This document provides an overview of plant tissue culture. It discusses the basics, including definitions and history. The key facilities, materials, and equipment needed are described. The composition and role of various media components such as macronutrients, micronutrients, carbon sources, and growth regulators are summarized. Different types of cultures and their applications are listed. The general steps involved in plant tissue culture are outlined.

1. BAPATLA COLLEGE OF PHARMACY
PLANT TISSUE
CULTURE
UNDER GUIDANCE OF:
Dr. J V Suresh M.Pharm PhD
Department of Pharmacognosy
PRESENTED BY:
A Sowmya
14101R0041

2. PLANT TISSUE CULTURE:
Broadly, plant tissue culture refers to “in vitro cultivation of all
plant parts, whether a single cell, a tissue or an organ under aseptic
conditions”. This is a technique with which “the plant cells, tissues or
organs are on an artificial nutrient medium, either static or liquid, under
aseptic and controlled conditions”

3. Year Researcher Studies Species
1892 Klercher First attempt to
isolate protoplast
-
1902 Haberlandt First experiment
with plant cell
Tradescantia
1934 White First permanent
root culture which
terminated in1988
Lycopersicum
1946 Ball (Father of
micro propagation)
Development of
transplantable whole
plants from leaf
primoda
Lupinus and
Trapaeolum
1957 Skoog and Miller Demonstrated the
role of auxin and
cytokinin on root
and shoot formation
Nicotiana
1962 Murashige and
Skoog
Introduced the
medium
Nicotiana
HISTORY :

4. BASICS PRINCIPLES OF PTC:
1. Totipotency
2. Plasticity
3. Explants
4. Callus
5. Plant Regeneration
• Somatic embryogenesis
• Organogenesis

5. Essential
facilities
Incubator
Autoclave
Refrigerators
and freezers
Microscope
Washing
Equipment
Oven
Centrifuge
Shakers
Culture room
Laminar air
flow cabinet
Beneficial
facilities
Laminar air
flow hood
Cell counter
Vacuum pump
CO2 incubator
Osmometer
Densitometer
Phase contrast
and fluorescent
microscope
Magnetic stirrer
Useful
facilities
Colony counter
Glassware
washing
machines
FACILITIES AND REQUIREMENTS :

6. PLANT TISSUE CULTURE LABORATORY:
Well designed PTC lab allows free movement of supplies, people, cultures
and finished products. In order to keep air borne contaminants to a minimum,
the laboratory has very few entry sites and generally has sufficient areas
designated as clean rooms with purified air flowing under positive pressure. The
basic areas of any PTC is as shown below
Media
Storage
Transfer
Room
Culture
Room
Office Lockers
Cold
storage
Shipping
area

7. MATERIALS OF PTC:
•Explants
•Aseptic environment
•Tissue culture media
Composition of PTC media
 Macronutrients
 Micronutrients
 Carbon and energy source
 Vitamins
 Amino acids
 Other complex organic supplements
 Plant growth regulators
 Gelling or solidifying agents
 pH regulators
•Aeration

8. EXPLANT:
• Detached portion of cell or tissues from plant organ to start in vitro
cultures is called Explants.
• Age and type of explants plays a vital role in formation of callus.
• It may be obtained from bud, root , nodal segments , apical meristem, seed,
embryo , anthers, pollens and protoplast cells.

9. ASEPTIC ENVIRONMENT:
•During culture , aseptic environment is required.
•Contaminants can easily overgrow in PTC
•All equipments and materials must be sterilized
•Laminar air flow is mandatory prerequisite for any tissue culture
technique.

10. TISSUE CULTURE MEDIA:
• The type and composition of culture media very strongly govern the growth
and morphogenesis of plant tissues
• The choice of culture media is based on the nutritional requirements of the
whole plant
• The composition of culture ,medium can be arranged into two groups
• Components which are indispensible for growth
• Those which are not required but are beneficial
• Several media have been formulated and modified from time to time to suit
nutrition of particular tissues such as callus culture, cell suspension cultures ,
somatic embryos or protoplasts.

