Plant tissue culture is a technique of growing plant cells, tissues, organs, seeds, or other plant parts in a sterile environment on a nutrient medium. Tissue culture had its origins at the beginning of the 20th century with the work of Gottlieb Haberlandt (plants). WHY PLANT TISSUE CULTURES ARE DONE ?? The production of clones of plants that produce particularly good flowers, fruits, or have other desirable traits. To quickly produce mature plants. The production of multiples of plants in the absence of seeds or necessary pollinators to produce seeds. The regeneration of whole plants from plant cells that have been genetically modified. The production of plants in sterile containers reduces disease transmission Allows production of plants from seeds that otherwise have very low chances of germinating and growing, i.e.: orchids and Nepenthes. To clean particular plants of viral and other infections and to quickly multiply these plants as 'cleaned stock' for horticulture and agriculture. ***For PTC, the laboratory must have the following facilities: Washing facility for glassware and ovens for drying glassware.  Medium preparation room with autoclave, electronic balance and pH meter. Transfer area sterile room with laminar air-flow bench and a positive pressure ventilation unit called High Efficiency Particulate Air (HEPA) filter to maintain aseptic condition. Culture facility: Growing the explant inoculated into culture tubes at 22-28° C with illumination of light 2400 lux, with a photoperiod of 8-16 hours and a relative humidity of about 60%. *****Based on the explants some other plant tissue culture types are: 1.     Organ culture 2.     Meristem culture 3.     Protoplast culture 4.     Cell culture.

1. PLANT
TISSUE CULTURE
PREPARED BY:
RAVINDRA KUMAR KACHHAP ORAON
Biotechnologist, Entrepreneur, Researcher
Education Qualification: B.Sc.(H) Biotechnology,
M.Sc. Biotechnology

2. Plant tissue culture: what is it?
 Plant tissue culture is a technique of growing plant cells, tissues, organs, seeds, or other plant parts in a sterile
environment on a nutrient medium.
 Tissue culture had its origins at the beginning of the 20th century with the work of Gottlieb Haberlandt
(plants).
INTRODUCTION

3. WHY PLANT TISSUE CULTURES ARE DONE ??
 The production of clones of plants that produce particularly good flowers,
fruits, or have other desirable traits.
 To quickly produce mature plants.
 The production of multiples of plants in the absence of seeds or necessary pol
linators to produce seeds.
 The regeneration of whole plants from plant cells that have been genetically
modified.
 The production of plants in sterile containers reduces disease transmission
 Allows production of plants from seeds that otherwise have very low chances
of germinating and growing, i.e.: orchids and Nepenthes.
 To clean particular plants of viral and other infections and to quickly multiply
these plants as 'cleaned stock' for horticulture and agriculture.

4. Laboratory Facilities for PTC
For PTC, the laboratory must have the following facilities:
Washing facility for glassware and ovens for drying glassware.
Medium preparation room with autoclave, electronic balance and pH
meter.
Transfer area sterile room with laminar air-flow bench and a positive
pressure ventilation unit called High Efficiency Particulate Air (HEPA)
filter to maintain aseptic condition.
Culture facility: Growing the explant inoculated into culture tubes at 22-
28° C with illumination of light 2400 lux, with a photoperiod of 8-16 hours
and a relative humidity of about 60%.
PTC LAB - 2
PTC LAB-1

5. Technique Involved in PTC
1. Sterilization:
Sterilization is the technique employed to get rid of microbes such as
bacteria and fungi in the culture medium, vessels and explants.
i. Maintenance of Aseptic Environment: During in vitro tissue culture
maintenance of aseptic environmental condition should be followed, i.e.,
sterilization of glassware, forceps, scalpels, and all accessories in wet steam
sterilization by autoclaving at 15 psi (121°C) for 15 to 30 minutes or
dipping in 70% ethanol followed by flaming and cooling.
ii. Sterilization of culture room: Floor and walls are washed first with
detergent and then with 2% sodium hypochlorite or 95% ethanol. The
cabinet of laminar airflow is sterilized by clearing the work surface with
95% ethanol and then exposure of UV radiation for 15 minutes.
Autoclave Hot Air Sterilizer

