaine Seeds/MS+kinetin (0.47 M) 2,4-D (4.52 M or
0.45 M), sucrose (3%)/incubated at 25°C
Pinellia ternataa Guanosine. inosine, trigonelline Tubercles, shoots/MS+NAA (0.5 ppm), 6-BA
(1.0 ppm); NAA (02 ppm), 6-BA (1.0ppm)/cultured
under 16 h photoperiod providing 135 mol/m2/s light
intensity at 25°C
Przewalskia tanguticac
(Solanaceae)
Tropane alkaloids Seeds, germinated seedlings, leaves liquid MS;
genetic transformation-MS+AS
(100 mol/l) 30% sucrose 3.0 g/l phytogel/250 mg/l;
germination of seeds at 25 0.5°C in dark conditions.
Bacterial preparation carried out in orbital shaker at 180
rpm at 28°C for 30 min, culturing of hairy roots at 110
rpm at 25 1°C
Rauwolfia serpentineb
(Apocynaceae)
Reserpine Leaves
MS+PABA (1 ppm)+NAA (4 ppm); temp -25 2°C,
photoperiod of 16/8 light and dark cycle; some leaf
explants incubated under total dark conditions to
observe root growth.
Schizanthus hookerib
(Solanaceae)
Pyrrolidine derivatives to tropane
esters derived from angelic acid,
tiglic acid, seneciocic acid and
methyl mesaconic acid,
tropane alkaloids-3 -methylmesc
aonyloxytropane
Roots, callus for generating shoots MS+
NAA(2.69 M), BA(2.22 M)
BA(4.44 M)+NAA (0.54 M); temp -22 1°C, light
regimen of 14 h at 48 mol/m2/s
Solanum nigrumb
(Solanaceae)
Solasodine Seeds, germinated plantlets
MS 2,4-D (0.5 ppm), Kin (0.5 ppm); temp - 25°C
light/20°C dark, photoperiod of 16 h by photon flux
density of 100 mmol/m2/S
Ahmad et al., 2012

36. Solanum tuberosumb
(Solanaceae)
Glycoalkaloids Tubers
MS+BA(0.5 ppm), IAA(2 ppm) Kinetin; temp
- 26°C 16/8 light/dark cycle
Taxus (Taxaceae) Taxane, paclitaxel, baccatin III,
10-deacetyl-baccatin
Roots
MS+l-phenylalanine (1 M); temp -25°C in dark
conditions on a gyrorotary shaker at 122 rpm
Taxus globosaa
(Taxaceae)
Baccatin III, paclitaxel Stem, internodes, leaves, meristmatic tissues
modified gamborg B5 methyl jasmonate
(0.1, 1.0, 10, 100 M)/incubated at 25 1°C under dark
conditions or 16 h photoperiod, light is provided by
fluorescent lighting of 1500 W
Thalictrum minusa
(Ranunculaceae)
Berberin Leaf segments/LS+NAA (60 M), BA (10 M)/ cultures
were agitated on reciprocal shaker at speed of 100
strokes/min at 25°C in dark
Young leaves
Vernonia cinereab
(Asteraceae)
Alkaloids MS+NAA (1, 1.5 ppm), BA (1, 5 ppm)/cultures incubated
at 25 2°C with 16/8 h (light/dark) photoperiod under
cool white fluorescent tubes
Withania coagulansc
(Solanaceae)
WithaferinA MS+IBA (0.2 ppm), sucrose (3%); provided with
continuous white light illumination at 25°C on rotary
shaker at 80 rpm
Withania somniferad
(Solanaceae)
Withaferin A, withanolideD Single shoot tips/MS+BA (1 mg/l), sucrose (3%)/
incubated at 16 h/8 h (light/dark) photoperiod with rel.
humidity of 55-60% maintained at temp -25 2°C
Ahmad et al., 2012

37. Advantages :
The nutrients can be continually adjusted.
This system can be scaled for largescale
production of the cells.
A whole plant can be regenerated fromasingle
plant cell.
Disadvantages :
The productivity of suspension cultures decreases
over extended subculture periods.
Slow growth and low productivity of plant
cells.
Cells may get damaged by shear conditions.