11. COMPOSITION OF PTC MEDIUM:
• Macronutrients
• Micronutrients
• Iron source
• Carbon and energy source
• Vitamins
• Amino acids
• Other complex organic supplements
• Plant growth regulators
• Gelling or solidifying agents
• pH regulators

12. MACRONUTRIENTS:
•Major inorganic nutrients
• First mineral nutrient media was prepared by Knop based on six macronutrients in
three salt formulae:
•Calcium nitrate
•Potassium phosphate
•Magnesium sulphate
•Functions :
Organic nitrogen (proline and
glutamine)
Enhances somatic embryogenesis and
organogenesis
Inorganic nitrogen (ammonium salts) Nitrogen source
Calcium Organization of middle lamella of cell
wall
Magnesium Constitution of chlorophyll
Phosphorous Structural element of nucleic acids
Others Catalytic processes of enzymes

13. MICRONUTRIENTS:
• Apart from the six major elements , the plants need additional nutrients in small
quantities.
• A media lacking these trace elements does not support healthy and wholesome
growth and the plant exhibits deficiency symptoms such as
Occurrence of pigmentation
Absence of vessels
Presence of narrow cambial zone
Cellular hypertrophy
Symptoms of Fe and S
•Examples of micronutrients:
Boron BO- Nickel
Manganese Mn++ Aluminium
Zinc Zn++ Cobalt Co++
Copper Cu+ ,Cu ++ Iodine
Molybdenum MoO4
- Sodium
Chlorine Cl-

14. IRON SOURCE:
• The PTC media contains iron in the form of ferric
sulphate (FeSO4 , 7H2O )
• A chelated iron source (Fe – EDTA) is preferred due to
its less easy depletion
• Iron is required for chlorophyll synthesis as well as in
oxidation reduction reactions.

15. CARBON AND ENERGY SOURCE:
•Sugar is added as an energy source because callus is incapable of
producing energy
•The most preferred carbon source is sucrose at concentration of 20 –
60 g / L
•Glucose is also equally good , while fructose is less efficient.
•Sucrose or glucose at 2-4 % is most suitable carbon source
•Other sources like fructose, maltose, galactose , raffinose and sorbitol
also supports the growth of various plant cells

16. VITAMINS:
• Healthy growth of tissue in the culture media requires vitamins
• Water soluble vitamins such as vitamin B complex plays an
important catalytic role in the cell metabolism .
• Among all vitamins Thiamine is required for most tissue cultures.
• Vitamin C is sometimes required as antioxidant
• Myo- ionositol is a growth accelerator
• Vitamins are synthesized in the plants in sub optimal quantities
and therefore addition may not be necessary unless specified.

17. AMINO ACIDS:
•Added to media to stimulate cell growth in protoplast cultures and
establishing cell lines
•They induce and maintain somatic embryogenesis.
•They are more readily taken up the plant cells
•They also act as a source of reduced nitrogen and acidifies the medium
•Examples:
Casein hydrolysate
 L – Arginine
 L – Glutamine
 L – asparginine
 L – Glycine
 L – cysteine
Aspartic acid
Alanine
 Glutamic acid
Proline
Tyrosine

18. OTHER COMPLEX ORGANIC SUPPLEMENTS:
• Coconut milk
•Casein hydrolysate (edamin)
•Peptone
•Yeast extract
•Ground banana
•Watermelon juice
•Malt extract
•Potato extract
•Ripe tomato juice
•Orange juice
•Coconut water

19. PLANT GROWTH REGULATORS (PGRs):
•They are the critical media components in determining the developmental pathway
of plant cells.
•These stimulate cell division and regulate the growth and differentiation of shoot
and roots.
•The most commonly used PGRs are phytohormones and their synthetic analogues.
•The concentration and ratio may vary from plant to plant .
•The PGRs used in cultures fall into 5 main classes, namely
Auxins
Cytokinins
Gibberellins
Abscisic acid
Ethylene
Activated charcoal

