1. The document outlines the general steps of plant tissue culture which include preparation and sterilization of explants, production of callus from explants, proliferation through sub-culturing, and suspension culture. 2. It discusses the nutritional requirements for plant tissue culture media including macronutrients, micronutrients, vitamins, organic nutrients and growth regulators. Common media such as MS and B5 are described. 3. The roles of various elements, vitamins, carbon sources, and growth regulators used in plant tissue culture media are explained. The structures of some common growth regulators are also shown.

1. General Steps of Plant Tissue Culture and Nutritional
Requirements, Growth and Their Maintenance.
Academica In-Charge, HOD,
Pritam Juvatkar
Mobile :
Email : pritamjuvatkar@gmail.Com
9987779536
Department of Pharmacognosy and
Phytochemistry
Konkan Gyanpeeth Rahul Dharkar College of
Pharmacy and Research Institute, karjat

2. General
procedure
involve in plant
tissue culture:
Preparation and sterilization of explants
Production of callus from explants
Proliferation
Suspension culture
Sterilization of glasswares and vessels:
01
02
03
05
04

3. 01. Sterilization of glasswares and
vessels
1. Glasswares ------------ overnight dipped in ------------------ sodium dichromate – sulphuric
acid------for removal of dirt, or any microorganisms or any waxy materials.
2. Then further -------- cleaned -------- running tap water -------followed by double distilled
water.
3. Drying ------- Glasswares ------- hot air oven -------at temp about 121°C for 30 minutes.
4. For plastic materials -------- sterilization is -------- with non-abrasive detergent -------- by
cleaning under running tap water -------- rinsed with acetone for drying.
5. All the stainless steel equipment's, scalpels, forceps -------- sterilized with flame sterilization
-------- wrapped with aluminum foil after cooling -------- kept in dust free cupboard.

4. 02. Preparation and sterilization of
explants
1. Explants -------- any part of the plants such as -------- young leaves, roots, flowers, stems,
cambium, anthers etc.
2. The young part -------- removed -------- with sharp knife -------- sterilization is carried out.
3. Types of sterilant -------- chromic acid (3-5 min), 0.1% mercuric chloride (3-10 min), 1-2% sodium
or calcium hypochlorite (5-15 min) and 70% alcohol for 3 to 10 sec -------- followed by washing
with double distilled water.
4. Leaves are sterilized -------- 0.1% mercuric chloride solution -------- followed by sterile
water.
5. Stems are sterilized -------- 2% sodium hypochlorite solution -------- 15-20 minutes --------
followed by washing with sterile double distilled water.
6. Seeds are sterilized by -------- ethanol for 10 second -------- followed by rinsed with purified
water -------- then 10% calcium hypochlorite solution for 15 minutes.

5. 03. Production of callus from explants
1. Sterile explants -------- transferred aseptically in to the sterilized medium -------- incubated
into BOD incubator for necessary growth.
2. Temperature is maintained -------- at 25°C ± 2°C kept for -------- 2-3 weeks.

6. 04. Proliferation
1. Developed callus -------- cut into small pieces with sterile scalpel -------- transferred into
another fresh medium for further proliferation.
2. This process is also known as -------- sub-culturing.
3. This method is carried -------- out at an interval of 4-5 weeks -------- based on the growth of
the callus.

7. 05. Suspension culture:
1. It contains a uniform suspension of separate cells in liquid medium.
2. For this callus -------- transferred in to liquid medium and agitated continuously (80-150 rpm) in BOD incubator
for cells separate.
3. After cell growth, -------- again sub-culturing is carried out.
Culture media:
1. Culture media plays a vital role for the in-vitro growth and morphogenesis of plant tissues because it should
contain the same nutrients for growth as required by the whole plant.
2. The composition of the culture media is primarily dependent on two parameters viz. the particular species of the
plant and the type of material used for culture.
3. The media used are solid (solid medium) or liquid (liquid medium) in nature which is dependent on the better
response of a culture.
4. There are several culture media developed such as White’s medium, Murashige and Skoog (MS) medium, B-5 medium, N-6
medium, Nitsch’s medium

8. White’s medium:
• This culture media is developed for root culture of tomato.
• This is one of the earliest media with low salt formulation. .
Culture
media:
.
A
B
C
D
E
Murashige and Skoog (MS) medium
• Originally formulated a medium to induce organogenesis and
regeneration of plants in cultured tissues.
• MS medium is widely used for many types of culture systems.
B5 medium:
• Developed by Gamborg, B-5 medium is prepared for cell
suspension and callus cultures.
• At present with certain modifications, this medium is used for
protoplast culture.
N-6 medium
• Chu formulated this medium and it is used for cereal anther culture.
• This medium is used to improve the formation, growth and
differentiation of pollen callus in rice
Nitsch’s medium
• This medium was developed by Nitsch and Nitsch for anther cultures.
• This medium is used in the production of haploid plants of various
species of Nicotiana raised from pollen grains.