38. Single Cell Culture: Meaning, Principle, Factors and
Importance | Plant Tissue Culture
What is the Meaning of Single Cell Culture?
Single cell culture is a method of growing isolated single cell
aseptically on a nutrient medium under controlled condition.
Principle of Single Cell Culture:
The basic principle of single cell culture is the isolation of large
number of intact living cells and cultures them on a suitable
nutrient medium for their requisite growth and development. Single
cells can be isolated from a variety of tissue and organ of green
plant as well as from callus tissue and cell suspension. Single cells
from the intact plant tissue (leaf, stem, root cladode etc.) are
isolated either mechanically or enzymatically.

39. • Single cells are traditionally isolated from the established
friable callus tissue and cell suspension culture
• Mechanically, isolated from cell suspension or friable callus
with a needle or fine glass capillary.
• Alternatively, the friable tissue is transferred to liquid medium
and the medium is continuously agitated by a shaker.
Agitation of liquid medium breaks and dispenses the single cells
and cell clumps in the medium (makes a cell suspension)
The cell suspension is first filtered to remove cell clumps and
the filtrate is then centrifuged to collect the single cells from the
pellete.

40. • The isolated single cell can be cultured either in liquid medium or on
solid medium
• Five basic methods that are used for culturing single cells such as
paper raft nurse technique the petri dish plating technique, the
micro-chamber technique, the micro-droplet technique, the plating
with nurse tissue technique. In culture, the single cells divide re-divide
to form a callus tissue. Such callus tissue also retains the capacity to
regenerate the plantlets through organogenesis and embryogenesis.

42. Single cell culture technique is very important for the fundamental
and mutation studies and it has a wide industrial application.
1. Single cell culture could be used successfully to obtain single
cell clones.
2. Plants could be regenerated from the callus tissue derived from
the single cell clones
3. The occurrence of high degree of spontaneous variability in the
cultured tissue and their exploitation through single cell culture are
very important in relation to crop improvement programmes.
4. One of the major problems of mutation breeding in higher plants
is the formation of chimeras following the mutagenic treatment of
multi-cellular organism. In this respect single cell culture method
are more efficient. Isolated single cells can be handled as a
microbial system for the treatment of mutagens and for mutant
selection.
Importance of Single Cell Culture:

43. 5. Many plants synthesize various important natural compounds in
the form of alkaloids, steroids etc. Some of these natural com-
pounds are highly medicinally important. From the commercial
point of view, single cell culture in large-scale could become a
valuable technique for industrial production of important natural
compound.(Several workers have reported the synthesis of several
times higher amounts of alkaloid by cell culture than the alkaloid
content in the intact plant)
6. Biotransformation means the cellular conversion of an
exogenously supplied substrate compound not available in the
cell or the precursor of a particular cellular compound to a
new compound or the known

44. 7. Induction of polyploidy has been found to be very useful
for plant breeding to overcome the problem of sterility
associated with hybrids of unrelated plants. Polyploidy can
easily be achieved by single cell culture.
A large-number of genetically sterile hybrids exist in the
genus Saccharum. When cell culture of such sterile hybrid is
treated with 50 mg/L colchicine for 4 days, it has been found
that about 48% of such treated cells become uniformly
polyploid. These polyploid cells are then induced to
regenerate a large-number of fertile plants. In this regard, cell
culture is very useful with other crops also.(Cheavegatti et al.,
2011)

