This document discusses different culture systems used for the growth and maintenance of plant tissue cultures. It describes callus culture, where callus masses grow on solidified media, and suspension culture, where cells or cell aggregates grow in liquid media. It provides details on initiating and maintaining both callus and suspension cultures, including selecting explants, media, incubation, sub-culturing, and different suspension culture techniques like batch, semi-continuous, and continuous systems.

1. GROWTH AND
MAINTENANCE
OF PLANT
TISSUE
CULTURE
Prepared by
G.Hemaharshini
U21BP019​

2. INTRODUCTION
For the growth establishment and maintenance of various
types of plant tissue cultures, there are three main culture
systems, selected on the basis of the objective.
• Growth of callus masses on solidified media (callus
culture also known as static culture).
• Growth in liquid media (suspension culture) consists of
mixture of single cells or cell aggregates.
• Protoplast culture: Callus culture (static tissue culture)
or
Suspension culture
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3. CALLUS CULTURE
• Callus is an amorphous aggregate of loosely arranged parenchyma cells, which
proliferate from mother cells. Cultivation of callus usually on a solidified nutrient
medium under aseptic conditions is known as callus culture.
Initiation of callus culture
• (a) Selection and preparation of explant Selection: For the preparation of callus culture,
organ or culture is selected such as segments of root or stem, leaf primordia, flower structure
or fruit, etc.
Preparation:
• 1. Excised parts of the plant organ are first washed with tap water, and then sterilized with 0.1%
of mercuric chloride (HgCl2 ) or 2% w/v, sodium hypochlorite (NaOCl) solution for 15 min. In the
case of plant organ containing waxy layer, the material is either pretreated with wetting agents
[ethanol 70–90%; tween 20 (polyoxyethylene sorbitan monolaurate): 1–20 drops into 100 ml
distilled water]; or other detergents are added to the sterilization solution to reduce the water
repulsion.
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4. (b) Selection of culture medium
• The organ is to be cultured in well-defined nutrient medium containing
inorganic and organic nutrients and vitamins. The culture of the medium
depends on the species of the plant and the objective of the experiment. The
MS medium is quite suitable for dicot tissues because of relatively high
concentration of nitrate, potassium and ammonium ions in comparison to
other media.
• Growth hormones (auxin, cytokinin) are adjusted in the medium according to
the objective of the culture. For example, auxins, 1BA and NAA are widely
used in medium for rooting and in combination with cytokinin for shoot
proliferation. 2, 4-D and 2, 4, 5-T are effective for good growth of the callus
culture.
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5. (c) Transfer of explant
• Surface sterilized organs (explant) from stem, root or tuber or leaf, etc., are
transferred aseptically into the vessel containing semisolid culture medium.
(d) Incubation of culture
• The inoculated vessels are transferred into BOD incubator with auto controlled
device. Incubate at 25–28°C using light and dark cycles for 12-h duration.
Nutrient medium is supplemented with auxin to induce cell division. After three
to four weeks, callus should be about five times the size of the explant. Many
tissue explants possess some degree of polarity with the result that the callus
is formed most early at one surface. In stem segment, callus is formed
particularly from that surface which in vivo is directed towards the root.
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6. • Callus is formed through three stages of development, such as:
1. Induction
2. Cell division and
3. Cell differentiation.
Induction:
• During this stage, metabolic activities of the cell will increase; with the result,
the cell accumulates organic contents and finally divides into a number of cells.
The length of this phase depends upon the functional potential of the explant
and the environmental conditions of the cell division stage.
Cell division:
• This is the phase of active cell division as the explant cells revert to
meristematic state.
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7. MAINTENANCE OF CALLUS CULTURE
Cell differentiation:
• This is the phase of cellular differentiation, i.e. morphological and physiological
differentiation occur leading to the formation of secondary metabolites.
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• After sufficient time of callus growth on the same medium following change will
occur, i.e.
1. Depletion of nutrients in the medium
2. Gradual loss of water
3. Accumulation of metabolic toxins.
• Hence for the maintenance of growth in callus culture it becomes necessary to
sub-culture the callus into a fresh medium. Healthy callus tissue of sufficient size
(5–10 mm in diameter) and weight 20–100 mg) is transferred under aseptic
conditions to fresh medium; sub-culturing should be repeated after even four to
five weeks.