9. 01
Nitrogen
Functions of Elements in Plant Tissue Culture
02
Calcium
Important for synthesis of cell wall and cell functioning
03
Potassium
It regulates osmotic potential
04
Magnesium
It is component of chlorophyll
F u n c t i o n s o f
E l e m e n t s i n
P l a n t T i s s u e
C u l t u r e

10. 05
PHOSPHORUS
t is component of nucleic acid
06
SULPHUR
It is component of amino acids
07
MAGANESE
It is cofactor for certain enzyme and helps in cell
elongation
08
IRON
It helps in electron transfer reaction
10
COBALT
It is component of vitamin-B12
11
ZINC
It requires for chlorophyll biosynthesis
12
MOLYBDENUM
It is component of certain enzymes and
helps in cell elongation
13
CHLORINE
it helps in photosynthesis
09
COPPER
14
BORON
It involves in electron transfer reaction It is used for retardation of cell division and
cell elongation
Functions
of
Elements
in
Plant
Tissue
Culture

11. V i t a m i n s
A
Plants can produce their
requirements of vitamins.
However, plant cell cultures
need to be supplemented with
certain vitamins like
• Thiamine (vit B1),
• Niacin (vit B3), Pyridoxine
(vit B6),
• Myo-inositol
• (Member of the vit. B
complex).
Organic Nutrients
V i t a m i n s
A
• Thiamine – Involved in the
direct biosynthesis of
certain amino acids and
• essential co-factor of
carbohydrates metabolism.
• Vit E – Antioxidants.
• Vit C- To prevent blacking
during explant isolation.
• Vit D- Growth regulatory
effect
A m i n o A c i d s
B
Glycine- has little benefit in the
growth of plant. They may be
directly utilized by plant own be
provided as N2 source.
C a r b o n
S o u r c e s
C
Sucrose (is most commonly
used carbon source) at a
concentration of 3%, glucose
and fructose also known to
support plant
growth. Sucrose in the medium
is necessary for various
metabolic activities.

12. A u x i n
D
Auxin are involved in cell
division and elongation and
in cell wall synthesis. IAA,
IBA, NAA, 2, 4-D are the most
frequently used auxin in plant
tissue culture. The principal
naturally occurring auxin, the
IAA is not often used in the
tissue culture, because it is
unstable. IBA is slightly more
potent than IAA and is not
easily broken down. Hormones
of this group are involved with
elongation of stems and
inter nodes, tropism, apical
dominance abscission,
rooting etc.
Organic Nutrients :- Growth Regulators
C y t o k i n i n
E
These hormones, are concerned
with cell division, modification
of apical dominance, shoot
differentiation etc. Most
commonly used cytokinins are
BAP, BA, Kinetin, and Zeatin.
They usually promote cell
division if added together with an
auxin. Of these, BAP is the most
effective cytokinins for
stimulating axillary shoot
proliferation.
G i b b e r e l l i n s
F
There are over 20 known
gibberellins. Of these, generally,
GA3 is used. They are rarely
used and reported to stimulate
normal development of
plantlets from in vitro formed
adventives embryos.
O t h e r s
G
Abscisic acid is most often
required for normal growth and
development of somatic
embryos and only in its
presence they resemble zygotic
embryos.

13. Organic Nutrients :- Structures of various growth regulators

14. A g a r
H
This is obtained from red
algae, especially Gelidium
amansii.
Complex mixture of related
polysaccharides built up from
the sugar,
galactose. These include the
natural polymer fractions,
agarose, which gives strength
to the gel and the highly
charged anionic
polysaccharides
Agaro pectins which give agar
its viscosity.
Agar is used at varying
concentration from 0.8 to 1%.
Organic Nutrients :- Gelling Agent :-
p H m e d i u m
I
G e l r i t e
J
There are over 20 known
gibberellins. Of these, generally,
GA3 is used. They are rarely
used and reported to stimulate
normal development of plantlets
from in vitro formed adventives
embryos.
O t h e r s
K
Abscisic acid is most often
required for normal growth and
development of somatic
embryos and only in its
presence they resemble zygotic
embryos.
The optimal pH for most
tissue cultures is in the range
of 5.0-6.0. The pH generally
falls by 0.3-0.5 units after
autoclaving. At a pH higher
than 7.0 and lower than 4.5,
the plant cells stop growing in
cultures. In general, pH
above 6.0, the medium
became hard whereas pH
below 5.0 does not allow
gelling of the medium