20. GELLING AGENTS OR SOLIDIFYING AGENTS:
•Media can be in either liquid or solid form
•Most desirable property of a gelling agent is that it should
withstand sterilization temperatures
•Agar is the most common gelling agent.
•It is used in the concentration range of 0.5 % - 1 %
•Alternate gelling agents are gelrite , gellan and phytagel or
agargel

21. pH REGULATORS:
•Correct pH is important for the growth of culture
•It is generally in range of 5.6 – 6.0 before sterilization
•Highly acidic or alkaline pH drastically affects the
uptake of the nutrients.
•It is generally regulated by phosphate buffer.

22. MOST COMMONLY USED MEDIA:
Constituent Concentration in culture media (mg/L)
MS SH B5
A) Macronutrients
KNO3 1900 2500 2500
NH4NO3 1650 - -
NH4H2PO4 - 300 -
(NH4)2 SO4 - - 134
MgSO4 .7H2O 370 400 250
CaCl2 .2H2O 440 200 150
KH2PO4 170 - -
NaH2PO4.7H2O - - 150
B) Micronutrients
MnSO4.H2O - 10 10
MnSO4.4H2O 22.3 - -
KI 0.83 1.0 .75
H3 BO3 6.2 5.0 3.0
ZnSO4 .7H2O 8.6 1.0 2.0
CuSO4 .5H2O 0.025 0.2 0.025
C) Iron source
FeSO4 .7H2O 27.8 15.0 27.8
D) Vitamins
Nicotinic acid 0.5 5.0 1.0
Pyridoxine HCl 0.5 0.5 1.0
Thiamine HCl 0.1 5.0 10.0
E) Growth regulators
F) Sucrose 30,000 30,000 20,000
G) Agar 10,000 8000 6800

23. TYPES OF
CULTURES
Meristem
culture
Protoplast
fusion
Protoplasts
culture
Embryo
culture
Seed culture
Ovary
culture
Anther
culture
Shoot tip
culture
Root
culture
Hairy root
culture

24. Transfer of Plantlets to green House or Green Field
Hardening
Regeneration
Sub culturing
Incubation
Inoculation of Explants
Sterilization of explants
Isolation of explants
Selection of plant
GENERAL STEPS INVOLVES
IN PTC:

25. APPLICATIONS OF PTC:
1. To study Respiration and Metabolism
2. To study Proliferation and Organ Function
3. Single cell culture of higher plants
4. Genetic transformation
5. Production of disease resistant plants
6. Germplasm Storage
7. Embryo rescue
8. Somaclonal Modification
9. Production of Haploids
10.Production of artificial seeds
11.Clonal propagation
12.Micro propagation
13.Mutant selection
14.Somatic hybridization
15.Cloning
16.Production of secondary Metabolites

26. REFERENCES:
1) C. K. Kokate; A. P. Purohit; S. B. Gokhale “PHARMACOGNOSY”
Nirali Prakashan 53rd Edition Pg.no: 5.1 – 5.17
2) Dr. G. S. Kumar; Dr. K. N. Jayaveera “ A TEXTBOOK OF
PHARMACOGNOSY AND PHYTOCHEMISTRY” S. Chand
Publishers Pg. no: 131 – 135
3) P Suresh Narayana; D Varalakshmi; T Pullaiah “TEXTBOOK OF
PHARMACOGNOSY” Vol III CBS Publishers & Distributors Pvt
Ltd Pg.no: 133 – 181
4) S. L. Deore ; S. S. Khadabadi ; B. A. Baviskar “PHARMACOGNOSY
AND PHYTOCHEMISTRY A COMPREHENSIVE APPROACH”
PharmaMed Press Pg.no: 635 – 659
5) Vinod D Rangari “PHARMACOGNOSY & PHYTOCHEMISTRY”
Vol II Career Publications Pg no: 346 – 415.