This document discusses the establishment and maintenance of plant tissue cultures, including callus culture and suspension culture. It provides details on the initiation and various growth phases of callus culture and suspension culture. The key steps for callus culture include selection of explant, preparation of culture medium, transfer of explant, and incubation. For suspension culture, callus fragments or single cells are transferred to liquid medium with agitation to keep cells separate. The growth of plant tissue cultures can be determined through methods like cell counting, packed cell volume, fresh cell weight, and viable cell tests. Subculturing is needed every 4-5 weeks to maintain callus growth due to nutrient depletion and toxin accumulation in the medium.

1. ASSIGNMENT
ON
GROWTH AND MAINTAINANCE OF
PLANT TISSUE CULTURE
SUBMITTED BY: SUBMITTED TO:
MARY MELNA KUJUR Dr. K SRINIVASA RAO SIR
ENROLL NO:
1801024022
B.Pharm IV sem

2. ESTABLISHMENT AND MAINTAINANCE OF VARIOUS
CULTURES
 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)
 Suspension Culture

3. CALLUS CULTURE
 Callus is an amorphous aggregates of loosely arranged
parenchyma cells,which proliferate from mother cells.
 Cultivation of callus culture usually on a solidified
nutrient medium under aseptic condition is known as
callus culture;unlike tumor tissue ,the cell division takes
place preclinically.
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.

4. 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.
2. Wash the sterilized explants with sterile glass distilled
water and cut aseptically into small segments (2–5 mm).

5. . (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.

6. (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 autocontrolled 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.

7. Callus is formed through three stages of development, such
as:
• Induction
• Cell division and
• 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.

8. Cell differentiation :
This is the phase of cellular differentiation, i.e.
morphological and physiological differentiation occur
leading to the formation of secondary metabolites.
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. After sufficient numbers
of cells are produced, subculturing can be done in fresh
liquid medium. Single cellscan also be obtained from fresh
plant organ (leaf).

9. Initiation of suspension culture
(a)Isolation of single cell from callus culture:
Healthy
• 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. 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:
Mechanical method
Enzymatic method

10. 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.
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, washed 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.

11. GROWTH PROFILE OF PLANT TISSUE CULTURE
They are classified as :
Single cell culture
Callus culture
SINGLE CELL CULTURE :
The single cell culture exhibits various stages of growth.
a) Lag phase: Tissue starts to grow.
b) Exponential phase: This phase is characterized by rapid
cell multiplication.
c) Linear phase: The growth follows a linear pattern with
respect to time.

12. d) Progressive declaration phase:
Aging of culture increase cell division decreases.
e) Stationary phase:
No growth of cells occur
Rate of production of cells = rate of their death.
f) Senescent phase:
Cell death occurs to lack of nutrients.

13. IN CALLUS CULTURE
a) Lag phase:
In this phase cell trying to adjust the new environment condition.
b) Exponential phase :
By utilizing nutrients rapid multiplication occurs.
c) Decline phase :
Due to starvation some cells leads to decline in the callus culture.
d) Stationary phase :
No growth is evident, requires sub culturing

14. GROWTH DETERMINATION
Methods used to determine are..
CELL NUMBER
• By counting the cell number in haemocytometer under a microscope.
•Suspension culture is preferable.

15.  PACKED CELL VOLUME
•Cell suspension is transfer to graduated centrifuge.
•Centrifuged at 2000 rpm for 5mints.
•Cell will form pellets called biomass volume, expressed by ml-1.

16. FRESH CELL WEIGHT
•When cells increase in number, the liquid will be turbid.
•As a result optical density altered, detected by colorimeter.

17.  VIABLE CELL TEST
•The staining method such as fluorescein di-acetate is used for accessing the cell
viability.
•Dead cells appear as fluorescein red.

18. Maintenance
• After sufficient time of callus growth on the same medium
following changes will occur :
 Depletion of nutrient in the medium
 Gradual loss of water
 Accumulation of metabolic toxins
• Hence for maintenance of growth in callus it is necessary to
subculture the callus.
• Subculture should be repeated after 4-5 weeks.