45. 1. The composition of the medium for the growth of single
cell culture is generally more complex than callus and cell
suspension culture. For example, Convolvulus cells require
a cytokinin and amino acids that are not necessary for the
callus culture of that species.
2. Induction of division of single cells using paper raft
technique indicates that isolated cells get the exact essential
nutrient from the callus mass. It has been suggested that
the callus mass leaches out the essential nutrient through
plasma membrane of the cells.
3. In case of petri dish plating technique the initial plating
cell density is very critical.
Factors Affecting Single Cell Culture:

46. Date palm accumulates a wide range of secondary metabolites
high in nutritional and therapeutic value. In the present study, date
palm (Phoenix dactylifera L., cv. Shaishi) shoot-tip-induced callus
was used to establish cell suspension cultures in Murashige and
Skoog (MS) liquid medium containing 1.5 mg L-1 2-
isopentenyladenine (2iP) and 10 mg L-1 naphthaleneacetic acid
(NAA).

47. • The 11-week-old culture was found highest in the
production of biomass (62.9 g L-1 fresh weight and 7.6 g
L-1 dry weight) and polyphenols (catechin-155.9 μg L-1,
caffeic acid- 162.7 μg L-1, kaempferol-89.7 μg L-1, and
apigenin-242.7 μg L-1) from the cell suspension cultures
• This is the first report on the production of
polyphenols from the cell suspension culture of date
palm
• This study facilitates further development of large-scale
production of polyphenols and the utilization of
bioreactors

49. Anthraquinones (AQs) and phenolic compounds are important
phytochemicals that are biosynthesized in cell suspension
cultures of Polygonum multiflorum.
The production of secondary metabolites by field-grown intact
plants has various disadvantages, such as low yields, slow growth
cycles, fluctuations in quantity due to unfavorable environmental
conditions, infestation, and disease.
Plant cell suspension culture, an attractive alternative system for
uniform phytochemical synthesis, can continuously offer high-value
medicines, foods, and healthy ingredients, independent of
geographical, climatic, or environmental variations and constraints
(Wilson et al., 2014)

50. • First time, cell suspension cultures of P. multiflorum for the
production of AQs and phenolic compounds was established
• It could be concluded that the flask cell suspension
cultures of P. multiflorum have the potential for scale-up
studies on a commercial level by pharmaceutical industries
• Higher amounts of biomass accumulation and AQ synthesis
were observed in the cell suspension culture when a liquid
medium with standardized concentrations of PGRs (MS + 4%
sucrose + 1 mg/L 2,4-D + 0.5 mg/L TDZ + 100 M L-
glutamine) was combined with elicitors, 100 M JA or SA. The
levels of phenolic groups such as flavonols and
hydroxybenzoic and hydroxycinnamic acids were higher in the
JA- and SA-elicited culture cells than in the control cell
cultures

51. Significant progress has been achieved in recent years in the
cultivation of plant cells on an industrial scale.
Cell suspension Culture provides stable conditions, with
manufacturing capabilities for a wide range of therapeutic
compounds, including plant-derived pharmaceuticals and using
various types of plant cells

53. • Cai, T.; Daly, B.; Butler, L. Callus induction and plant regeneration from shoot portions of mature
embryos of high tannin sorghums. Plant Cell Tissue Organ Cult. 1987, (9):245–252
• Chourey, P.S.; Sharpe, D.Z. Callus formation from protoplasys of Sorghum cell suspension cultures.
Plant Sci. 1985, (39) 171–175
• Evans, D.E.; Coleman, J.O.D.; Kearns, A. Plant Cell Culture; BIOS Scientific Publishers: London,
UK, 2003.
• Wilson, S.A.; Roberts, S.C. Metabolic engineering approaches for production of biochemicals in
food and medicinal plants. Curr. Opin. Biotechnol. 2014, (26):174–182
• https://www.researchgate.net/publication/332290593_Callus_and_Cell_Suspension_Culture
• Bhojwani S.S and Razdan M.K.; 2006; Plant Tissue Culture : Theory and Practise, a Revised
Edition
• FAOSTAT. 2017. Available online: http://www.fao.org (accessed on 22 March 2019).
References