6. Technique Involved in PTC
iii. Sterilization of Nutrient Media: Culture media are dispensed in glass
containers, plugged with non-absorbent cotton or sealed with plastic closures
and then sterilized using autoclave at 15 psi (121°C) for 15 to 30 minutes.
The plant extracts, vitamins, amino acids and hormones are sterilized by
passing through Millipore filter with 0.2 mm pore diameter and then added to
sterilized culture medium inside Laminar Airflow Chamber under sterile
condition.
iv. Sterilization of Explants: The plant materials to be used for tissue culture
should be surface sterilized by first exposing the material in running tap water
and then treating it in surface sterilization agents like 0.1% mercuric chloride,
70% ethanol under aseptic condition inside the Laminar Air Flow Chamber.
HOT AIR OVEN
LAMINAR AIR FLOW

7. Types of Plant tissue cultures
Based on the explants some other plant tissue culture types are:
1. Organ culture
2. Meristem culture
3. Protoplast culture
4. Cell culture.
1. Organ culture
The culture of embryos, anthers, ovaries, roots, shoots,
or other organs of plants on culture media.
2. Meristem Culture:
The culture of any plant meristematic tissue on culture
media.
Organ Culture
Meristem Culture

8. Types of Plant tissue cultures
3. Protoplast Culture:
Protoplasts are cells without a cell wall, but bounded by a cell me
mbrane or plasma membrane. Using protoplasts, it is possible to re
generate whole plants from single cells and also develop somatic
hybrids.
4. Cell Suspension Culture
The growing of cells including the culture of single cells or small a
ggregates of cells in vitro in liquid medium is known as cell suspensi
on culture. The cell suspension is prepared by transferring a porti
on of callus to the liquid medium and agitated using rotary shake
r instrument. The cells are separated from the callus tissue and used
for cell suspension culture.
Protoplast Culture

9. COMMON EXPLANT STERILIZATION AGENTS
a) Mercuric chloride: Due to its severe toxicity to both plants and people, it is rarely used in laboratories. After being
exposed to mercuric chloride, the explants must be thoroughly rinsed with sterile water to get rid of all chemical traces.
For explant surface sterilization, it is the most popular option.
b) Sodium hypochlorite constitutes 5.25 percent of commercial laundry bleach. Bleach is diluted to a final concentration
of between 0.5 and 1.0% when employed in tissue culture, which is between 10 and 20 percent.
c) Calcium hypochlorite: It is available in powder form, and before using it for sterilization, it must be dissolved in water.
d) Hydrogen Peroxide: It is a chemical for surface sterilization that is rarely used. If you are employing the chemical to
sterilize the explants` surfaces, exercise extreme caution. Although 70% ethanol is a common sterilizing agent in tissue
culture facilities, it is also very phytotoxic.

10. COMMON EXPLANT STERILIZATION AGENTS
Sterilizing Agent Concn used Ease of Removal Treatment time(min) Remarks
Sodium hypochlorite
Calcium hypochlorite
Hydrogen peroxide
Bromine water
Silver nitrate
Mercuric chloride
Antibiotics
1-1.4%
9-10%
10-12%
1-2%
1%
0.01-1%
4-50 mg/l
+++
+++
+++++
+++
+
+
++
5-30
5-30
5-15
2-10
5-30
2-10
30-60
Very effective
Very effective
Effective
Very effective
Effective
Satisfactory
Effective
*Common usage rate is 20% v/v of the commercial solution

11. The stock solution of MS-Medium
The following four main elements are present in the media:
a) Inorganic nutrient: Mineral salts that are necessary for the growth and development of plants are included in inorganic
nutrients. Macronutrients (Calcium, Magnesium, and Nitrogen) and micronutrients are divided into two categories
(copper, iron, and zinc).
a) Organic nutrients: These are essential for the development and differentiation of cultures, and the mostly consist of
vitamins and amino acids.
b) Growth Hormones: Auxins, cytokinin, and gibberellins are growth hormones. It is necessary for the expansion and
maturation of tissues and organs.
c) Gelling agents: Agar and gelatine are used as gelling agents. It offers the cultures assistance for their development.