8. SUSPENSION CULTURE
• Suspension culture contains a uniform suspension of separate cells in liquid
medium. For the preparation of suspension culture, callus fragments is
transferred to liquid medium (without agar), which is agitated continuously to
keep the cells separate. Agitation can be achieved by rotary shaker system
attached within the BOD incubator at a rate of 50–150 rpm.
Initiation of suspension culture
(a)Isolation of single cell from callus culture:
• Callus tissue is selected and placed in a petridish on a sterile filter paper and
cut into small pieces with the help of sterile scalpel.
• Selected small piece of callus fragment about 300–500 mg and transferred into
flask containing about 60ml of liquid nutrient media (i.e. defined nutrient
medium without gelling agent), the flasks is agitated at 50–150 rpm to make
the separation of the cells in the medium.
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9. • Decant the medium and resuspend residue by gently rotating the flask, and
finally transfer 1/4th of the entire residue to fresh medium, followed by sieving
the medium to obtain the degree of uniformity of cells.
(b) Isolation of single cell from plant organ:
• From the plant organ (leaf tissue) single cell can be isolated by any of the
following methods:
1. Mechanical method
2. Enzymatic method.
• Mechanical method: The surface sterilized fresh leaves are grinded in (1:4)
grinding medium (20 μmol sucrose; 10 μmol MgCl2 , 20 μmol tris-HCl buffer,
pH 7.8) in glass pestle mortar. The homogenate is passed through muslins (two
layers) cloth, washed with sterile distilled water, centrifuged with culture
medium, sieved and placed on culture dish for inoculation.
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10. • Enzymatic method:
• Leaves are taken from 60- to 80-day-old plant and sterilized by immersing
them in 70% ethanol solution followed by hypochlorite solution treatment, was
with sterile double distilled water, placed on sterile tile and peeled off the lower
surface with sterile forceps. Cut the peeled surface area of the leaves into
small pieces (4 cm2 ). Transfer them (2 g leaves) into an Erlenmeyer flask (100
ml) containing about 20 ml of filtered sterilized enzyme solution (macerozyme
0.5% solution, 0.8% mannitol and 1% potassium dextran sulphate). Incubate
the flask at 25°C for 2 h. During incubation, change the enzyme solution with
the fresh one at every 30 min, wash the cell twice with culture medium and
place them in culture dish.
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11. MAINTENANCE OF SUSPENSION CULTURE 11

12. (i) Batch suspension culture:
• In this technique, the cells are allowed to multiply in liquid medium, which is
continuously agitated to break up cell aggregates. The system is closed with
respect to additions or removal of culture, except for circulation of air.
• In this technique to commence the growth again on the stationary phase,
more amount of nutrient medium is added to the original culture or the cells
are to be transferred into fresh medium. Each fresh medium containing culture
(suspension) constitutes a batch.
• Such cultures are grown again and again for the purpose of experiment to
achieve certain specific objectives. In batch culture there is no steady state of
growth; hence, it is not ideal for commercial production of secondary
metabolites.
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13. (ii) Semicontinuous suspension culture:
• In this type, the system is open. It is designed for periodic removal of culture and
addition of fresh medium. Hence, the growth is continuously maintained.
(iii) Continuous suspension culture:
• Here, the volume of culture remains constant and fresh medium and culture are
continuously added and withdrawn respectively. The important feature of the
continuous culture is the proliferation of cell occurs under constant conditions. In
this very suspension culture technique, a steady state is achieved by adding
medium in which single nutrient has been adjusted so as to be growth limiting.
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14. • In the closed type, addition of fresh medium is balanced by the outflow of spent
medium. The cell passing through the outgoing medium are separated
mechanically and reintroduced into the culture for the continuous growth of the cell
biomass.
• Open continuous system involves regulated new medium and balancing harvest of
equal volume of culture.
• The opensystem is further of two types depending upon regulation technique:
chemostat and turbidostat.
• In chemostat, the desired rate of growth is maintained by adjusting the level of
concentration of nutrient by constant inflow of fresh medium.
• In turbidostat, on the contrary, the input of medium is intermittent, and it is mainly
required to maintain the cell density in the culture.
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15. THANK YOU
Reference:
Textbook of Pharmacognosy and
phytochemistry. Pg.448-451
Biren Shah and A.K.Seth.
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