12. The stock solution of MS-Medium
S.N. Ingredients Stock solution (mg/100ml) Volume used for final Concn
(ml L-1)
1.
2.
3.
MACRONUTRIENTS
Stock solution I
CaCl2.2H2O
Stock solution II
MgSO4.7H2O
KH2PO4
Stock solution III
FeSO4.7H2O
Na2-EDTA
4400
3700
1700
2785
3725
10
10
10

13. The stock solution of MS-Medium
S.N. Ingredients Stock solution
(mg/100ml)
Volume used for final Concn
(ml L-1)
4.
MICRONUTRIENTS
Stock solution IV
H3BO3
MnSO4.4H2O
ZnSO4.4H2O
KI
Na2MoO4.2H2O
CuSO4.5H2O
CoCl2.6H2O
620
2230
860
83
25
2.5
2.5
1

14. The stock solution of MS-Medium
S.N. Ingredients Stock solution (mg/
100ml)
Volume used for final Concn
(ml L-1)
5.
VITAMINS
Stock solution V
Nicotinic acid
Thiamine-HCL
Pyridoxine-HCL
Glycine
50
10
50
200 1
1.
2.
3.
4.
Directly used Macronutrients
NH4NO3
KNO3
Sucrose
Myo-inositol
1,600
1,900
30,000
100

15. Stock solution of Growth Hormones
PGR Solvent for dissolving Distilled water
(ml)
Concentration used in
mg/ml
Stock (mg/ml)
2,4-D 50% ethanol 100 100 1
α- NAA 1N NaOH 100 100 1
Kinetin 1N NaOH 100 100 1
IAA 1N NaOH 100 100 1
BAP 1N NaOH 100 100 1
GA 50% ethanol 100 100 1
TDZ DMSO/KOH/50% ethanol 100 100 1

16. Stages of Plant Tissue Culture
Following are the four steps that make up the commercial manufacturing of tissue
culture plantlets:
STAGE I: Initiation; In this stage, plant tissues are established in the appropriate in
vitro environment. In addition, the material must be sterilized under sterilized
conditions. After sterilization, the material is introduced to the tissue culture.
STAGE II: Phase of multiplication; This phase involves multiplication. A new division
is made in vitro, followed by placing it in a plant growth medium. In this case, the plant
material is also supported by growth regulators that allow multiple shoots to grow. This
process needs to be repeated depending on the total number of plants required.
STAGE III: Establishment of roots; As roots begin to form, this phase is called the
rooting phase. The hormones stimulate plant growth, resulting in complete plantlets. The
plants are then transported to greenhouses after completing these three phases.
STAGE IV: Hardening or Acclimatization; In the fourth stage, acclimatization takes pl
ace. As a result, they become accustomed to living outside the strict confines of the labo
ratory. Use quality tissue culture kits for DIY tissue culture if you're interested in using
tissue culture at home.
STAGE I
Initiation Phase
STAGE II
Mutiplication
Phase
STAGE III
Rooting Phase
STAGE IV
Hardening
or
Acclimitization

17. STAGES OF PLANT TISSUE CULTURE

18. APPLICATIONS
Plant tissue culture techniques have several applications such as:
 Improved hybrids production through somatic hybridization.
 Somatic embryoids can be encapsulated into synthetic seeds (synseeds).
These encapsulated seeds or synthetic seeds help the conservation of plant
biodiversity.
 Production of disease-resistant plants through meristem and shoot tip
culture.
 Production of stress-resistant plants like herbicide-tolerant, and heat-tolerant
plants.
 Micropropagation technique to obtain large numbers of plantlets of both
crop and tree species useful in forestry within a short span of time and all
through the year.
 Production of secondary metabolites from cell culture utilized in
pharmaceutical, cosmetic, and food industries.

19. REFERENCES
Introduction to plant tissue culture MK Razdan
Introduction to biotechnology H.S. Chawla
Biotechnology Expanding Horizons B.D.Singh
Plant Biotechnology Laboratory Manual for Plant Biotechnology H.S.Chawla